0	Molecular Biology of the Cell, otherFeat=[]-->, belongsTo=nota_cab_pie
1	Vol. 19, 2424 ?2432, June 2008, otherFeat=[]-->, belongsTo=nota_cab_pie
2	A Novel Protein Phosphatase is a Binding Partner for the, otherFeat=[]-->, belongsTo=title
3	Protein Kinase Domains of UNC-89 (Obscurin) in, otherFeat=[]-->, belongsTo=title
4	Caenorhabditis elegans, otherFeat=[]-->, belongsTo=title
5	Hiroshi Qadota,* Lee Anne McGaha,* Kristina B. Mercer,* Thomas J. Stark,*, otherFeat=[]-->, belongsTo=title
6	Tracey M. Ferrara,* and Guy M. Benian*, otherFeat=[]-->, belongsTo=title
7	*Department of Pathology, Graduate Division of Biological and Biomedical Sciences, Emory University,, otherFeat=[]-->, belongsTo=title
8	Atlanta, GA 30322, otherFeat=[]-->, belongsTo=title
9	Submitted January 22, 2008; Revised March 3, 2008; Accepted March 5, 2008, otherFeat=[]-->, belongsTo=parrnote
10	Monitoring Editor: Erika Holzbaur, otherFeat=[]-->, belongsTo=parrnote
11	Mutation of the Caenorhabditis elegans gene unc-89 results in disorganization of muscle A-bands. unc-89 encodes a giant, otherFeat=[]-->, belongsTo=parr
12	polypeptide (900 kDa) containing two protein kinase domains, PK1 and PK2. Yeast two-hybrid screening using a portion, otherFeat=[]-->, belongsTo=parr
13	of UNC-89 including PK2, yielded SCPL-1 (small CTD phosphatase-like-1), which contains a C terminal domain (CTD), otherFeat=[]-->, belongsTo=parr
14	phosphatase type domain. In addition to the PK2 domain, interaction with SCPL-1 required the putative autoinhibitory, otherFeat=[]-->, belongsTo=parr
15	sequence, and immunoglobulin (Ig) and fibronectin type 3 (Fn3) domains lying N-terminal of the kinase domain. SCPL-1, otherFeat=[]-->, belongsTo=parr
16	also interacts with PK1, and it similarly requires the kinase domain and upstream Fn3 and Ig domains. Analogous regions, otherFeat=[]-->, belongsTo=parr
17	from the two other giant kinases of C. elegans, twitchin and TTN-1, failed to interact with SCPL-1. The interaction between, otherFeat=[]-->, belongsTo=parr
18	SCPL-1 and either Ig-Fn3-PK2 or Fn3-Ig-PK1 was confirmed by biochemical methods. The scpl-1b promoter is expressed, otherFeat=[]-->, belongsTo=parr
19	in the same set of muscles as unc-89. Antibodies to SCPL-1 localize to the M-line and a portion of the I-band. Bacterially, otherFeat=[]-->, belongsTo=parr
20	expressed SCPL-1 proteins have phosphatase activity in vitro with properties similar to previously characterized members, otherFeat=[]-->, belongsTo=parr
21	of the CTD phosphatase family. RNA interference knockdown results in a defect in the function of egg-laying muscles., otherFeat=[]-->, belongsTo=parr
22	These studies suggest a new role for the CTD phosphatase family, that is, in muscle giant kinase signaling., otherFeat=[]-->, belongsTo=parr
23	INTRODUCTION, otherFeat=[]-->, belongsTo=parr
24	The sarcomere is the molecular machine that performs the, otherFeat=[]-->, belongsTo=parr
25	work of contraction in striated muscle. It is comprised of, otherFeat=[]-->, belongsTo=parr
26	overlapping, interacting thin and thick filaments and their, otherFeat=[]-->, belongsTo=parr
27	attachment structures, the M-lines and Z-disks. The assem-, otherFeat=[]-->, belongsTo=parr
28	bly and maintenance of the sarcomere involves specific in-, otherFeat=[]-->, belongsTo=parr
29	teractions between hundreds of different proteins. Sarco-, otherFeat=[]-->, belongsTo=parr
30	meres are unusually enriched for very large polypeptides,, otherFeat=[]-->, belongsTo=parr
31	generally 700,000 Da, including nebulin and titin. These, otherFeat=[]-->, belongsTo=parr
32	giant proteins are primarily composed of multiple inexact, otherFeat=[]-->, belongsTo=parr
33	copies of small domains, typically 35?100 residues. From the, otherFeat=[]-->, belongsTo=parr
34	now extensive knowledge about the many binding partners, otherFeat=[]-->, belongsTo=parr
35	of the 3-MDa polypeptide titin in vertebrate muscle, several, otherFeat=[]-->, belongsTo=parr
36	themes have emerged (Lange et al., 2006). First, depending, otherFeat=[]-->, belongsTo=parr
37	on its location within the titin polypeptide chain, the same, otherFeat=[]-->, belongsTo=parr
38	type of domain, the immunoglobulin domain, can interact, otherFeat=[]-->, belongsTo=parr
39	with many different partners. Second, some titin interactors, otherFeat=[]-->, belongsTo=parr
40	are located only at the sarcomere, whereas others are located, otherFeat=[]-->, belongsTo=parr
41	at the sarcomere and the nucleus. One of the newest muscle, otherFeat=[]-->, belongsTo=parr
42	giant modular proteins to be identified is UNC-89 in Caeno-, otherFeat=[]-->, belongsTo=parr
43	rhabditis elegans (Ferrara et al., 2005)., otherFeat=[]-->, belongsTo=parr
44	The nematode C. elegans is an excellent system in which to, otherFeat=[]-->, belongsTo=parr
45	study the organization, assembly, and function of striated, otherFeat=[]-->, belongsTo=parr
46	muscle in a whole organism (Waterston, 1988; Moerman and, otherFeat=[]-->, belongsTo=parr
47	Fire, 1997; Moerman and Williams, 2006). The gene unc-89, otherFeat=[]-->, belongsTo=parr
48	was originally identified in genetic screens for worms that, otherFeat=[]-->, belongsTo=parr
49	are both slow moving or paralyzed and that have disorga-, otherFeat=[]-->, belongsTo=parr
50	nization of their myofilament lattice (Waterston et al., 1980),, otherFeat=[]-->, belongsTo=parr
51	and then molecularly cloned (Benian et al., 1996). Obscurin, otherFeat=[]-->, belongsTo=parr
52	was originally identified through a two-hybrid screen by, otherFeat=[]-->, belongsTo=parr
53	using portions of titin as bait (Bang et al., 2001; Young et al.,, otherFeat=[]-->, belongsTo=parr
54	2001), and identified as a homologue of UNC-89 (Young et, otherFeat=[]-->, belongsTo=parr
55	al., 2001). The body wall muscle of unc-89 mutants shows an, otherFeat=[]-->, belongsTo=parr
56	especial disorganization of the A-band, and for most mutant, otherFeat=[]-->, belongsTo=parr
57	alleles, lacks M-lines, the structures in the middle of the, otherFeat=[]-->, belongsTo=parr
58	A-band at which thick filaments are cross-linked (Waterston, otherFeat=[]-->, belongsTo=parr
59	et al., 1980; Benian et al., 1999). Similarly, obscurin has been, otherFeat=[]-->, belongsTo=parr
60	shown to have a role in the assembly or maintenance of, otherFeat=[]-->, belongsTo=parr
61	A-bands: overexpression of a C-terminal portion of obscurin, otherFeat=[]-->, belongsTo=parr
62	in primary skeletal myotubes results in a disorganization of, otherFeat=[]-->, belongsTo=parr
63	sarcomeric myosin (Kontrogianni-Konstantopoulos et al.,, otherFeat=[]-->, belongsTo=parr
64	2004)., otherFeat=[]-->, belongsTo=parr
65	unc-89 is a complex gene, which through the use of three, otherFeat=[]-->, belongsTo=parr
66	different promoters expressed in different sets of muscles,, otherFeat=[]-->, belongsTo=parr
67	and alternative splicing, generates at least six major protein, otherFeat=[]-->, belongsTo=parr
68	isoforms UNC-89-A?F (Benian et al., 1996; Small et al., 2004;, otherFeat=[]-->, belongsTo=parr
69	Ferrara et al., 2005). The largest of these isoforms, UNC-89-B, otherFeat=[]-->, belongsTo=parr
70	and UNC-89-F, which are each 900,000 Da, contain 52, otherFeat=[]-->, belongsTo=parr
71	immunoglobulin (Ig) domains; two fibronectin type 3 (Fn3), otherFeat=[]-->, belongsTo=parr
72	domains; a triplet of Src homology 3 (SH3), Dbl homology, otherFeat=[]-->, belongsTo=parr
73	(DH), and pleckstrin homology (PH) domains near their N, otherFeat=[]-->, belongsTo=parr
74	termini; and two protein kinase domains near their C ter-, otherFeat=[]-->, belongsTo=parr
75	mini. Two somewhat smaller isoforms, UNC-89-A and, otherFeat=[]-->, belongsTo=parr
76	UNC-89-E, lack the kinase domains at their C termini. The, otherFeat=[]-->, belongsTo=parr
77	smallest isoforms, UNC-89-C and UNC-89-D, begin with, otherFeat=[]-->, belongsTo=parr
78	partial first kinase domains, and each is directed by its own, otherFeat=[]-->, belongsTo=parr
79	tissue-specific promoter. The human obscurin gene similarly, otherFeat=[]-->, belongsTo=parr
80	This article was published online ahead of print in MBC in Press, otherFeat=[]-->, belongsTo=parrnote
81	(http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E08 ? 01? 0053), otherFeat=[]-->, belongsTo=parrnote
82	on March 12, 2008., otherFeat=[]-->, belongsTo=parrnote
83	Address correspondence to: Guy M. Benian (pathgb@emory.edu)., otherFeat=[]-->, belongsTo=parrnote
84	2424, otherFeat=[]-->, belongsTo=nota_cab_pie
85	? 2008 by The American Society for Cell Biology, otherFeat=[]-->, belongsTo=nota_cab_pie
86	has 3 -most exons encoding two protein kinase domains,, otherFeat=[]-->, belongsTo=parr
87	with differential splicing producing obscurin A, which lacks, otherFeat=[]-->, belongsTo=parr
88	the kinase domains (like UNC-89-A and -E); obscurin B (like, otherFeat=[]-->, belongsTo=parr
89	UNC-89-B and -F), which contains the kinase domains; and, otherFeat=[]-->, belongsTo=parr
90	two smaller obscurins, one obscurin of which contains intact, otherFeat=[]-->, belongsTo=parr
91	first and second kinase domains (Bang et al., 2001; Russell et, otherFeat=[]-->, belongsTo=parr
92	al., 2002; Fukuzawa et al., 2005). Although obscurin contains, otherFeat=[]-->, belongsTo=parr
93	all the same domains as nematode UNC-89, a difference is, otherFeat=[]-->, belongsTo=parr
94	that the SH3, DH, PH trio is located near the C terminus, otherFeat=[]-->, belongsTo=parr
95	rather than near the N terminus as it is in UNC-89. For C., otherFeat=[]-->, belongsTo=parr
96	elegans UNC-89, homology modeling indicates that the first, otherFeat=[]-->, belongsTo=parr
97	kinase domain (PK1) is inactive, but that the second kinase, otherFeat=[]-->, belongsTo=parr
98	domain (PK2) may have kinase activity (Small et al., 2004)., otherFeat=[]-->, belongsTo=parr
99	So far, however, neither substrates nor phosphotransferase, otherFeat=[]-->, belongsTo=parr
100	activity has been demonstrated. Antibodies generated to, otherFeat=[]-->, belongsTo=parr
101	three distinct regions of UNC-89 localize the proteins exclu-, otherFeat=[]-->, belongsTo=parr
102	sively to the M-lines by immunofluorescence microscopy,, otherFeat=[]-->, belongsTo=parr
103	and more recently, by immuno-electron microscopy (A., otherFeat=[]-->, belongsTo=parr
104	Reedy, G. Benian, and P. Hoppe, unpublished data). Verte-, otherFeat=[]-->, belongsTo=parr
105	brate obscurin is located at the peripheries of both M-lines, otherFeat=[]-->, belongsTo=parr
106	and Z-disks (Kontrogianni-Konstantopoulos et al., 2003)., otherFeat=[]-->, belongsTo=parr
107	However, the situation is more complex, with obscurin A, otherFeat=[]-->, belongsTo=parr
108	(lacking kinase domains) located at the M-line, obscurin B, otherFeat=[]-->, belongsTo=parr
109	located at the A/I junction and additional isoforms located, otherFeat=[]-->, belongsTo=parr
110	at the Z-disk and Z/I junction (Bowman et al., 2007)., otherFeat=[]-->, belongsTo=parr
111	To help understand how UNC-89 performs its functions,, otherFeat=[]-->, belongsTo=parr
112	we wanted to identify its binding partners. To begin, we, otherFeat=[]-->, belongsTo=parr
113	have focused on the C-terminal protein kinase domains. We, otherFeat=[]-->, belongsTo=parr
114	expected that binding partners of the protein kinase do-, otherFeat=[]-->, belongsTo=parr
115	mains would include kinase substrate(s), activator/inhibitor, otherFeat=[]-->, belongsTo=parr
116	of the kinases or coregulators. Using the yeast two-hybrid, otherFeat=[]-->, belongsTo=parr
117	method, we found that a novel protein phosphatase binds to, otherFeat=[]-->, belongsTo=parr
118	each of the protein kinase domains of UNC-89., otherFeat=[]-->, belongsTo=parr
119	MATERIALS AND METHODS, otherFeat=['U']-->, belongsTo=parr
120	Worm Strains, Culture, and RNA Interference (RNAi), otherFeat=[]-->, belongsTo=parr
121	Nematodes were grown at 20?C on NGM plates with Escherichia coli strain, otherFeat=[]-->, belongsTo=parrnote
122	OP50 as food source (Brenner, 1974). Bristol N2 was the wild-type strain, and, otherFeat=[]-->, belongsTo=parrnote
123	mutants included unc-89 (su75) (Small et al., 2004), unc-89 (tm752) (Ferrara et, otherFeat=[]-->, belongsTo=parrnote
124	al., 2005), and two intragenic deletions from the C. elegans Gene Knockout, otherFeat=[]-->, belongsTo=parrnote
125	Consortium, scpl-1(ok1080) and scpl-1(gk283). RNAi for scpl-1 was performed, otherFeat=[]-->, belongsTo=parrnote
126	on wild type and unc-89 mutants by using a feeding procedure essentially as, otherFeat=[]-->, belongsTo=parrnote
127	described in Kamath and Afhringer (2003). Full-length cDNAs for scpl-1a and, otherFeat=[]-->, belongsTo=parrnote
128	scpl-1b were cloned into pPD129.36, and these plasmids or the empty vector, otherFeat=[]-->, belongsTo=parrnote
129	were transformed into HT115 (DE3) bacteria before feeding the worms., otherFeat=[]-->, belongsTo=parrnote
130	Plasmid Construction, otherFeat=[]-->, belongsTo=parr
131	For the screening of the two-hybrid library with Ig-Fn3-PK2 kinase, this, otherFeat=[]-->, belongsTo=parrnote
132	region of UNC-89 was cloned into the bait plasmid pGBDU-C1 by using, otherFeat=[]-->, belongsTo=parrnote
133	BamHI and SalI sites of the vector. This region of UNC-89, called 15/14 in, otherFeat=[]-->, belongsTo=parrnote
134	Figure 1A, was polymerase chain reaction (PCR) amplified from the RB2, otherFeat=[]-->, belongsTo=parrnote
135	random primed cDNA library (a gift from Robert Barstead, Oklahoma Med-, otherFeat=[]-->, belongsTo=parrnote
136	ical Research Foundation), by using primers NTSY-15 and NTSY-14 (see, otherFeat=[]-->, belongsTo=parrnote
137	Supplemental Table 1 for all primer sequences)., otherFeat=[]-->, belongsTo=parrnote
138	To test for interaction between SCPL-1a or -1b prey and various segments, otherFeat=[]-->, belongsTo=parrnote
139	of UNC-89 surrounding the PK2 kinase, we first made a series of three baits, otherFeat=[]-->, belongsTo=parrnote
140	in the pGBDU-C1 vector, with the same N terminus lying just after the end of, otherFeat=[]-->, belongsTo=parrnote
141	the Fn3 domain and varying amounts of sequence C-terminal of the PK2, otherFeat=[]-->, belongsTo=parrnote
142	kinase catalytic core called 11/12, 11/13, and 11/14 (Figure 1A). PCR was, otherFeat=[]-->, belongsTo=parrnote
143	used to amplify the corresponding coding sequences from the RB2 cDNA, otherFeat=[]-->, belongsTo=parrnote
144	library, by using the same 5 primer, NTSY-11, with added BamHI site, and, otherFeat=[]-->, belongsTo=parrnote
145	three different 3 primers with added SalI site, NTSY-12, NTSY-13, or NTSY-, otherFeat=[]-->, belongsTo=parrnote
146	14. Two additional PK2 region baits were also made similarly, called 15/12, otherFeat=[]-->, belongsTo=parrnote
147	and 15/13 (Figure 1A), by using the primers NTSY-15, -12, and -13., otherFeat=[]-->, belongsTo=parrnote
148	To construct two-hybrid plasmids expressing various fragments of SCPL-1,, otherFeat=[]-->, belongsTo=parrnote
149	PCR-amplified fragments of scpl-1 were cloned into the plasmid pGAD-C1 by, otherFeat=[]-->, belongsTo=parrnote
150	using EcoRI and SalI sites of the vector. The primers used for amplification are, otherFeat=[]-->, belongsTo=parrnote
151	as follows: SCPL-1-1 and SCPL-1-3 for full length (1-345 amino acids aa )of, otherFeat=[]-->, belongsTo=parrnote
152	SCPL-1a; SCPL-1-2 and SCPL-1-3 for full length (1-491 aa) of SCPL-1b; SCPL-, otherFeat=[]-->, belongsTo=parrnote
153	1-1 and SCPL-1-4 for amino acids 1-156 of SCPL-1a; SCPL-1-2 and SCPL-1-4, otherFeat=[]-->, belongsTo=parrnote
154	for amino acids 1-302 of SCPL-1b; SCPL-1-2 and SCPL-1-5 for amino acids, otherFeat=[]-->, belongsTo=parrnote
155	1-146 of SCPL-1b; and SCPL-1-11 and SCPL-1-3 for amino acids 157-345 of, otherFeat=[]-->, belongsTo=parrnote
156	SCPL-1a (amino acids 303-491 of SCPL-1b). The prey plasmid harboring, otherFeat=[]-->, belongsTo=parrnote
157	amino acids 245-491 of SCPL-1b was isolated from the library during two-, otherFeat=[]-->, belongsTo=parrnote
158	hybrid screening., otherFeat=[]-->, belongsTo=parrnote
159	To test whether the Ig-Fn3-kinase regions from twitchin and TTN-1 interact, otherFeat=[]-->, belongsTo=parrnote
160	with SCPL-1a, -1b,-2, -3a, or -4, these regions of twitchin and TTN-1 were first, otherFeat=[]-->, belongsTo=parrnote
161	cloned into the bait plasmid pGBDU-C1 by using the BamHI and SalI sites of, otherFeat=[]-->, belongsTo=parrnote
162	the vector. The relevant regions of twitchin were PCR amplified from the RB2, otherFeat=[]-->, belongsTo=parrnote
163	cDNA library by using primers TWI-F and TWI-R, and for TTN-1, the primers, otherFeat=[]-->, belongsTo=parrnote
164	were TTN-F and TTN-R., otherFeat=[]-->, belongsTo=parrnote
165	Figure 1. (A) Yeast two-hybrid assays demonstrate the specificity, otherFeat=[]-->, belongsTo=fig_caption
166	of the interaction of UNC-89 PK2 with SCPL-1. Left, schematic, otherFeat=[]-->, belongsTo=fig_caption
167	representation of baits used to test full-length SCPL-1a and -b prey., otherFeat=[]-->, belongsTo=fig_caption
168	Right, images of yeast growth on Ade plates. Note that for UNC-, otherFeat=[]-->, belongsTo=fig_caption
169	89, interaction requires the catalytic core (PK2) plus the N-terminal, otherFeat=[]-->, belongsTo=fig_caption
170	Ig and Fn3 domains, and the C-terminal autoinhibitory domain., otherFeat=[]-->, belongsTo=fig_caption
171	Note that comparable regions from the two other giant kinases in, otherFeat=[]-->, belongsTo=fig_caption
172	the worm, twitchin and TTN-1, fail to interact with SCPL-1. (B) The, otherFeat=[]-->, belongsTo=fig_caption
173	PK1 region of UNC-89 also interacts with SCPL-1 in the yeast, otherFeat=[]-->, belongsTo=fig_caption
174	two-hybrid assay. Domain mapping indicates that interaction of the, otherFeat=[]-->, belongsTo=fig_caption
175	PK1 region with SCPL-1 requires, in addition to the catalytic core, otherFeat=[]-->, belongsTo=fig_caption
176	(PK1), the Fn3 and Ig domains. , growth and , no growth on, otherFeat=[]-->, belongsTo=fig_caption
177	Ade plates. (C) Only the phosphatase domain of SCPL-1 is re-, otherFeat=[]-->, belongsTo=fig_caption
178	quired for interaction to the PK regions of UNC-89 in yeast two-, otherFeat=[]-->, belongsTo=fig_caption
179	hybrid assays. The indicated portions of SCPL-1a and -1b were, otherFeat=[]-->, belongsTo=fig_caption
180	tested as bait against Fn3-Ig-PK1 (PK1) or Ig-Fn3-PK2 (PK2) prey. ,, otherFeat=[]-->, belongsTo=fig_caption
181	growth and , no growth on Ade plates. The colored bar indicates, otherFeat=[]-->, belongsTo=fig_caption
182	the minimal region of SCPL-1a/b required for interaction., otherFeat=[]-->, belongsTo=fig_caption
183	Novel Phosphatase Binds Giant Protein Kinase, otherFeat=[]-->, belongsTo=nota_cab_pie
184	Vol. 19, June 2008, otherFeat=[]-->, belongsTo=nota_cab_pie
185	2425, otherFeat=[]-->, belongsTo=nota_cab_pie
186	To determine whether SCPL-1a or -1b preys interact with PK1 region baits,, otherFeat=[]-->, belongsTo=parrnote
187	baits called AB, EB, FB, and AG (Figure 1B), were made in pGBDU-C1 by, otherFeat=[]-->, belongsTo=parrnote
188	using the SmaI and SalI sites, by insertion of PCR-amplified fragments by, otherFeat=[]-->, belongsTo=parrnote
189	using primers U89-PK1-A, U89-PK1-B, U89-PK1-E, U89-PK1-F, and U89-, otherFeat=[]-->, belongsTo=parrnote
190	PK1-G., otherFeat=[]-->, belongsTo=parrnote
191	To test whether UNC-89 Ig-Fn3-PK2 of UNC-89 or comparable regions of, otherFeat=[]-->, belongsTo=parrnote
192	twitchin or TTN-1 interact with SCPL-2, -3a, and -4, full-length cDNAs for, otherFeat=[]-->, belongsTo=parrnote
193	these other phosphatases were PCR amplified by using the following primers,, otherFeat=[]-->, belongsTo=parrnote
194	and then they were inserted into the prey plasmid pGAD-C1 by using BamHI, otherFeat=[]-->, belongsTo=parrnote
195	and SalI: for SCPL-2, SCPL-2-F and -R; for SCPL-3a, SCPL-3-F and -R; and for, otherFeat=[]-->, belongsTo=parrnote
196	SCPL-4, SCPL-4-F and -R., otherFeat=[]-->, belongsTo=parrnote
197	To express hemagglutinin (HA)-tagged UNC-89 fragments in yeast, we, otherFeat=[]-->, belongsTo=parrnote
198	prepared the following plasmids. The PCR-amplified fragments of the rele-, otherFeat=[]-->, belongsTo=parrnote
199	vant region of UNC-89 PK2 by using the primers U89-PK2-1 and -2 for, otherFeat=[]-->, belongsTo=parrnote
200	fragment 11/12; U89-PK2-1 and -3 for fragment 11/13; U89-PK2-1 and -4 for, otherFeat=[]-->, belongsTo=parrnote
201	11/14; U89-PK2-5 and -2 for 15/12; U89-PK2-5 and -3 for 15/13; and U89-, otherFeat=[]-->, belongsTo=parrnote
202	PK2-5 and -4 for 15/14 were cloned into pKS-HA8(XbaI) (three HA-tagged, otherFeat=[]-->, belongsTo=parrnote
203	vectors) by using the EcoRV site of the vector. The PCR-amplified fragments, otherFeat=[]-->, belongsTo=parrnote
204	from the relevant region of UNC-89 PK1 by using the primers as described, otherFeat=[]-->, belongsTo=parrnote
205	above were cloned into pKS-HA8(Nhex2) (three HA-tagged vectors) by using, otherFeat=[]-->, belongsTo=parrnote
206	EcoRV and SalI sites. HA-tagged UNC-89 11/12, 11/13, 11/14, 15/12, 15/13,, otherFeat=[]-->, belongsTo=parrnote
207	and 15/14, AB were cut out with XbaI or NheI from HA-tagged vectors and, otherFeat=[]-->, belongsTo=parrnote
208	cloned into pGAP-C-Nhe (yeast expression vector, TRP1 marker) by using the, otherFeat=[]-->, belongsTo=parrnote
209	NheI site of the vector., otherFeat=[]-->, belongsTo=parrnote
210	To express myc-tagged SCPL-1 in yeast, we prepared the following plas-, otherFeat=[]-->, belongsTo=parrnote
211	mids. The PCR-amplified fragments of full-length SCPL-1a and SCPL-1b by, otherFeat=[]-->, belongsTo=parrnote
212	using the primers as described above, were cloned into pKK51 (myc-tagged, otherFeat=[]-->, belongsTo=parrnote
213	vector) by using EcoRI and XhoI sites of the vector. Myc-tagged SCPL-1a and, otherFeat=[]-->, belongsTo=parrnote
214	myc-tagged SCPL-1b fragments were cut out with SpeI from myc-tagged, otherFeat=[]-->, belongsTo=parrnote
215	vector and cloned into pGAPU-C-Nhe (yeast expression vector, URA3, otherFeat=[]-->, belongsTo=parrnote
216	marker) by using the NheI site of the vector., otherFeat=[]-->, belongsTo=parrnote
217	To express glutathione-S transferase (GST) or maltose-binding protein, otherFeat=[]-->, belongsTo=parrnote
218	(MBP) fusions of SCPL-1 in bacteria, we prepared the following. The full-, otherFeat=[]-->, belongsTo=parrnote
219	length fragments of SCPL-1a and SCPL-1b were PCR amplified by using, otherFeat=[]-->, belongsTo=parrnote
220	primers as described above and cloned into pMAL-KK-1 by using EcoRI and, otherFeat=[]-->, belongsTo=parrnote
221	XhoI sites. The fragments of SCPL-1 (1-156 aa of SCPL-1a, 1-302 aa of SCPL-, otherFeat=[]-->, belongsTo=parrnote
222	1b, and 1-146 aa of SCPL-1b) were PCR amplified by using the primers, otherFeat=[]-->, belongsTo=parrnote
223	described above. Amplified fragments were cloned into pGEX-KK-1 and, otherFeat=[]-->, belongsTo=parrnote
224	pMAL-KK-1 by using EcoRI and XhoI sites of the vectors. The plasmids, otherFeat=[]-->, belongsTo=parrnote
225	derived from pGEX-KK-1 were used for production of GST fusion proteins,, otherFeat=[]-->, belongsTo=parrnote
226	whereas the plasmids derived from pMAL-KK-1 were used for production of, otherFeat=[]-->, belongsTo=parrnote
227	MBP fusion proteins., otherFeat=[]-->, belongsTo=parrnote
228	Yeast Two-Hybrid Screens and Assays, otherFeat=[]-->, belongsTo=parr
229	Two-hybrid screening was performed as described in Miller et al. (2006). Use, otherFeat=[]-->, belongsTo=parrnote
230	of the two-hybrid method to study protein?protein interaction was performed, otherFeat=[]-->, belongsTo=parrnote
231	as described in Mackinnon et al. (2002)., otherFeat=[]-->, belongsTo=parrnote
232	Purification of Bacterially Expressed Proteins, otherFeat=[]-->, belongsTo=parr
233	GST or MBP fusions proteins were prepared as described in Mercer et al., otherFeat=[]-->, belongsTo=parrnote
234	(2006)., otherFeat=[]-->, belongsTo=parrnote
235	Assays to Confirm Interaction between SCPL-1 and, otherFeat=[]-->, belongsTo=parr
236	UNC-89 PK2 or PK1, otherFeat=[]-->, belongsTo=parr
237	To confirm an interaction between SCPL-1 and the PK2 region of UNC-89, we, otherFeat=[]-->, belongsTo=parrnote
238	expressed in the same yeast cell myc tagged SCPL-1b together with HA-, otherFeat=[]-->, belongsTo=parrnote
239	tagged versions of one of the portions of UNC-89 containing PK2 as shown in, otherFeat=[]-->, belongsTo=parrnote
240	Figure 1A (11/12, 11/13, 11/14, 15/12, 15/13, 15/14), as described above., otherFeat=[]-->, belongsTo=parrnote
241	Yeast were grown in 50 ml of minimal media plus dextrose, casamino acids,, otherFeat=[]-->, belongsTo=parrnote
242	and adenine (SD CA) to an optical density of 0.6 at 600 nm, pelleted, and, otherFeat=[]-->, belongsTo=parrnote
243	frozen at 80?C. Each yeast pellet was resuspended in 500 l of yeast lysis, otherFeat=[]-->, belongsTo=parrnote
244	buffer (50 mM HEPES, pH 7.5, 150 mM KCl, 1 mM EGTA, 1 mM EDTA, and, otherFeat=[]-->, belongsTo=parrnote
245	complete Mini protease inhibitors, Roche, Indianapolis, IN). The yeast slurries, otherFeat=[]-->, belongsTo=parrnote
246	were transferred to 1.5-ml Eppendorf tubes that contained 250 l of glass, otherFeat=[]-->, belongsTo=parrnote
247	beads (425? 600 m in diameter; G-8772, Sigma-Aldrich, St. Louis, MO). Yeast, otherFeat=[]-->, belongsTo=parrnote
248	were lysed by vortexing at maximum speed, six bursts of 30 s each, separated, otherFeat=[]-->, belongsTo=parrnote
249	by 1 min on ice. After pelleting the beads by centrifugation in a microfuge at, otherFeat=[]-->, belongsTo=parrnote
250	3000 rpm for 5 min, the supernatant was transferred to a fresh tube, and, otherFeat=[]-->, belongsTo=parrnote
251	cellular debris was pelleted by centrifugation in a microfuge at 13,200 rpm for, otherFeat=[]-->, belongsTo=parrnote
252	30 min. The resulting supernatant was transferred to a new tube, and its total, otherFeat=[]-->, belongsTo=parrnote
253	protein concentration was determined by Bradford assay (Bradford 1976). The, otherFeat=[]-->, belongsTo=parrnote
254	HA-tagged PK2 proteins were immunoprecipitated in a total volume of 500 l, otherFeat=[]-->, belongsTo=parrnote
255	for 1 h with shaking at 4?C from 120 g of protein in immunoprecipitation, otherFeat=[]-->, belongsTo=parrnote
256	(IP) buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1 mM EGTA, 1 mM EDTA,, otherFeat=[]-->, belongsTo=parrnote
257	0.25% gelatin, complete mini protease inhibitors, and 0.1% NP-40) and 50 l, otherFeat=[]-->, belongsTo=parrnote
258	of a 1:1 slurry of agarose beads conjugated monoclonal antibodies to HA, otherFeat=[]-->, belongsTo=parrnote
259	(A2095, Sigma-Aldrich). Beads were pelleted by centrifugation and washed, otherFeat=[]-->, belongsTo=parrnote
260	two times with IP buffer and once with IP buffer lacking NP-40. A final hard, otherFeat=[]-->, belongsTo=parrnote
261	spin for 5 min was performed, and as much supernatant was aspirated off as, otherFeat=[]-->, belongsTo=parrnote
262	possible. Laemmli sample buffer (2 ) was added to the pellet, and then the, otherFeat=[]-->, belongsTo=parrnote
263	mixture was vortexed 5 s, heated at 95?C for 5 min, vortexed for 20 s, and, otherFeat=[]-->, belongsTo=parrnote
264	Figure 2. Verification of interaction between SCPL-1 and UNC-89, otherFeat=[]-->, belongsTo=fig_caption
265	PK1 or PK2 regions. (A) In the same yeast cell, myc-tagged SCPL-1b, otherFeat=[]-->, belongsTo=fig_caption
266	and individual HA-tagged derivatives of the UNC-89 PK2 region, otherFeat=[]-->, belongsTo=fig_caption
267	were expressed. 11/12, 11/13 . . . refer to the derivatives presented, otherFeat=[]-->, belongsTo=fig_caption
268	in Figure 1A. Total protein lysates were incubated with agarose, otherFeat=[]-->, belongsTo=fig_caption
269	beads coated with antibodies to the HA tag, washed, and eluted,, otherFeat=[]-->, belongsTo=fig_caption
270	and portions were run on two gels and blotted. One blot was, otherFeat=[]-->, belongsTo=fig_caption
271	reacted with anti-HA to detect the presence of the PK2 derivatives, otherFeat=[]-->, belongsTo=fig_caption
272	(top). As shown above, appropriately sized proteins were detected, otherFeat=[]-->, belongsTo=fig_caption
273	from each PK2 derivative. The other blot was reacted with anti-myc, otherFeat=[]-->, belongsTo=fig_caption
274	to detect the presence of SCPL-1 that might have been brought, otherFeat=[]-->, belongsTo=fig_caption
275	down with the PK2 protein (bottom). Only derivative 15/14 (Ig-, otherFeat=[]-->, belongsTo=fig_caption
276	Fn3-PK2-autoinhibitory region) is coimmunoprecipitated. An arrow, otherFeat=[]-->, belongsTo=fig_caption
277	designates the position on the blot of myc-SCPL-1b from the lysate., otherFeat=[]-->, belongsTo=fig_caption
278	(B) Bacterially expressed MBP-SCPL-1a interacts with yeast ex-, otherFeat=[]-->, belongsTo=fig_caption
279	pressed HA-PK1 or PK2. Total protein extracts were prepared from, otherFeat=[]-->, belongsTo=fig_caption
280	yeast expressing either HA-PK2 15/14 (see Figure 1A) or HA-PK1, otherFeat=[]-->, belongsTo=fig_caption
281	AB (see Figure 1B), incubated with agarose beads coated with, otherFeat=[]-->, belongsTo=fig_caption
282	antibodies to HA, washed, incubated with purified, bacterially ex-, otherFeat=[]-->, belongsTo=fig_caption
283	pressed MBP or MBP-SCPL-1a, and washed. The proteins were, otherFeat=[]-->, belongsTo=fig_caption
284	eluted, and portions of each sample were run on two gels and, otherFeat=[]-->, belongsTo=fig_caption
285	blotted. One blot was reacted with anti-HA to detect the presence of, otherFeat=[]-->, belongsTo=fig_caption
286	the yeast expressed protein, the other blot was reacted with anti-, otherFeat=[]-->, belongsTo=fig_caption
287	MBP to detect possible binding with MBP or MBP-SCPL-1a. (C), otherFeat=[]-->, belongsTo=fig_caption
288	Coomassie stained gel of purified MBP-SCPL-1a and MBP-SCPL-1b., otherFeat=[]-->, belongsTo=fig_caption
289	In each case, the top band is likely to be the full-length fusion, otherFeat=[]-->, belongsTo=fig_caption
290	protein; the second band may have resulted from degradation., otherFeat=[]-->, belongsTo=fig_caption
291	H. Qadota et al., otherFeat=[]-->, belongsTo=nota_cab_pie
292	Molecular Biology of the Cell, otherFeat=[]-->, belongsTo=nota_cab_pie
293	2426, otherFeat=[]-->, belongsTo=nota_cab_pie
294	centrifuged for 5 min to pellet the beads. The supernatant was then carefully, otherFeat=[]-->, belongsTo=parrnote
295	transferred to a fresh tube. Two 12% SDS-polyacrylamide gels were run, otherFeat=[]-->, belongsTo=parrnote
296	containing either 10 or 40 l of supernatant, and they were transblotted to, otherFeat=[]-->, belongsTo=parrnote
297	nitrocellulose. The blot that had 10 l of supernatant was reacted with, otherFeat=[]-->, belongsTo=parrnote
298	anti-HA (rabbit antibodies, affinity purified, 1:1000; H6908, Sigma-Aldrich),, otherFeat=[]-->, belongsTo=parrnote
299	and the blot that had 40 l of supernatant was reacted with anti-myc (mouse, otherFeat=[]-->, belongsTo=parrnote
300	monoclonal purified immunoglobulin, 1:400; M4439, Sigma-Aldrich), reacted, otherFeat=[]-->, belongsTo=parrnote
301	with appropriate horseradish peroxidase (HRP)-conjugated secondary anti-, otherFeat=[]-->, belongsTo=parrnote
302	bodies, and detected by enhanced chemiluminescence (ECL). Controls in-, otherFeat=[]-->, belongsTo=parrnote
303	cluded no added yeast lysate, and 10 g of total protein from any of the yeast, otherFeat=[]-->, belongsTo=parrnote
304	to verify the expression of the myc-tagged SCPL-1b., otherFeat=[]-->, belongsTo=parrnote
305	To confirm an interaction between SCPL-1 and the PK1 region of UNC-89, otherFeat=[]-->, belongsTo=parrnote
306	and to further verify an interaction with the PK2 region of UNC-89, the, otherFeat=[]-->, belongsTo=parrnote
307	following procedure was used. Yeast expressing either an HA-tagged PK1, otherFeat=[]-->, belongsTo=parrnote
308	region (segment AB in Figure 1B) or HA-tagged PK2 region (15/14 in Figure, otherFeat=[]-->, belongsTo=parrnote
309	1A) were grown; yeast lysates were prepared; and beginning with 300 gof, otherFeat=[]-->, belongsTo=parrnote
310	total protein, the HA-tagged UNC-89 segments were immunoprecipitated by, otherFeat=[]-->, belongsTo=parrnote
311	using anti-HA agarose beads, and washed, as described above. A binding, otherFeat=[]-->, belongsTo=parrnote
312	reaction was set up in which the HA-tagged UNC-89 proteins attached to, otherFeat=[]-->, belongsTo=parrnote
313	beads were incubated at 4?C for 1 h with shaking in a total volume of 500 l, otherFeat=[]-->, belongsTo=parrnote
314	with either bacterially expressed and purified MBP (10 g) or MBP-SCPL-1a, otherFeat=[]-->, belongsTo=parrnote
315	(10 g). The beads were washed three times with IP buffer, transferred to, otherFeat=[]-->, belongsTo=parrnote
316	clean Eppendorf tubes, centrifuged for 5 min, and as much supernatant was, otherFeat=[]-->, belongsTo=parrnote
317	removed as possible. The proteins on the pelleted beads were eluted in, otherFeat=[]-->, belongsTo=parrnote
318	Laemmli buffer as described above, and two 10% SDS-PAGE gels were run, otherFeat=[]-->, belongsTo=parrnote
319	and transferred to nitrocellulose-- one gel contained 5 l of eluted protein per, otherFeat=[]-->, belongsTo=parrnote
320	lane, and the other gel contained 40 l/lane. The blot that had the 5- l, otherFeat=[]-->, belongsTo=parrnote
321	samples was reacted with anti-HA (described above), followed by anti rabbit, otherFeat=[]-->, belongsTo=parrnote
322	Ig-conjugated HRP, and detection by ECL. The blot containing 40- l samples, otherFeat=[]-->, belongsTo=parrnote
323	was incubated with HRP-conjugated anti-MBP (1:5000; New England Biolabs,, otherFeat=[]-->, belongsTo=parrnote
324	Beverly, MA) and detected by ECL., otherFeat=[]-->, belongsTo=parrnote
325	Expression Pattern of scpl-1b Promoter, otherFeat=[]-->, belongsTo=parr
326	A 7.5-kb genomic segment that includes the initiator ATG, 5 -untranslated, otherFeat=[]-->, belongsTo=parrnote
327	region and putative promoter sequence upstream of scpl-1b was produced by, otherFeat=[]-->, belongsTo=parrnote
328	PCR and cloned into pPD95.77 (a gift from A. Fire, Stanford University,, otherFeat=[]-->, belongsTo=parrnote
329	Stanford, CA), by using the SphI and BamHI sites. The primers used were, otherFeat=[]-->, belongsTo=parrnote
330	SCPL-1b-promo-F and SCPL-1b-promo-R. The promoter-gfp expression plas-, otherFeat=[]-->, belongsTo=parrnote
331	mid was injected into wild-type strain N2 worms, and one transgenic line was, otherFeat=[]-->, belongsTo=parrnote
332	recovered. Green fluorescent protein (GFP) fluorescence images of adults, otherFeat=[]-->, belongsTo=parrnote
333	were obtained as described in Qadota et al., 2007., otherFeat=[]-->, belongsTo=parrnote
334	SCPL-1 Antibodies, otherFeat=[]-->, belongsTo=parr
335	For production of anti-SCPL-1b and anti-SCPL-1a, MBP fusion of residues, otherFeat=[]-->, belongsTo=parrnote
336	1-302 of SCPL-1b and MBP fusion of residues 1-156 of SCPL-1a were used as, otherFeat=[]-->, belongsTo=parrnote
337	immunogens. For affinity purification of anti-SCPL-1b and anti-SCPL-1a, GST, otherFeat=[]-->, belongsTo=parrnote
338	fusion of residues 1-146 of SCPL-1b and GST fusion of residues 1-156 of, otherFeat=[]-->, belongsTo=parrnote
339	SCPL-1a were used, respectively. Affinity purification was carried out as, otherFeat=[]-->, belongsTo=parrnote
340	described in Mercer et al. (2006)., otherFeat=[]-->, belongsTo=parrnote
341	Western Blot and Immunofluorescence Microscopy, otherFeat=[]-->, belongsTo=parr
342	Procedures for preparing worm protein lysates and Western blots were, otherFeat=[]-->, belongsTo=parrnote
343	described previously (Mercer et al., 2006). The following worm strains were, otherFeat=[]-->, belongsTo=parrnote
344	used to make lysates: wild type (N2), scpl-1(ok1080) and scpl-1(gk283). The blot, otherFeat=[]-->, belongsTo=parrnote
345	also contained lysates from yeast expressing myc tagged SCPL-1a and -1b., otherFeat=[]-->, belongsTo=parrnote
346	Yeast lysates were prepared by suspending a yeast pellet froma2ml over-, otherFeat=[]-->, belongsTo=parrnote
347	night culture in Laemmli buffer and heating at 95o for 5 min. Affinity-purified, otherFeat=[]-->, belongsTo=parrnote
348	anti-SCPL-1a was used at 1:200 dilution, and affinity purified anti-SCPL-1b, otherFeat=[]-->, belongsTo=parrnote
349	was used at 1:200 dilution. Wild type adult worms were immunostained after, otherFeat=[]-->, belongsTo=parrnote
350	fixation by the method described by Nonet et al. (1993). Anti-SCPL-1b was, otherFeat=[]-->, belongsTo=parrnote
351	used at 1:100 dilution, and anti-UNC-89 (MH42) was used at 1:200 dilution., otherFeat=[]-->, belongsTo=parrnote
352	Secondary antibodies and confocal microscopy were as described in Qadota et, otherFeat=[]-->, belongsTo=parrnote
353	al. (2007)., otherFeat=[]-->, belongsTo=parrnote
354	SCPL-1 Phosphatase Activity Assays, otherFeat=[]-->, belongsTo=parr
355	Hydrolysis of phosphate from p-nitrophenyl phosphate (pNPP) was assayed, otherFeat=[]-->, belongsTo=parrnote
356	in a 1-ml reaction mixture containing 50 mM Tris acetate, pH 5.0, 10 mM, otherFeat=[]-->, belongsTo=parrnote
357	MgCl2,20mM pNPP, and MBP or MBP-SCPL-1b (typically 10 g) as de-, otherFeat=[]-->, belongsTo=parrnote
358	scribed in Zheng et al. (2005). The amount of protein, the pH, and inclusion of, otherFeat=[]-->, belongsTo=parrnote
359	different divalent cations was as described in Zheng et al. (2005), and the use, otherFeat=[]-->, belongsTo=parrnote
360	of various phosphatase inhibitors was as described in Kamenski et al. (2004)., otherFeat=[]-->, belongsTo=parrnote
361	Egg-Laying Assays, otherFeat=[]-->, belongsTo=parr
362	Examination of drug response to egg laying was performed as described in, otherFeat=[]-->, belongsTo=parrnote
363	Trent et al. (1983). Briefly, 96-well plates with 100 l of M9 containing 5, otherFeat=[]-->, belongsTo=parrnote
364	mg/ml serotonin or 0.75 mg/ml imipramine in each well were prepared., otherFeat=[]-->, belongsTo=parrnote
365	Single adult worms were soaked in each well for 1 h (serotonin) or 1.5 h, otherFeat=[]-->, belongsTo=parrnote
366	(imipramine) at room temperature. After incubation, the numbers of laid eggs, otherFeat=[]-->, belongsTo=parrnote
367	were counted., otherFeat=[]-->, belongsTo=parrnote
368	RESULTS, otherFeat=[]-->, belongsTo=parr
369	With the goal of identifying binding partners or possible, otherFeat=[]-->, belongsTo=parr
370	substrates for the protein kinase domains of UNC-89, a, otherFeat=[]-->, belongsTo=parr
371	portion of the protein containing PK2 was used to screen a, otherFeat=[]-->, belongsTo=parr
372	yeast two-hybrid library. The bait contained the domains, otherFeat=[]-->, belongsTo=parr
373	Ig-Fn3-PK2 (Figure 1A, 15/14), including the putative auto-, otherFeat=[]-->, belongsTo=parr
374	Figure 3. The SCPL family of proteins in C., otherFeat=[]-->, belongsTo=fig_caption
375	elegans and demonstration that UNC-89 PK2, otherFeat=[]-->, belongsTo=fig_caption
376	region specifically interacts with SCPL-1. (A), otherFeat=[]-->, belongsTo=fig_caption
377	Schematic representation of domain organiza-, otherFeat=[]-->, belongsTo=fig_caption
378	tion of proteins containing CTD type (or, otherFeat=[]-->, belongsTo=fig_caption
379	FCPH) phosphatase domains. C. elegans has, otherFeat=[]-->, belongsTo=fig_caption
380	five genes that encode proteins with FCPH, otherFeat=[]-->, belongsTo=fig_caption
381	domains: one domain called CeFCP-1 is more, otherFeat=[]-->, belongsTo=fig_caption
382	closely related to FCP proteins in that it has, otherFeat=[]-->, belongsTo=fig_caption
383	both phosphatase and BRCT domains. The, otherFeat=[]-->, belongsTo=fig_caption
384	others, including SCPL-1, which interacts with, otherFeat=[]-->, belongsTo=fig_caption
385	UNC-89, are more closely related to small, otherFeat=[]-->, belongsTo=fig_caption
386	CTD phosphatases (called SCPs), and they are, otherFeat=[]-->, belongsTo=fig_caption
387	designated SCP-L. The percentages indicate, otherFeat=[]-->, belongsTo=fig_caption
388	the percentage of identical amino acids in the, otherFeat=[]-->, belongsTo=fig_caption
389	200-residue phosphatase domains, com-, otherFeat=[]-->, belongsTo=fig_caption
390	pared with the phosphatase in CeB0379.4a, otherFeat=[]-->, belongsTo=fig_caption
391	(SCPL-1). (B) By yeast two-hybrid assays, the, otherFeat=[]-->, belongsTo=fig_caption
392	UNC-89 PK2 region interacts with SCPL-1, but, otherFeat=[]-->, belongsTo=fig_caption
393	not SCPL-2, -3, or -4. The comparable regions, otherFeat=[]-->, belongsTo=fig_caption
394	of twitchin and TTN-1 fail to interact with any, otherFeat=[]-->, belongsTo=fig_caption
395	of the SCPL proteins. , growth and ,no, otherFeat=[]-->, belongsTo=fig_caption
396	growth on Ade plates., otherFeat=[]-->, belongsTo=fig_caption
397	Novel Phosphatase Binds Giant Protein Kinase, otherFeat=[]-->, belongsTo=nota_cab_pie
398	Vol. 19, June 2008, otherFeat=[]-->, belongsTo=nota_cab_pie
399	2427, otherFeat=[]-->, belongsTo=nota_cab_pie
400	inhibitory domain. After screening 8.1 106 colonies, 42, otherFeat=[]-->, belongsTo=parr
401	positives were confirmed after retransformation, and all of, otherFeat=[]-->, belongsTo=parr
402	them represented portions of a single gene, B0379.4. As pre-, otherFeat=[]-->, belongsTo=parr
403	dicted on WormBase and confirmed by sequencing cDNAs,, otherFeat=[]-->, belongsTo=parr
404	this gene encodes two transcripts, B0379.4a and B0379.4b, by, otherFeat=[]-->, belongsTo=parr
405	alternative splicing in the 5 half of the gene. Our prey clones, otherFeat=[]-->, belongsTo=parr
406	included representatives of both splice isoforms. The pro-, otherFeat=[]-->, belongsTo=parr
407	teins encoded by B0379.4a and .4b are 345 and 491 residues,, otherFeat=[]-->, belongsTo=parr
408	respectively. The only recognizable domain found in these, otherFeat=[]-->, belongsTo=parr
409	proteins is a protein phosphatase domain ( 170 ?200 aa) in, otherFeat=[]-->, belongsTo=parr
410	the C-terminal half of each isoform (Figures 1C and 3A). For, otherFeat=[]-->, belongsTo=parr
411	reasons explained below, we have renamed B0379.4 as, otherFeat=[]-->, belongsTo=parr
412	SCPL-1., otherFeat=[]-->, belongsTo=parr
413	To determine which portions of the UNC-89 bait are min-, otherFeat=[]-->, belongsTo=parr
414	imally required to interact with SCPL-1, deletion derivatives, otherFeat=[]-->, belongsTo=parr
415	of the segment Ig-Fn3-PK2 were tested by two-hybrid, otherFeat=[]-->, belongsTo=parr
416	against SCPL-1a and -1b full-length prey. As shown in Fig-, otherFeat=[]-->, belongsTo=parr
417	ure 1A, all the domains are required for interaction, includ-, otherFeat=[]-->, belongsTo=parr
418	ing the C-terminal autoinhibitory region. Because the pat-, otherFeat=[]-->, belongsTo=parr
419	tern of domains, Ig-Fn3-kinase, is conserved among the, otherFeat=[]-->, belongsTo=parr
420	giant protein kinases, we tested the comparable regions, otherFeat=[]-->, belongsTo=parr
421	from the two other giant kinases in worm muscle, twitchin, otherFeat=[]-->, belongsTo=parr
422	and TTN-1. As can be seen in Figure 1A, these giant kinases, otherFeat=[]-->, belongsTo=parr
423	failed to interact with SCPL-1a or -1b., otherFeat=[]-->, belongsTo=parr
424	Given that UNC-89 has a second protein kinase domain,, otherFeat=[]-->, belongsTo=parr
425	PK1, which is predicted to be catalytically silent (Small et al.,, otherFeat=[]-->, belongsTo=parr
426	2004), we wondered whether it, too, might interact with, otherFeat=[]-->, belongsTo=parr
427	SCPL-1. Therefore, fragment Fn3-Ig-PK1 was used as bait to, otherFeat=[]-->, belongsTo=parr
428	check for interaction with SCPL-1 by two-hybrid analysis., otherFeat=[]-->, belongsTo=parr
429	As indicated in Figure 1B, interaction was found, and all the, otherFeat=[]-->, belongsTo=parr
430	domains, Fn3, Ig, and PK1 need to be present to obtain, otherFeat=[]-->, belongsTo=parr
431	interaction of the PK1 region with either SCPL-1a or -1b. To, otherFeat=[]-->, belongsTo=parr
432	determine which portion of SCPL-1a or -1b is required for, otherFeat=[]-->, belongsTo=parr
433	interaction with either PK1 or PK2 full-length regions, dele-, otherFeat=[]-->, belongsTo=parr
434	tion derivatives of SCPL-1a and -1b were tested by two-, otherFeat=[]-->, belongsTo=parr
435	hybrid analysis. As shown in Figure 1C, the minimal region, otherFeat=[]-->, belongsTo=parr
436	required is the phosphatase domain., otherFeat=[]-->, belongsTo=parr
437	To provide additional evidence that SCPL-1 interacts with, otherFeat=[]-->, belongsTo=parr
438	the PK2 and PK1 regions of UNC-89, further experiments, otherFeat=[]-->, belongsTo=parr
439	were performed. Although each isoform of SCPL-1 could be, otherFeat=[]-->, belongsTo=parr
440	expressed in bacteria as MBP or GST fusions, segments of, otherFeat=[]-->, belongsTo=parr
441	UNC-89, either Ig-Fn3-PK2, or Fn3-Ig-PK1 as GST or His-, otherFeat=[]-->, belongsTo=parr
442	tagged fusions, could not be expressed. In fact, the PK1 or, otherFeat=[]-->, belongsTo=parr
443	PK2 proteins seemed to significantly reduce the growth rate, otherFeat=[]-->, belongsTo=parr
444	of E. coli. Therefore, in the same yeast cell, myc-tagged, otherFeat=[]-->, belongsTo=parr
445	SCPL-1b and individual HA-tagged derivatives of the PK2, otherFeat=[]-->, belongsTo=parr
446	region corresponding to those depicted in Figure 1A were, otherFeat=[]-->, belongsTo=parr
447	expressed. Total protein lysates were prepared and incu-, otherFeat=[]-->, belongsTo=parr
448	bated with agarose beads coated with antibodies to the HA, otherFeat=[]-->, belongsTo=parr
449	tag, washed, and eluted, and portions were run on two gels, otherFeat=[]-->, belongsTo=parr
450	and blotted. One blot was reacted with anti-HA to detect the, otherFeat=[]-->, belongsTo=parr
451	presence of the PK2 derivatives. As shown in Figure 2A,, otherFeat=[]-->, belongsTo=parr
452	appropriately sized proteins were detected from each PK2, otherFeat=[]-->, belongsTo=parr
453	derivative. The second blot was reacted with anti-myc to, otherFeat=[]-->, belongsTo=parr
454	Figure 4. Expression pattern of the SCPL-1b promoter and char-, otherFeat=[]-->, belongsTo=fig_caption
455	acterization of antibodies to SCPL-1. (A) SCPL-1b is expressed in the, otherFeat=[]-->, belongsTo=fig_caption
456	same muscle cells as UNC-89. The exon/intron structure of the, otherFeat=[]-->, belongsTo=fig_caption
457	SCPL-1b gene is shown, as predicted on WormBase. 7.5 kb of DNA, otherFeat=[]-->, belongsTo=fig_caption
458	sequence upstream of the initiator methionine together with codons, otherFeat=[]-->, belongsTo=fig_caption
459	for a few amino acids of the first exon of SCPL-1b were fused, otherFeat=[]-->, belongsTo=fig_caption
460	in-frame with GFP, and used to create transgenic animals, and the, otherFeat=[]-->, belongsTo=fig_caption
461	sites of GFP expression were recorded. This promoter is expressed, otherFeat=[]-->, belongsTo=fig_caption
462	in pharyngeal (left), vulval (center), and body wall muscle (right)., otherFeat=[]-->, belongsTo=fig_caption
463	(B) Anti-SCPL-1b and anti-SCPL-1a antibodies specifically recog-, otherFeat=[]-->, belongsTo=fig_caption
464	nize SCPL-1b and SCPL-1a, respectively. Above is shown immu-, otherFeat=[]-->, belongsTo=fig_caption
465	nogens used to generate rabbit antibodies that were later affinity, otherFeat=[]-->, belongsTo=fig_caption
466	purified using the same regions. Below are Western blots reacted, otherFeat=[]-->, belongsTo=fig_caption
467	with the designated antibodies. In each case, the left-most three, otherFeat=[]-->, belongsTo=fig_caption
468	lanes are worm extracts, and the right-most lanes are yeast extracts., otherFeat=[]-->, belongsTo=fig_caption
469	WT, wild type; scpl-1(ok1080) and scpl-1(gk283) are intragenic dele-, otherFeat=[]-->, belongsTo=fig_caption
470	tions of the scpl-1 gene. Vector, Myc-SCPL-1a, and Myc-SCPL-1b are, otherFeat=[]-->, belongsTo=fig_caption
471	yeast harboring either the empty vector or myc-tagged SCPL-1, otherFeat=[]-->, belongsTo=fig_caption
472	isoforms. (C) Anti-SCPL-1 localize to M-lines and I-bands in body, otherFeat=[]-->, belongsTo=fig_caption
473	wall muscle. Anti-SCPL-1b and anti-UNC-89 were coincubated with, otherFeat=[]-->, belongsTo=fig_caption
474	wild-type worms, and the muscle was imaged by immunofluores-, otherFeat=[]-->, belongsTo=fig_caption
475	cence microscopy. The images show a portion of one body wall, otherFeat=[]-->, belongsTo=fig_caption
476	muscle cell. Weak labeling of the M-line and a portion of the I-band, otherFeat=[]-->, belongsTo=fig_caption
477	is seen. UNC-89 is a marker of the M-line. Bar, 10 m., otherFeat=[]-->, belongsTo=fig_caption
478	H. Qadota et al., otherFeat=[]-->, belongsTo=nota_cab_pie
479	Molecular Biology of the Cell, otherFeat=[]-->, belongsTo=nota_cab_pie
480	2428, otherFeat=[]-->, belongsTo=nota_cab_pie
481	detect the presence of any SCPL-1b that might have been, otherFeat=[]-->, belongsTo=parr
482	brought down with the PK2 protein. As shown in Figure 2A, otherFeat=[]-->, belongsTo=parr
483	(bottom), only derivative 15/14 (Ig-Fn3-PK2-autoinhibitory, otherFeat=[]-->, belongsTo=parr
484	region) was coimmunoprecipitated, consistent with the do-, otherFeat=[]-->, belongsTo=parr
485	main mapping two-hybrid experiment shown in Figure 1A., otherFeat=[]-->, belongsTo=parr
486	A variation of this experiment was conducted in which, otherFeat=[]-->, belongsTo=parr
487	yeast was used to express the PK2 or PK1 segments, and E., otherFeat=[]-->, belongsTo=parr
488	coli was used to express SCPL-1. Protein extracts were pre-, otherFeat=[]-->, belongsTo=parr
489	pared from yeast expressing either HA-PK2 15/14 (Figure, otherFeat=[]-->, belongsTo=parr
490	1A) or HA-PK1 AB (Figure 1B), and these UNC-89 segments, otherFeat=[]-->, belongsTo=parr
491	were immunoprecipitated with anti-HA antibody-conju-, otherFeat=[]-->, belongsTo=parr
492	gated agarose beads. After washing, these beads were incu-, otherFeat=[]-->, belongsTo=parr
493	bated with purified, bacterially expressed MBP or MBP-, otherFeat=[]-->, belongsTo=parr
494	SCPL-1a (Figure 2C), washed, and the proteins were eluted., otherFeat=[]-->, belongsTo=parr
495	Portions of each sample were run on two gels and blotted., otherFeat=[]-->, belongsTo=parr
496	One blot was reacted with anti-HA to detect the presence of, otherFeat=[]-->, belongsTo=parr
497	the yeast expressed protein, the other blot was reacted with, otherFeat=[]-->, belongsTo=parr
498	anti-MBP to detect possible binding with MBP or MBP-, otherFeat=[]-->, belongsTo=parr
499	SCPL-1a. As shown in Figure 2B, this method demonstrates, otherFeat=[]-->, belongsTo=parr
500	interaction between MBP-SCPL-1a and either Ig-Fn3-PK2, or, otherFeat=[]-->, belongsTo=parr
501	Fn3-Ig-PK1. Using MBP-SCPL-1b, we obtained similar re-, otherFeat=[]-->, belongsTo=parr
502	sults (data not shown)., otherFeat=[]-->, belongsTo=parr
503	Application of BLAST and Pfam reveals that the phospha-, otherFeat=[]-->, belongsTo=parr
504	tase domain of B0379.4 (SCPL-1) is most closely related to, otherFeat=[]-->, belongsTo=parr
505	the protein phosphatase domains of the CTD phosphatase, otherFeat=[]-->, belongsTo=parr
506	family. The founding member of this family is FCP1 (TFIIF-, otherFeat=[]-->, belongsTo=parr
507	associating component of CTD phosphatase), which is con-, otherFeat=[]-->, belongsTo=parr
508	served from yeast to humans, and it is known to dephos-, otherFeat=[]-->, belongsTo=parr
509	phorylate the C-terminal domain (CTD) of the largest, otherFeat=[]-->, belongsTo=parr
510	subunit of RNA polymerase II. This phosphatase family has, otherFeat=[]-->, belongsTo=parr
511	no sequence similarity to other phosphatases, except for the, otherFeat=[]-->, belongsTo=parr
512	motif DXDX(T/V) that is common to both phosphotrans-, otherFeat=[]-->, belongsTo=parr
513	ferases and phosphohydrolases (Collet et al., 1998). In addi-, otherFeat=[]-->, belongsTo=parr
514	tion to a phosphatase domain, FCP proteins have a Brca1, otherFeat=[]-->, belongsTo=parr
515	C-terminal (BRCT) domain. Other members of this family, otherFeat=[]-->, belongsTo=parr
516	are generally smaller proteins that lack the BRCT domain,, otherFeat=[]-->, belongsTo=parr
517	and they are called small CTD phosphatases (SCPs). The, otherFeat=[]-->, belongsTo=parr
518	function of the SCPs is still being established (see Discus-, otherFeat=[]-->, belongsTo=parr
519	sion). Our analysis has revealed that C. elegans has five genes, otherFeat=[]-->, belongsTo=parr
520	that encode proteins with FCP1 homology (FCPH) or CTD-, otherFeat=[]-->, belongsTo=parr
521	type phosphatase domains (Figure 3A). One of them is more, otherFeat=[]-->, belongsTo=parr
522	closely related to FCP (in sequence and the presence of both, otherFeat=[]-->, belongsTo=parr
523	phosphatase and BRCT domains); thus, it is called CeFCP-1., otherFeat=[]-->, belongsTo=parr
524	The others, including B0379.4 (SCPL-1), are more closely, otherFeat=[]-->, belongsTo=parr
525	related to SCPs; therefore, we call these proteins SCP-like, otherFeat=[]-->, belongsTo=parr
526	(SCPL). It seems noteworthy that the phosphatase domain of, otherFeat=[]-->, belongsTo=parr
527	worm SCPL-1 is more closely related to the phosphatase, otherFeat=[]-->, belongsTo=parr
528	domains of human SCP1, SCP2, and SCP3 (64 ? 67% identical, otherFeat=[]-->, belongsTo=parr
529	amino acids over a span of 200 amino acids), than it is to, otherFeat=[]-->, belongsTo=parr
530	the other SCPs in C. elegans (ranging from 23 to 38% iden-, otherFeat=[]-->, belongsTo=parr
531	tity). It is also clear that the phosphatase domain of SCPL-1, otherFeat=[]-->, belongsTo=parr
532	is least similar to the phosphatase domains from worm,, otherFeat=[]-->, belongsTo=parr
533	yeast, and human FCPs (16 ?19% identity)., otherFeat=[]-->, belongsTo=parr
534	To determine whether the PK2 and PK1 regions of, otherFeat=[]-->, belongsTo=parr
535	UNC-89 interact specifically with SCPL-1, prey plasmids, otherFeat=[]-->, belongsTo=parr
536	were generated specifying the complete coding sequences, otherFeat=[]-->, belongsTo=parr
537	for nematode SCPL-2, SCPL-3, and SCPL-4, and they were, otherFeat=[]-->, belongsTo=parr
538	tested by two-hybrid for interaction. As shown in Figure 3B,, otherFeat=[]-->, belongsTo=parr
539	when the Ig-Fn3-PK2 segment was tested against all the, otherFeat=[]-->, belongsTo=parr
540	SCPL proteins, interaction was found only with SCPL-1. In, otherFeat=[]-->, belongsTo=parr
541	addition, as shown in Figure 3B, when the comparable ki-, otherFeat=[]-->, belongsTo=parr
542	nase-containing regions of twitchin and TTN-1 were tested,, otherFeat=[]-->, belongsTo=parr
543	they failed to interact with any of the SCPL proteins., otherFeat=[]-->, belongsTo=parr
544	Because the two-hybrid library contains cDNAs made, otherFeat=[]-->, belongsTo=parr
545	from mRNA prepared from whole worms (including all, otherFeat=[]-->, belongsTo=parr
546	tissues and cell types), it was important to determine, otherFeat=[]-->, belongsTo=parr
547	whether scpl-1 is expressed in muscle. To do this, we fused, otherFeat=[]-->, belongsTo=parr
548	in-frame with GFP, 7.5 kb of DNA sequence upstream of the, otherFeat=[]-->, belongsTo=parr
549	predicted initiator methionine together with codons for a, otherFeat=[]-->, belongsTo=parr
550	few amino acids of the first exon of scpl-1b. This DNA was, otherFeat=[]-->, belongsTo=parr
551	used to create transgenic animals, and the sites of GFP, otherFeat=[]-->, belongsTo=parr
552	expression were recorded. As shown in Figure 4A, scpl-1b is, otherFeat=[]-->, belongsTo=parr
553	expressed in pharyngeal, vulval, and body wall muscle, the, otherFeat=[]-->, belongsTo=parr
554	same muscle cells that express unc-89 (Small et al., 2004)., otherFeat=[]-->, belongsTo=parr
555	This result bolsters the likelihood that SCPL-1 and UNC-89, otherFeat=[]-->, belongsTo=parr
556	interact in vivo., otherFeat=[]-->, belongsTo=parr
557	To determine where the SCPL-1 proteins are expressed, otherFeat=[]-->, belongsTo=parr
558	and localized, we generated affinity-purified rabbit antibod-, otherFeat=[]-->, belongsTo=parr
559	ies that specifically recognize SCPL-1a or SCPL-1b. As, otherFeat=[]-->, belongsTo=parr
560	shown in Figure 4B, by Western blot, anti-SCPL-1b recog-, otherFeat=[]-->, belongsTo=parr
561	nizes yeast-expressed myc-tagged SCPL-1b, but not SCPL-, otherFeat=[]-->, belongsTo=parr
562	1a, and anti-SCPL-1a recognizes yeast-expressed myc-, otherFeat=[]-->, belongsTo=parr
563	tagged SCPL-1a but not SCPL-1b. These antibodies, otherFeat=[]-->, belongsTo=parr
564	recognize the expected sized proteins from C. elegans. Anti-, otherFeat=[]-->, belongsTo=parr
565	SCPL-1b reacts with an 70-kDa protein from wild-type, otherFeat=[]-->, belongsTo=parr
566	worms likely to be SCPL-1b, and with truncated proteins, otherFeat=[]-->, belongsTo=parr
567	from the intragenic deletion mutants, ok1080 and gk283., otherFeat=[]-->, belongsTo=parr
568	Similarly, anti-SCPL-1a reacts with an 50-kDa protein, otherFeat=[]-->, belongsTo=parr
569	Table 1. Loss of function of scpl-1 results in a defect in egg laying, otherFeat=[]-->, belongsTo=tab_caption
570	Strain, otherFeat=[]-->, belongsTo=tab_cell
571	RNAi, otherFeat=[]-->, belongsTo=tab_cell
572	Line, otherFeat=[]-->, belongsTo=tab_cell
573	Serotonin, otherFeat=[]-->, belongsTo=tab_cell
574	Imipramine, otherFeat=[]-->, belongsTo=tab_cell
575	( )( ), otherFeat=[]-->, belongsTo=tab_cell
576	( )( ), otherFeat=[]-->, belongsTo=tab_cell
577	N2, otherFeat=[]-->, belongsTo=tab_cell
578	scpl-1a full, otherFeat=[]-->, belongsTo=tab_cell
579	a, otherFeat=[]-->, belongsTo=tab_cell
580	5, otherFeat=[]-->, belongsTo=tab_cell
581	3, otherFeat=[]-->, belongsTo=tab_cell
582	2, otherFeat=[]-->, belongsTo=tab_cell
583	0, otherFeat=[]-->, belongsTo=tab_cell
584	8, otherFeat=[]-->, belongsTo=tab_cell
585	2, otherFeat=[]-->, belongsTo=tab_cell
586	0, otherFeat=[]-->, belongsTo=tab_cell
587	0, otherFeat=[]-->, belongsTo=tab_cell
588	b7, otherFeat=[]-->, belongsTo=tab_cell
589	1, otherFeat=[]-->, belongsTo=tab_cell
590	0, otherFeat=[]-->, belongsTo=tab_cell
591	1, otherFeat=[]-->, belongsTo=tab_cell
592	9, otherFeat=[]-->, belongsTo=tab_cell
593	0, otherFeat=[]-->, belongsTo=tab_cell
594	1, otherFeat=[]-->, belongsTo=tab_cell
595	0, otherFeat=[]-->, belongsTo=tab_cell
596	N2, otherFeat=[]-->, belongsTo=tab_cell
597	scpl-1b full, otherFeat=[]-->, belongsTo=tab_cell
598	a, otherFeat=[]-->, belongsTo=tab_cell
599	7, otherFeat=[]-->, belongsTo=tab_cell
600	1, otherFeat=[]-->, belongsTo=tab_cell
601	1, otherFeat=[]-->, belongsTo=tab_cell
602	0, otherFeat=[]-->, belongsTo=tab_cell
603	9, otherFeat=[]-->, belongsTo=tab_cell
604	1, otherFeat=[]-->, belongsTo=tab_cell
605	0, otherFeat=[]-->, belongsTo=tab_cell
606	0, otherFeat=[]-->, belongsTo=tab_cell
607	b8, otherFeat=[]-->, belongsTo=tab_cell
608	1, otherFeat=[]-->, belongsTo=tab_cell
609	1, otherFeat=[]-->, belongsTo=tab_cell
610	0, otherFeat=[]-->, belongsTo=tab_cell
611	10, otherFeat=[]-->, belongsTo=tab_cell
612	0, otherFeat=[]-->, belongsTo=tab_cell
613	0, otherFeat=[]-->, belongsTo=tab_cell
614	0, otherFeat=[]-->, belongsTo=tab_cell
615	N2, otherFeat=[]-->, belongsTo=tab_cell
616	empty vector, otherFeat=[]-->, belongsTo=tab_cell
617	a, otherFeat=[]-->, belongsTo=tab_cell
618	10, otherFeat=[]-->, belongsTo=tab_cell
619	0, otherFeat=[]-->, belongsTo=tab_cell
620	0, otherFeat=[]-->, belongsTo=tab_cell
621	0, otherFeat=[]-->, belongsTo=tab_cell
622	10, otherFeat=[]-->, belongsTo=tab_cell
623	0, otherFeat=[]-->, belongsTo=tab_cell
624	0, otherFeat=[]-->, belongsTo=tab_cell
625	0, otherFeat=[]-->, belongsTo=tab_cell
626	b9, otherFeat=[]-->, belongsTo=tab_cell
627	1, otherFeat=[]-->, belongsTo=tab_cell
628	0, otherFeat=[]-->, belongsTo=tab_cell
629	0, otherFeat=[]-->, belongsTo=tab_cell
630	8, otherFeat=[]-->, belongsTo=tab_cell
631	2, otherFeat=[]-->, belongsTo=tab_cell
632	0, otherFeat=[]-->, belongsTo=tab_cell
633	0, otherFeat=[]-->, belongsTo=tab_cell
634	unc-89 (tm752), otherFeat=[]-->, belongsTo=tab_cell
635	scpl-1b full, otherFeat=[]-->, belongsTo=tab_cell
636	a, otherFeat=[]-->, belongsTo=tab_cell
637	7, otherFeat=[]-->, belongsTo=tab_cell
638	2, otherFeat=[]-->, belongsTo=tab_cell
639	1, otherFeat=[]-->, belongsTo=tab_cell
640	0, otherFeat=[]-->, belongsTo=tab_cell
641	10, otherFeat=[]-->, belongsTo=tab_cell
642	0, otherFeat=[]-->, belongsTo=tab_cell
643	0, otherFeat=[]-->, belongsTo=tab_cell
644	0, otherFeat=[]-->, belongsTo=tab_cell
645	b9, otherFeat=[]-->, belongsTo=tab_cell
646	0, otherFeat=[]-->, belongsTo=tab_cell
647	1, otherFeat=[]-->, belongsTo=tab_cell
648	0, otherFeat=[]-->, belongsTo=tab_cell
649	10, otherFeat=[]-->, belongsTo=tab_cell
650	0, otherFeat=[]-->, belongsTo=tab_cell
651	0, otherFeat=[]-->, belongsTo=tab_cell
652	0, otherFeat=[]-->, belongsTo=tab_cell
653	unc-89 (tm752), otherFeat=[]-->, belongsTo=tab_cell
654	empty vector, otherFeat=[]-->, belongsTo=tab_cell
655	a, otherFeat=[]-->, belongsTo=tab_cell
656	9, otherFeat=[]-->, belongsTo=tab_cell
657	1, otherFeat=[]-->, belongsTo=tab_cell
658	0, otherFeat=[]-->, belongsTo=tab_cell
659	0, otherFeat=[]-->, belongsTo=tab_cell
660	9, otherFeat=[]-->, belongsTo=tab_cell
661	1, otherFeat=[]-->, belongsTo=tab_cell
662	0, otherFeat=[]-->, belongsTo=tab_cell
663	0, otherFeat=[]-->, belongsTo=tab_cell
664	b, otherFeat=[]-->, belongsTo=tab_cell
665	10, otherFeat=[]-->, belongsTo=tab_cell
666	0, otherFeat=[]-->, belongsTo=tab_cell
667	0, otherFeat=[]-->, belongsTo=tab_cell
668	0, otherFeat=[]-->, belongsTo=tab_cell
669	10, otherFeat=[]-->, belongsTo=tab_cell
670	0, otherFeat=[]-->, belongsTo=tab_cell
671	0, otherFeat=[]-->, belongsTo=tab_cell
672	0, otherFeat=[]-->, belongsTo=tab_cell
673	unc-89 (su75), otherFeat=[]-->, belongsTo=tab_cell
674	scpl-1b full, otherFeat=[]-->, belongsTo=tab_cell
675	a, otherFeat=[]-->, belongsTo=tab_cell
676	8, otherFeat=[]-->, belongsTo=tab_cell
677	1, otherFeat=[]-->, belongsTo=tab_cell
678	0, otherFeat=[]-->, belongsTo=tab_cell
679	1, otherFeat=[]-->, belongsTo=tab_cell
680	9, otherFeat=[]-->, belongsTo=tab_cell
681	1, otherFeat=[]-->, belongsTo=tab_cell
682	0, otherFeat=[]-->, belongsTo=tab_cell
683	0, otherFeat=[]-->, belongsTo=tab_cell
684	b7, otherFeat=[]-->, belongsTo=tab_cell
685	2, otherFeat=[]-->, belongsTo=tab_cell
686	1, otherFeat=[]-->, belongsTo=tab_cell
687	0, otherFeat=[]-->, belongsTo=tab_cell
688	10, otherFeat=[]-->, belongsTo=tab_cell
689	0, otherFeat=[]-->, belongsTo=tab_cell
690	0, otherFeat=[]-->, belongsTo=tab_cell
691	0, otherFeat=[]-->, belongsTo=tab_cell
692	unc-89 (su75), otherFeat=[]-->, belongsTo=tab_cell
693	empty vector, otherFeat=[]-->, belongsTo=tab_cell
694	a, otherFeat=[]-->, belongsTo=tab_cell
695	8, otherFeat=[]-->, belongsTo=tab_cell
696	2, otherFeat=[]-->, belongsTo=tab_cell
697	0, otherFeat=[]-->, belongsTo=tab_cell
698	0, otherFeat=[]-->, belongsTo=tab_cell
699	10, otherFeat=[]-->, belongsTo=tab_cell
700	0, otherFeat=[]-->, belongsTo=tab_cell
701	0, otherFeat=[]-->, belongsTo=tab_cell
702	0, otherFeat=[]-->, belongsTo=tab_cell
703	b9, otherFeat=[]-->, belongsTo=tab_cell
704	1, otherFeat=[]-->, belongsTo=tab_cell
705	0, otherFeat=[]-->, belongsTo=tab_cell
706	0, otherFeat=[]-->, belongsTo=tab_cell
707	10, otherFeat=[]-->, belongsTo=tab_cell
708	0, otherFeat=[]-->, belongsTo=tab_cell
709	0, otherFeat=[]-->, belongsTo=tab_cell
710	0, otherFeat=[]-->, belongsTo=tab_cell
711	, more than 8 eggs; ( ), 4 ?7 eggs; ( ), 1?3 eggs; and , 0 eggs., otherFeat=[]-->, belongsTo=parrnote
712	Novel Phosphatase Binds Giant Protein Kinase, otherFeat=[]-->, belongsTo=nota_cab_pie
713	Vol. 19, June 2008, otherFeat=[]-->, belongsTo=nota_cab_pie
714	2429, otherFeat=[]-->, belongsTo=nota_cab_pie
715	from wild-type worms likely to be SCPL-1a, and this protein, otherFeat=[]-->, belongsTo=parr
716	is missing from the intragenic deletion mutants., otherFeat=[]-->, belongsTo=parr
717	Anti-SCPL-1b and anti-UNC-89 were coincubated with, otherFeat=[]-->, belongsTo=parr
718	wild-type worms, and the muscle was imaged by immuno-, otherFeat=[]-->, belongsTo=parr
719	fluorescence microscopy. The image presented in Figure 4C, otherFeat=[]-->, belongsTo=parr
720	shows a portion of one body wall muscle cell. Labeling of, otherFeat=[]-->, belongsTo=parr
721	the M-line (marked by UNC-89) and a portion of the I-band, otherFeat=[]-->, belongsTo=parr
722	is seen. In experiments not shown, the I-band location was, otherFeat=[]-->, belongsTo=parr
723	confirmed by use of known I-band or dense body markers., otherFeat=[]-->, belongsTo=parr
724	Similar results were obtained using anti-SCPL-1a (data not, otherFeat=[]-->, belongsTo=parr
725	shown). Thus, the partial colocalization of SCPL-1 with, otherFeat=[]-->, belongsTo=parr
726	UNC-89 at the M-line adds further credibility to a functional, otherFeat=[]-->, belongsTo=parr
727	interaction between SCPL-1 and UNC-89., otherFeat=[]-->, belongsTo=parr
728	Genome-wide RNAi screening (on WormBase) has re-, otherFeat=[]-->, belongsTo=parr
729	ported that scpl-1(RNAi) results in a reduction of egg laying, otherFeat=[]-->, belongsTo=parr
730	or Egl phenotype. We had a difficult time discerning this, otherFeat=[]-->, belongsTo=parr
731	subtle effect. Therefore, we chose to assay the response to, otherFeat=[]-->, belongsTo=parr
732	drugs that induce egg laying as described in Trent et al., otherFeat=[]-->, belongsTo=parr
733	(1983) and shown in Table 1. Compared with wild type,, otherFeat=[]-->, belongsTo=parr
734	scpl-1(RNAi) shows a weak response to serotonin, suggest-, otherFeat=[]-->, belongsTo=parr
735	ing a defect in function of egg laying muscles. unc-89 mu-, otherFeat=[]-->, belongsTo=parr
736	tants showed normal responses to serotonin, unc-89; scpl-, otherFeat=[]-->, belongsTo=parr
737	1(RNAi) double showed the same defect as scpl-1(RNAi),, otherFeat=[]-->, belongsTo=parr
738	suggesting that scpl-1 is epistatic to unc-89. Thus, scpl-1 may, otherFeat=[]-->, belongsTo=parr
739	function downstream of unc-89 in egg laying muscles., otherFeat=[]-->, belongsTo=parr
740	To determine whether SCPL-1 has phosphatase catalytic, otherFeat=[]-->, belongsTo=parr
741	activity, bacterially expressed MBP fusion proteins of, otherFeat=[]-->, belongsTo=parr
742	SCPL-1a and SCPL-1b (Figure 2C) were tested in vitro for, otherFeat=[]-->, belongsTo=parr
743	ability to remove the phosphate from the nonspecific sub-, otherFeat=[]-->, belongsTo=parr
744	strate p-nitrophenyl phosphate. Results are shown in Figure, otherFeat=[]-->, belongsTo=parr
745	5A for SCPL-1b; similar results were obtained for SCPL-1a;, otherFeat=[]-->, belongsTo=parr
746	MBP itself showed no activity. As shown in Figure 5A, the, otherFeat=[]-->, belongsTo=parr
747	amount of product generated was proportional to the, otherFeat=[]-->, belongsTo=parr
748	amount of added protein. The enzymatic properties are very, otherFeat=[]-->, belongsTo=parr
749	similar to those of previously characterized FCPs and SCPs, otherFeat=[]-->, belongsTo=parr
750	(Kamenski et al. (2004); Zheng et al. (2005)), including a pH, otherFeat=[]-->, belongsTo=parr
751	optimum of 5.0, a preference for Mg 2 as divalent cation,, otherFeat=[]-->, belongsTo=parr
752	and unusual response to small molecule inhibitors. Figure, otherFeat=[]-->, belongsTo=parr
753	5D shows that SCPL-1 is not inhibited by the typical phos-, otherFeat=[]-->, belongsTo=parr
754	phatase inhibitors NaF or Na3VO4, but it is inhibited by, otherFeat=[]-->, belongsTo=parr
755	BeF3 and AlF4., otherFeat=[]-->, belongsTo=note
756	DISCUSSION, otherFeat=[]-->, belongsTo=parr
757	We have shown that both the putatively catalytically active, otherFeat=[]-->, belongsTo=parr
758	(PK2) and catalytically inactive (PK1) kinase domains of, otherFeat=[]-->, belongsTo=parr
759	UNC-89 interact with a novel protein phosphatase, SCPL-1., otherFeat=[]-->, belongsTo=parr
760	For UNC-89, this requires the upstream Ig and Fn3 domains,, otherFeat=[]-->, belongsTo=parr
761	as well as the autoinhibitory region at least in PK2. For, otherFeat=[]-->, belongsTo=parr
762	SCPL-1, only the phosphatase domain itself is required for, otherFeat=[]-->, belongsTo=parr
763	interaction with UNC-89. There are two indications of the, otherFeat=[]-->, belongsTo=parr
764	specificity of this interaction: 1) SCPL-1 does not interact, otherFeat=[]-->, belongsTo=parr
765	with similar regions of the two other giant titin-like kinases, otherFeat=[]-->, belongsTo=parr
766	in the worm, twitchin and TTN-1. 2) The UNC-89 PK2, otherFeat=[]-->, belongsTo=parr
767	region does not interact with three other related proteins in, otherFeat=[]-->, belongsTo=parr
768	C. elegans: SCPL-2, -3, and -4, which SAGE data available on, otherFeat=[]-->, belongsTo=parr
769	WormBase suggest express in body wall muscle. The plau-, otherFeat=[]-->, belongsTo=parr
770	sibility of the UNC-89 ?SCPL-1 interaction is indicated by, otherFeat=[]-->, belongsTo=parr
771	finding the following: 1) The scpl-1b promoter is expressed in, otherFeat=[]-->, belongsTo=parr
772	the same sets of muscles (body wall, pharyngeal, and vulval), otherFeat=[]-->, belongsTo=parr
773	as unc-89. 2) By antibody staining of body wall striated, otherFeat=[]-->, belongsTo=parr
774	Figure 5. Biochemical properties of SCPL-1. SCPL-1b was ex-, otherFeat=[]-->, belongsTo=fig_caption
775	pressed as an MBP fusion protein in E. coli, and they are shown to, otherFeat=[]-->, belongsTo=fig_caption
776	have phosphatase activity toward a model substrate, p-nitrophenyl, otherFeat=[]-->, belongsTo=fig_caption
777	phosphate (A). Enzymatic properties were very similar to previ-, otherFeat=[]-->, belongsTo=fig_caption
778	ously characterized FCPs and SCPs, including a pH optimum of, otherFeat=[]-->, belongsTo=fig_caption
779	5.0 (B), preference for Mg 2 (C), and unusual response to small-, otherFeat=[]-->, belongsTo=fig_caption
780	molecule inhibitors (not inhibited by NaF or Na3VO4 but inhibited, otherFeat=[]-->, belongsTo=fig_caption
781	by BeF3 and AlF4; shown in D)., otherFeat=[]-->, belongsTo=fig_caption
782	H. Qadota et al., otherFeat=[]-->, belongsTo=nota_cab_pie
783	Molecular Biology of the Cell, otherFeat=[]-->, belongsTo=nota_cab_pie
784	2430, otherFeat=[]-->, belongsTo=nota_cab_pie
785	muscle, SCPL-1 is at least partially colocalized to the M-, otherFeat=[]-->, belongsTo=parr
786	lines, where it may interact with UNC-89 in situ. Although, otherFeat=[]-->, belongsTo=parr
787	knockdown of scpl-1 mRNA by RNAi has no obvious phe-, otherFeat=[]-->, belongsTo=parr
788	notype in the structure or function of body wall muscle, it, otherFeat=[]-->, belongsTo=parr
789	does show a mild defect in egg-laying muscles, as manifest, otherFeat=[]-->, belongsTo=parr
790	by an abnormal response to serotonin-induced egg laying., otherFeat=[]-->, belongsTo=parr
791	unc-89 mutants showed a normal response to serotonin,, otherFeat=[]-->, belongsTo=parr
792	whereas the unc-89;scpl-1 (RNAi) doubles show the same, otherFeat=[]-->, belongsTo=parr
793	deficit as spcl-1(RNAi), indicating that scpl-1 is epistatic to, otherFeat=[]-->, belongsTo=parr
794	unc-89. Therefore, scpl-1 functions downstream of unc-89 in, otherFeat=[]-->, belongsTo=parr
795	egg-laying muscles. Finally, we have shown that the phos-, otherFeat=[]-->, belongsTo=parr
796	phatase domain of SCPL-1 is an active phosphatase, because, otherFeat=[]-->, belongsTo=parr
797	phosphatase activity can be demonstrated in vitro with a, otherFeat=[]-->, belongsTo=parr
798	model substrate. Moreover, because the phosphatase activ-, otherFeat=[]-->, belongsTo=parr
799	ity is optimal in acidic pH conditions, this might have func-, otherFeat=[]-->, belongsTo=parr
800	tional implications. For example, in mammalian muscle,, otherFeat=[]-->, belongsTo=parr
801	repeated or excessive muscle activity leads to acidosis, and, otherFeat=[]-->, belongsTo=parr
802	this may stimulate SCPL-1 activity. Alternatively, the phos-, otherFeat=[]-->, belongsTo=parr
803	phatase might be recruited to a different cellular compart-, otherFeat=[]-->, belongsTo=parr
804	ment that is acidic (e.g., peroxisomes or autophagosomes)., otherFeat=[]-->, belongsTo=parr
805	What might be the biochemical consequences of this in-, otherFeat=[]-->, belongsTo=parr
806	teraction? There are at least five possibilities: 1) The UNC-89, otherFeat=[]-->, belongsTo=parr
807	putatively active kinase PK2 phosphorylates SCPL-1 to affect, otherFeat=[]-->, belongsTo=parr
808	its activity. This idea is consistent with SCPL-1 functioning, otherFeat=[]-->, belongsTo=parr
809	downstream of UNC-89, as suggested by the epistasis re-, otherFeat=[]-->, belongsTo=parr
810	sults. However, protein kinase assays using yeast-expressed, otherFeat=[]-->, belongsTo=parr
811	HA-tagged PK2 and bacterially expressed MBP-SCPL-1b, otherFeat=[]-->, belongsTo=parr
812	were negative (data not shown). 2) The SCPL-1 phosphatase, otherFeat=[]-->, belongsTo=parr
813	dephosphorylates UNC-89 active kinase PK2 to affect its, otherFeat=[]-->, belongsTo=parr
814	activity. Precedence for the regulatory affect of phosphory-, otherFeat=[]-->, belongsTo=parr
815	lation on a kinase domain in a giant protein kinase is the, otherFeat=[]-->, belongsTo=parr
816	tyrosine phosphorylation within the kinase domain of mam-, otherFeat=[]-->, belongsTo=parr
817	malian titin that is one step required in embryonic muscle to, otherFeat=[]-->, belongsTo=parr
818	relieve autoinhibition (Mayans et al., 1998). 3) Each enzyme,, otherFeat=[]-->, belongsTo=parr
819	UNC-89 kinase and SCPL-1 phosphatase, has the same sub-, otherFeat=[]-->, belongsTo=parr
820	strate, and the two proteins act antagonistically. Vertebrate, otherFeat=[]-->, belongsTo=parr
821	titin kinase (Grater et al., 2005; Lange et al., 2005), and per-, otherFeat=[]-->, belongsTo=parr
822	haps the other titin-like kinases such as twitchin, TTN-1, and, otherFeat=[]-->, belongsTo=parr
823	UNC-89, may be activated (from their autoinhibited states), otherFeat=[]-->, belongsTo=parr
824	by small mechanical forces that occur with each contraction/, otherFeat=[]-->, belongsTo=parr
825	relaxation cycle. Thus, it could be imagined that contraction, otherFeat=[]-->, belongsTo=parr
826	activates UNC-89 PK2 to phosphorylate the substrate, and, otherFeat=[]-->, belongsTo=parr
827	during relaxation when UNC-89 PK2 has returned to its, otherFeat=[]-->, belongsTo=parr
828	autoinhibited state, SCPL-1 phosphatase takes over and de-, otherFeat=[]-->, belongsTo=parr
829	phosphorylates the substrate. 4) The interaction of UNC-89, otherFeat=[]-->, belongsTo=parr
830	kinase regions and SCPL-1 forms a structure that recognizes, otherFeat=[]-->, belongsTo=parr
831	a substrate that neither of the separate enzymes can recog-, otherFeat=[]-->, belongsTo=parr
832	nize alone. 5) The interaction of UNC-89 kinase regions with, otherFeat=[]-->, belongsTo=parr
833	SCPL-1 recruits SCPL-1 to the M-line so that SCPL-1 can, otherFeat=[]-->, belongsTo=parr
834	dephoshorylate a substrate located there. This scenario does, otherFeat=[]-->, belongsTo=parr
835	not require that the UNC-89 kinase domains have phospho-, otherFeat=[]-->, belongsTo=parr
836	transferase activities, but instead act as protein?protein in-, otherFeat=[]-->, belongsTo=parr
837	teraction modules. This model is consistent with the finding, otherFeat=[]-->, belongsTo=parr
838	that both the predicted active PK2 and inactive PK1 bind to, otherFeat=[]-->, belongsTo=parr
839	SCPL-1. This model of PK1 or PK2 functioning to target, otherFeat=[]-->, belongsTo=parr
840	SCPL-1 to the M-line is further supported by our finding, otherFeat=[]-->, belongsTo=parr
841	that prior incubation of PK1 or PK2 with SCPL-1 under, otherFeat=[]-->, belongsTo=parr
842	conditions that are favorable for binding does not affect the, otherFeat=[]-->, belongsTo=parr
843	subsequent phosphatase activity of SCPL-1 (data not, otherFeat=[]-->, belongsTo=parr
844	shown). Further experiments will be required to sort out, otherFeat=[]-->, belongsTo=parr
845	these different scenarios., otherFeat=[]-->, belongsTo=parr
846	Our studies have pointed to a new function for the CTD, otherFeat=[]-->, belongsTo=parr
847	phosphatases, that is, in muscle giant protein signaling. The, otherFeat=[]-->, belongsTo=parr
848	founding member of the CTD phosphatase family is FCP,, otherFeat=[]-->, belongsTo=parr
849	found in all eukaryotes, and it is known to dephosphorylate, otherFeat=[]-->, belongsTo=parr
850	serines in the CTD of the largest subunit of RNA polymerase, otherFeat=[]-->, belongsTo=parr
851	II. This activity of FCP is required for RNA polymerase, otherFeat=[]-->, belongsTo=parr
852	recycling and global transcription (Yeo et al., 2003). FCP, otherFeat=[]-->, belongsTo=parr
853	proteins contain both a phosphatase domain (FCPH do-, otherFeat=[]-->, belongsTo=parr
854	main) and a BRCT domain. Higher eukaryotes also contain, otherFeat=[]-->, belongsTo=parr
855	small CTD phosphatases, proteins possessing the CTD phos-, otherFeat=[]-->, belongsTo=parr
856	phatase domain, but lacking the BRCT domain (Yeo et al.,, otherFeat=[]-->, belongsTo=parr
857	2003). In vitro, SCPs can dephosphorylate the CTD of RNA, otherFeat=[]-->, belongsTo=parr
858	polymerase II, but a definitive role as general regulators of, otherFeat=[]-->, belongsTo=parr
859	transcription has not been demonstrated. So far, there is, otherFeat=[]-->, belongsTo=parr
860	evidence that SCPs have more specialized functions in tran-, otherFeat=[]-->, belongsTo=parr
861	scriptional regulation. For example, SCP1-3 silence neuron-, otherFeat=[]-->, belongsTo=parr
862	specific gene expression in non-neuronal cells by being re-, otherFeat=[]-->, belongsTo=parr
863	cruited by a silencer factor complex (Yeo et al., 2005)., otherFeat=[]-->, belongsTo=parr
864	Recently, in mammalian cells and Xenopus embryos, SCP1-3,, otherFeat=[]-->, belongsTo=parr
865	but not FCP1, have been shown to dephosphorylate Smad, otherFeat=[]-->, belongsTo=parr
866	transcription factors and by doing so, regulate transforming, otherFeat=[]-->, belongsTo=parr
867	growth factor- and BMP signaling (Knockaert et al., 2006;, otherFeat=[]-->, belongsTo=parr
868	Sapkota et al., 2006; Wrighton et al., 2006). Our finding that, otherFeat=[]-->, belongsTo=parr
869	one of four SCPs in C. elegans specifically interacts with the, otherFeat=[]-->, belongsTo=parr
870	kinase domains of the giant protein UNC-89 demonstrates, otherFeat=[]-->, belongsTo=parr
871	yet another function for an SCP protein, a function probably, otherFeat=[]-->, belongsTo=parr
872	not related to transcriptional regulation. In addition, until, otherFeat=[]-->, belongsTo=parr
873	our findings, all SCPs have been reported to be nuclear (e.g.,, otherFeat=[]-->, belongsTo=parr
874	Yeo et al., 2003 for SCP1), whereas worm SCPL-1 is located, otherFeat=[]-->, belongsTo=parr
875	in myofibrils. It is possible that the kinase domains of ob-, otherFeat=[]-->, belongsTo=parr
876	scurin (UNC-89 in vertebrates) similarly interact with an, otherFeat=[]-->, belongsTo=parr
877	SCP protein. It is possible that the true vertebrate orthologue, otherFeat=[]-->, belongsTo=parr
878	for nematode SCPL-1 has not been identified; for example,, otherFeat=[]-->, belongsTo=parr
879	although searches of vertebrate proteomes have not re-, otherFeat=[]-->, belongsTo=parr
880	vealed an SCP protein with sequence homology outside the, otherFeat=[]-->, belongsTo=parr
881	phosphatase domain, it is possible that there is a functional, otherFeat=[]-->, belongsTo=parr
882	homologue of worm SCPL-1 that has an upstream region, otherFeat=[]-->, belongsTo=parr
883	that forms a similar structure, function, or both. Neverthe-, otherFeat=[]-->, belongsTo=parr
884	less, given the high degree of sequence identity between the, otherFeat=[]-->, belongsTo=parr
885	200-residue phosphatase domain of worm SCPL-1 to hu-, otherFeat=[]-->, belongsTo=parr
886	man SCP1, SCP2, and SCP3 (67, 64, and 64%, respectively),, otherFeat=[]-->, belongsTo=parr
887	even if these phosphatases target different substrate pro-, otherFeat=[]-->, belongsTo=parr
888	teins, the recognition sequences that immediately surround, otherFeat=[]-->, belongsTo=parr
889	the phophorylated serines/threonines might be very similar., otherFeat=[]-->, belongsTo=parr
890	ACKNOWLEDGMENTS, otherFeat=[]-->, belongsTo=parr
891	We thank Andy Fire for the promoter-less GFP vector, Robert Barstead for the, otherFeat=[]-->, belongsTo=parrnote
892	random primed nematode cDNA library, and Kozo Kaibuchi (Nagoya Uni-, otherFeat=[]-->, belongsTo=parrnote
893	versity) for vectors pMAL-KK1 and pKK51. Worm strains were provided by, otherFeat=[]-->, belongsTo=parrnote
894	the Caenorhabditis Genetics Center, which is supported by the National Center, otherFeat=[]-->, belongsTo=parrnote
895	for Research Resources of the National Institutes of Health. Support for this, otherFeat=[]-->, belongsTo=parrnote
896	work was provided by National Institute of Arthritis & Musculoskeletal &, otherFeat=[]-->, belongsTo=parrnote
897	Skin Diseases/National Institutes of Health grant AR-051466., otherFeat=[]-->, belongsTo=parrnote
898	REFERENCES, otherFeat=[]-->, belongsTo=parr
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954	integrin adhesion complexes. Curr. Biol. 12, 787?797., otherFeat=[]-->, belongsTo=parrnote
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978	filaments in Caenorhabditis elegans muscle. Mol. Biol. Cell 18, 4317? 4326., otherFeat=[]-->, belongsTo=parrnote
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980	Identification, tissue expression and chromosomal localization of human Ob-, otherFeat=[]-->, belongsTo=parrnote
981	scurin-MLCK, a member of the titin and Dbl families of myosin light chain, otherFeat=[]-->, belongsTo=parrnote
982	kinases. Gene 282, 237?246., otherFeat=[]-->, belongsTo=parrnote
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1009	dephosphorylates serine 5. Science 307, 596 ? 600., otherFeat=[]-->, belongsTo=parrnote
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1015	2432, otherFeat=[]-->, belongsTo=nota_cab_pie
==============================
0	Molecular Biology of the Cell-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->note	Molecular Biology of>>> Biology of the Cell
1	Vol. 19, 2424 ­2432, June 2008-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->note	Vol. 19, 2424 ­2432,>>>424 ­2432, June 2008
2	A Novel Protein Phosphatase is a Binding Partner for the-->id=1, page=0, size=25, fam=Times, col=#231f20, type=title, textLines=3--->[]--->title	A Novel Protein Phos>>>ding Partner for the
3	Protein Kinase Domains of UNC-89 (Obscurin) in-->id=1, page=0, size=25, fam=Times, col=#231f20, type=title, textLines=3--->[]--->title	Protein Kinase Domai>>>UNC-89 (Obscurin) in
4	Caenorhabditis elegans-->id=1, page=0, size=25, fam=Times, col=#231f20, type=title, textLines=3--->[]--->title	Caenorhabditis elega>>>enorhabditis elegans
5	Hiroshi Qadota,* Lee Anne McGaha,* Kristina B. Mercer,* Thomas J. Stark,* Tracey M. Ferrara,* and Guy M. Benian*-->id=3, page=0, size=17, fam=Times, col=#231f20, type=title, textLines=2--->[]--->note	Hiroshi Qadota,* Lee>>>* and Guy M. Benian*
6	*Department of Pathology, Graduate Division of Biological and Biomedical Sciences, Emory University,-->id=5, page=0, size=13, fam=Times, col=#231f20, type=title, textLines=2--->[]--->note	*Department of Patho>>>s, Emory University,
7	Atlanta, GA 30322-->id=5, page=0, size=13, fam=Times, col=#231f20, type=title, textLines=2--->[]--->note	Atlanta, GA 30322>>>Atlanta, GA 30322
8	Submitted January 22, 2008; Revised March 3, 2008; Accepted March 5, 2008-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->note	Submitted January 22>>>cepted March 5, 2008
9	Monitoring Editor: Erika Holzbaur-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->note	Monitoring Editor: E>>>itor: Erika Holzbaur
10	Mutation of the Caenorhabditis elegans gene unc-89 results in disorganization of muscle A-bands. unc-89 encodes a giant polypeptide (900 kDa) containing two protein kinase domains, PK1 and PK2. Yeast two-hybrid screening using a portion of UNC-89 including PK2, yielded SCPL-1 (small CTD phosphatase-like-1), which contains a C terminal domain (CTD) phosphatase type domain. In addition to the PK2 domain, interaction with SCPL-1 required the putative autoinhibitory sequence, and immunoglobulin (Ig) and fibronectin type 3 (Fn3) domains lying N-terminal of the kinase domain. SCPL-1 also interacts with PK1, and it similarly requires the kinase domain and upstream Fn3 and Ig domains. Analogous regions from the two other giant kinases of C. elegans, twitchin and TTN-1, failed to interact with SCPL-1. The interaction between SCPL-1 and either Ig-Fn3-PK2 or Fn3-Ig-PK1 was confirmed by biochemical methods. The scpl-1b promoter is expressed in the same set of muscles as unc-89. Antibodies to SCPL-1 localize to the M-line and a portion of the I-band. Bacterially expressed SCPL-1 proteins have phosphatase activity in vitro with properties similar to previously characterized members of the CTD phosphatase family. RNA interference knockdown results in a defect in the function of egg-laying muscles. These studies suggest a new role for the CTD phosphatase family, that is, in muscle giant kinase signaling.-->id=7, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=436--->[]--->parr	Mutation of the Caen>>>nt kinase signaling.
11	INTRODUCTION-->id=7, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=436--->[]--->parr	INTRODUCTION>>>INTRODUCTION
12	The sarcomere is the molecular machine that performs the work of contraction in striated muscle. It is comprised of overlapping, interacting thin and thick filaments and their attachment structures, the M-lines and Z-disks. The assembly and maintenance of the sarcomere involves specific interactions between hundreds of different proteins. Sarcomeres are unusually enriched for very large polypeptides, generally 700,000 Da, including nebulin and titin. These giant proteins are primarily composed of multiple inexact copies of small domains, typically 35­100 residues. From the now extensive knowledge about the many binding partners of the 3-MDa polypeptide titin in vertebrate muscle, several themes have emerged (Lange et al., 2006). First, depending on its location within the titin polypeptide chain, the same type of domain, the immunoglobulin domain, can interact with many different partners. Second, some titin interactors are located only at the sarcomere, whereas others are located at the sarcomere and the nucleus. One of the newest muscle giant modular proteins to be identified is UNC-89 in Caenorhabditis elegans (Ferrara et al., 2005).-->id=7, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=436--->[]--->parr	The sarcomere is the>>>rrara et al., 2005).
13	The nematode C. elegans is an excellent system in which to study the organization, assembly, and function of striated muscle in a whole organism (Waterston, 1988; Moerman and Fire, 1997; Moerman and Williams, 2006). The gene unc-89 was originally identified in genetic screens for worms that are both slow moving or paralyzed and that have disorganization of their myofilament lattice (Waterston et al., 1980), and then molecularly cloned (Benian et al., 1996). Obscurin was originally identified through a two-hybrid screen by using portions of titin as bait (Bang et al., 2001; Young et al., 2001), and identified as a homologue of UNC-89 (Young et al., 2001). The body wall muscle of unc-89 mutants shows an especial disorganization of the A-band, and for most mutant alleles, lacks M-lines, the structures in the middle of the A-band at which thick filaments are cross-linked (Waterston et al., 1980; Benian et al., 1999). Similarly, obscurin has been shown to have a role in the assembly or maintenance of A-bands: overexpression of a C-terminal portion of obscurin in primary skeletal myotubes results in a disorganization of sarcomeric myosin (Kontrogianni-Konstantopoulos et al., 2004).-->id=7, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=436--->[]--->parr	The nematode C. eleg>>>oulos et al., 2004).
14	unc-89 is a complex gene, which through the use of three different promoters expressed in different sets of muscles, and alternative splicing, generates at least six major protein isoforms UNC-89-A­F (Benian et al., 1996; Small et al., 2004; Ferrara et al., 2005). The largest of these isoforms, UNC-89-B and UNC-89-F, which are each 900,000 Da, contain 52 immunoglobulin (Ig) domains; two fibronectin type 3 (Fn3) domains; a triplet of Src homology 3 (SH3), Dbl homology (DH), and pleckstrin homology (PH) domains near their N termini; and two protein kinase domains near their C termini. Two somewhat smaller isoforms, UNC-89-A and UNC-89-E, lack the kinase domains at their C termini. The smallest isoforms, UNC-89-C and UNC-89-D, begin with partial first kinase domains, and each is directed by its own tissue-specific promoter. The human obscurin gene similarly has 3 -most exons encoding two protein kinase domains, with differential splicing producing obscurin A, which lacks the kinase domains (like UNC-89-A and -E); obscurin B (like UNC-89-B and -F), which contains the kinase domains; and two smaller obscurins, one obscurin of which contains intact first and second kinase domains (Bang et al., 2001; Russell et al., 2002; Fukuzawa et al., 2005). Although obscurin contains all the same domains as nematode UNC-89, a difference is that the SH3, DH, PH trio is located near the C terminus rather than near the N terminus as it is in UNC-89. For C. elegans UNC-89, homology modeling indicates that the first kinase domain (PK1) is inactive, but that the second kinase domain (PK2) may have kinase activity (Small et al., 2004). So far, however, neither substrates nor phosphotransferase activity has been demonstrated. Antibodies generated to three distinct regions of UNC-89 localize the proteins exclusively to the M-lines by immunofluorescence microscopy, and more recently, by immuno-electron microscopy (A. Reedy, G. Benian, and P. Hoppe, unpublished data). Vertebrate obscurin is located at the peripheries of both M-lines and Z-disks (Kontrogianni-Konstantopoulos et al., 2003). However, the situation is more complex, with obscurin A (lacking kinase domains) located at the M-line, obscurin B located at the A/I junction and additional isoforms located at the Z-disk and Z/I junction (Bowman et al., 2007).-->id=7, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=436--->[]--->parr	unc-89 is a complex >>>owman et al., 2007).
15	This article was published online ahead of print in MBC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E08 ­ 01­ 0053) on March 12, 2008.-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->parr	This article was pub>>>) on March 12, 2008.
16	Address correspondence to: Guy M. Benian (pathgb@emory.edu).-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->parr	Address corresponden>>> (pathgb@emory.edu).
17	2424-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->note	2424>>>2424
18	© 2008 by The American Society for Cell Biology-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->note	© 2008 by The Americ>>>ety for Cell Biology
19	To help understand how UNC-89 performs its functions, we wanted to identify its binding partners. To begin, we have focused on the C-terminal protein kinase domains. We expected that binding partners of the protein kinase domains would include kinase substrate(s), activator/inhibitor of the kinases or coregulators. Using the yeast two-hybrid method, we found that a novel protein phosphatase binds to each of the protein kinase domains of UNC-89.-->id=7, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=436--->[]--->parr	To help understand h>>>e domains of UNC-89.
20	MATERIALS AND METHODS-->id=7, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=436--->['U']--->title	MATERIALS AND METHOD>>>ATERIALS AND METHODS
21	Worm Strains, Culture, and RNA Interference (RNAi)-->id=7, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=436--->[]--->parr	Worm Strains, Cultur>>> Interference (RNAi)
22	Nematodes were grown at 20°C on NGM plates with Escherichia coli strain OP50 as food source (Brenner, 1974). Bristol N2 was the wild-type strain, and mutants included unc-89 (su75) (Small et al., 2004), unc-89 (tm752) (Ferrara et al., 2005), and two intragenic deletions from the C. elegans Gene Knockout Consortium, scpl-1(ok1080) and scpl-1(gk283). RNAi for scpl-1 was performed on wild type and unc-89 mutants by using a feeding procedure essentially as described in Kamath and Afhringer (2003). Full-length cDNAs for scpl-1a and scpl-1b were cloned into pPD129.36, and these plasmids or the empty vector were transformed into HT115 (DE3) bacteria before feeding the worms.-->id=773, page=1, size=8, fam=Times, col=#231f20, type=parrnote, textLines=317--->[]--->parr	Nematodes were grown>>>e feeding the worms.
23	Plasmid Construction-->id=7, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=436--->[]--->parr	Plasmid Construction>>>Plasmid Construction
24	For the screening of the two-hybrid library with Ig-Fn3-PK2 kinase, this region of UNC-89 was cloned into the bait plasmid pGBDU-C1 by using BamHI and SalI sites of the vector. This region of UNC-89, called 15/14 in Figure 1A, was polymerase chain reaction (PCR) amplified from the RB2 random primed cDNA library (a gift from Robert Barstead, Oklahoma Medical Research Foundation), by using primers NTSY-15 and NTSY-14 (see Supplemental Table 1 for all primer sequences).-->id=773, page=1, size=8, fam=Times, col=#231f20, type=parrnote, textLines=317--->[]--->parr	For the screening of>>>l primer sequences).
25	To test for interaction between SCPL-1a or -1b prey and various segments of UNC-89 surrounding the PK2 kinase, we first made a series of three baits in the pGBDU-C1 vector, with the same N terminus lying just after the end of the Fn3 domain and varying amounts of sequence C-terminal of the PK2 kinase catalytic core called 11/12, 11/13, and 11/14 (Figure 1A). PCR was used to amplify the corresponding coding sequences from the RB2 cDNA library, by using the same 5 primer, NTSY-11, with added BamHI site, and three different 3 primers with added SalI site, NTSY-12, NTSY-13, or NTSY14. Two additional PK2 region baits were also made similarly, called 15/12 and 15/13 (Figure 1A), by using the primers NTSY-15, -12, and -13.-->id=773, page=1, size=8, fam=Times, col=#231f20, type=parrnote, textLines=317--->[]--->parr	To test for interact>>>SY-15, -12, and -13.
26	To construct two-hybrid plasmids expressing various fragments of SCPL-1, PCR-amplified fragments of scpl-1 were cloned into the plasmid pGAD-C1 by using EcoRI and SalI sites of the vector. The primers used for amplification are as follows: SCPL-1-1 and SCPL-1-3 for full length (1-345 amino acids aa )of SCPL-1a; SCPL-1-2 and SCPL-1-3 for full length (1-491 aa) of SCPL-1b; SCPL1-1 and SCPL-1-4 for amino acids 1-156 of SCPL-1a; SCPL-1-2 and SCPL-1-4 for amino acids 1-302 of SCPL-1b; SCPL-1-2 and SCPL-1-5 for amino acids 1-146 of SCPL-1b; and SCPL-1-11 and SCPL-1-3 for amino acids 157-345 of SCPL-1a (amino acids 303-491 of SCPL-1b). The prey plasmid harboring amino acids 245-491 of SCPL-1b was isolated from the library during twohybrid screening.-->id=773, page=1, size=8, fam=Times, col=#231f20, type=parrnote, textLines=317--->[]--->parr	To construct two-hyb>>>twohybrid screening.
27	To test whether the Ig-Fn3-kinase regions from twitchin and TTN-1 interact with SCPL-1a, -1b,-2, -3a, or -4, these regions of twitchin and TTN-1 were first cloned into the bait plasmid pGBDU-C1 by using the BamHI and SalI sites of the vector. The relevant regions of twitchin were PCR amplified from the RB2 cDNA library by using primers TWI-F and TWI-R, and for TTN-1, the primers were TTN-F and TTN-R.-->id=773, page=1, size=8, fam=Times, col=#231f20, type=parrnote, textLines=317--->[]--->parr	To test whether the >>>ere TTN-F and TTN-R.
28	Figure 1. (A) Yeast two-hybrid assays demonstrate the specificity of the interaction of UNC-89 PK2 with SCPL-1. Left, schematic representation of baits used to test full-length SCPL-1a and -b prey. Right, images of yeast growth on Ade plates. Note that for UNC89, interaction requires the catalytic core (PK2) plus the N-terminal Ig and Fn3 domains, and the C-terminal autoinhibitory domain. Note that comparable regions from the two other giant kinases in the worm, twitchin and TTN-1, fail to interact with SCPL-1. (B) The PK1 region of UNC-89 also interacts with SCPL-1 in the yeast two-hybrid assay. Domain mapping indicates that interaction of the PK1 region with SCPL-1 requires, in addition to the catalytic core (PK1), the Fn3 and Ig domains. , growth and , no growth on Ade plates. (C) Only the phosphatase domain of SCPL-1 is required for interaction to the PK regions of UNC-89 in yeast twohybrid assays. The indicated portions of SCPL-1a and -1b were tested as bait against Fn3-Ig-PK1 (PK1) or Ig-Fn3-PK2 (PK2) prey. , growth and , no growth on Ade plates. The colored bar indicates the minimal region of SCPL-1a/b required for interaction.-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->capfig	Figure 1. (A) Yeast >>>red for interaction.
29	Novel Phosphatase Binds Giant Protein Kinase-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->note	Novel Phosphatase Bi>>>Giant Protein Kinase
30	Vol. 19, June 2008-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->note	Vol. 19, June 2008>>>Vol. 19, June 2008
31	2425-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->note	2425>>>2425
32	To determine whether SCPL-1a or -1b preys interact with PK1 region baits, baits called AB, EB, FB, and AG (Figure 1B), were made in pGBDU-C1 by using the SmaI and SalI sites, by insertion of PCR-amplified fragments by using primers U89-PK1-A, U89-PK1-B, U89-PK1-E, U89-PK1-F, and U89PK1-G.-->id=773, page=1, size=8, fam=Times, col=#231f20, type=parrnote, textLines=317--->[]--->parr	To determine whether>>>PK1-F, and U89PK1-G.
33	To test whether UNC-89 Ig-Fn3-PK2 of UNC-89 or comparable regions of twitchin or TTN-1 interact with SCPL-2, -3a, and -4, full-length cDNAs for these other phosphatases were PCR amplified by using the following primers, and then they were inserted into the prey plasmid pGAD-C1 by using BamHI and SalI: for SCPL-2, SCPL-2-F and -R; for SCPL-3a, SCPL-3-F and -R; and for SCPL-4, SCPL-4-F and -R.-->id=773, page=1, size=8, fam=Times, col=#231f20, type=parrnote, textLines=317--->[]--->parr	To test whether UNC->>>-4, SCPL-4-F and -R.
34	To express hemagglutinin (HA)-tagged UNC-89 fragments in yeast, we prepared the following plasmids. The PCR-amplified fragments of the relevant region of UNC-89 PK2 by using the primers U89-PK2-1 and -2 for fragment 11/12; U89-PK2-1 and -3 for fragment 11/13; U89-PK2-1 and -4 for 11/14; U89-PK2-5 and -2 for 15/12; U89-PK2-5 and -3 for 15/13; and U89PK2-5 and -4 for 15/14 were cloned into pKS-HA8(XbaI) (three HA-tagged vectors) by using the EcoRV site of the vector. The PCR-amplified fragments from the relevant region of UNC-89 PK1 by using the primers as described above were cloned into pKS-HA8(Nhex2) (three HA-tagged vectors) by using EcoRV and SalI sites. HA-tagged UNC-89 11/12, 11/13, 11/14, 15/12, 15/13, and 15/14, AB were cut out with XbaI or NheI from HA-tagged vectors and cloned into pGAP-C-Nhe (yeast expression vector, TRP1 marker) by using the NheI site of the vector.-->id=773, page=1, size=8, fam=Times, col=#231f20, type=parrnote, textLines=317--->[]--->parr	To express hemagglut>>> site of the vector.
35	To express myc-tagged SCPL-1 in yeast, we prepared the following plasmids. The PCR-amplified fragments of full-length SCPL-1a and SCPL-1b by using the primers as described above, were cloned into pKK51 (myc-tagged vector) by using EcoRI and XhoI sites of the vector. Myc-tagged SCPL-1a and myc-tagged SCPL-1b fragments were cut out with SpeI from myc-tagged vector and cloned into pGAPU-C-Nhe (yeast expression vector, URA3 marker) by using the NheI site of the vector.-->id=773, page=1, size=8, fam=Times, col=#231f20, type=parrnote, textLines=317--->[]--->parr	To express myc-tagge>>> site of the vector.
36	To express glutathione-S transferase (GST) or maltose-binding protein (MBP) fusions of SCPL-1 in bacteria, we prepared the following. The fulllength fragments of SCPL-1a and SCPL-1b were PCR amplified by using primers as described above and cloned into pMAL-KK-1 by using EcoRI and XhoI sites. The fragments of SCPL-1 (1-156 aa of SCPL-1a, 1-302 aa of SCPL1b, and 1-146 aa of SCPL-1b) were PCR amplified by using the primers described above. Amplified fragments were cloned into pGEX-KK-1 and pMAL-KK-1 by using EcoRI and XhoI sites of the vectors. The plasmids derived from pGEX-KK-1 were used for production of GST fusion proteins, whereas the plasmids derived from pMAL-KK-1 were used for production of MBP fusion proteins.-->id=773, page=1, size=8, fam=Times, col=#231f20, type=parrnote, textLines=317--->[]--->parr	To express glutathio>>>MBP fusion proteins.
37	Yeast Two-Hybrid Screens and Assays-->id=7, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=436--->[]--->parr	Yeast Two-Hybrid Scr>>>d Screens and Assays
38	Two-hybrid screening was performed as described in Miller et al. (2006). Use of the two-hybrid method to study protein­protein interaction was performed as described in Mackinnon et al. (2002).-->id=773, page=1, size=8, fam=Times, col=#231f20, type=parrnote, textLines=317--->[]--->parr	Two-hybrid screening>>>innon et al. (2002).
39	Purification of Bacterially Expressed Proteins-->id=7, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=436--->[]--->parr	Purification of Bact>>>y Expressed Proteins
40	GST or MBP fusions proteins were prepared as described in Mercer et al. (2006).-->id=773, page=1, size=8, fam=Times, col=#231f20, type=parrnote, textLines=317--->[]--->parr	GST or MBP fusions p>>>ercer et al. (2006).
41	Assays to Confirm Interaction between SCPL-1 and UNC-89 PK2 or PK1-->id=7, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=436--->[]--->parr	Assays to Confirm In>>>nd UNC-89 PK2 or PK1
42	To confirm an interaction between SCPL-1 and the PK2 region of UNC-89, we expressed in the same yeast cell myc tagged SCPL-1b together with HAtagged versions of one of the portions of UNC-89 containing PK2 as shown in Figure 1A (11/12, 11/13, 11/14, 15/12, 15/13, 15/14), as described above. Yeast were grown in 50 ml of minimal media plus dextrose, casamino acids, and adenine (SD CA) to an optical density of 0.6 at 600 nm, pelleted, and frozen at 80°C. Each yeast pellet was resuspended in 500 l of yeast lysis buffer (50 mM HEPES, pH 7.5, 150 mM KCl, 1 mM EGTA, 1 mM EDTA, and complete Mini protease inhibitors, Roche, Indianapolis, IN). The yeast slurries were transferred to 1.5-ml Eppendorf tubes that contained 250 l of glass beads (425­ 600 m in diameter; G-8772, Sigma-Aldrich, St. Louis, MO). Yeast were lysed by vortexing at maximum speed, six bursts of 30 s each, separated by 1 min on ice. After pelleting the beads by centrifugation in a microfuge at 3000 rpm for 5 min, the supernatant was transferred to a fresh tube, and cellular debris was pelleted by centrifugation in a microfuge at 13,200 rpm for 30 min. The resulting supernatant was transferred to a new tube, and its total protein concentration was determined by Bradford assay (Bradford 1976). The HA-tagged PK2 proteins were immunoprecipitated in a total volume of 500 l for 1 h with shaking at 4°C from 120 g of protein in immunoprecipitation (IP) buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1 mM EGTA, 1 mM EDTA, 0.25% gelatin, complete mini protease inhibitors, and 0.1% NP-40) and 50 l of a 1:1 slurry of agarose beads conjugated monoclonal antibodies to HA (A2095, Sigma-Aldrich). Beads were pelleted by centrifugation and washed two times with IP buffer and once with IP buffer lacking NP-40. A final hard spin for 5 min was performed, and as much supernatant was aspirated off as possible. Laemmli sample buffer (2 ) was added to the pellet, and then the mixture was vortexed 5 s, heated at 95°C for 5 min, vortexed for 20 s, and centrifuged for 5 min to pellet the beads. The supernatant was then carefully transferred to a fresh tube. Two 12% SDS-polyacrylamide gels were run containing either 10 or 40 l of supernatant, and they were transblotted to nitrocellulose. The blot that had 10 l of supernatant was reacted with anti-HA (rabbit antibodies, affinity purified, 1:1000; H6908, Sigma-Aldrich), and the blot that had 40 l of supernatant was reacted with anti-myc (mouse monoclonal purified immunoglobulin, 1:400; M4439, Sigma-Aldrich), reacted with appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies, and detected by enhanced chemiluminescence (ECL). Controls included no added yeast lysate, and 10 g of total protein from any of the yeast to verify the expression of the myc-tagged SCPL-1b.-->id=773, page=1, size=8, fam=Times, col=#231f20, type=parrnote, textLines=317--->[]--->parr	To confirm an intera>>> myc-tagged SCPL-1b.
43	Figure 2. Verification of interaction between SCPL-1 and UNC-89 PK1 or PK2 regions. (A) In the same yeast cell, myc-tagged SCPL-1b and individual HA-tagged derivatives of the UNC-89 PK2 region were expressed. 11/12, 11/13 . . . refer to the derivatives presented in Figure 1A. Total protein lysates were incubated with agarose beads coated with antibodies to the HA tag, washed, and eluted, and portions were run on two gels and blotted. One blot was reacted with anti-HA to detect the presence of the PK2 derivatives (top). As shown above, appropriately sized proteins were detected from each PK2 derivative. The other blot was reacted with anti-myc to detect the presence of SCPL-1 that might have been brought down with the PK2 protein (bottom). Only derivative 15/14 (IgFn3-PK2-autoinhibitory region) is coimmunoprecipitated. An arrow designates the position on the blot of myc-SCPL-1b from the lysate. (B) Bacterially expressed MBP-SCPL-1a interacts with yeast expressed HA-PK1 or PK2. Total protein extracts were prepared from yeast expressing either HA-PK2 15/14 (see Figure 1A) or HA-PK1 AB (see Figure 1B), incubated with agarose beads coated with antibodies to HA, washed, incubated with purified, bacterially expressed MBP or MBP-SCPL-1a, and washed. The proteins were eluted, and portions of each sample were run on two gels and blotted. One blot was reacted with anti-HA to detect the presence of the yeast expressed protein, the other blot was reacted with antiMBP to detect possible binding with MBP or MBP-SCPL-1a. (C) Coomassie stained gel of purified MBP-SCPL-1a and MBP-SCPL-1b. In each case, the top band is likely to be the full-length fusion protein; the second band may have resulted from degradation.-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->capfig	Figure 2. Verificati>>>ed from degradation.
44	H. Qadota et al.-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->note	H. Qadota et al.>>>H. Qadota et al.
45	Molecular Biology of the Cell-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->note	Molecular Biology of>>> Biology of the Cell
46	2426-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->note	2426>>>2426
47	To confirm an interaction between SCPL-1 and the PK1 region of UNC-89 and to further verify an interaction with the PK2 region of UNC-89, the following procedure was used. Yeast expressing either an HA-tagged PK1 region (segment AB in Figure 1B) or HA-tagged PK2 region (15/14 in Figure 1A) were grown; yeast lysates were prepared; and beginning with 300 gof total protein, the HA-tagged UNC-89 segments were immunoprecipitated by using anti-HA agarose beads, and washed, as described above. A binding reaction was set up in which the HA-tagged UNC-89 proteins attached to beads were incubated at 4°C for 1 h with shaking in a total volume of 500 l with either bacterially expressed and purified MBP (10 g) or MBP-SCPL-1a (10 g). The beads were washed three times with IP buffer, transferred to clean Eppendorf tubes, centrifuged for 5 min, and as much supernatant was removed as possible. The proteins on the pelleted beads were eluted in Laemmli buffer as described above, and two 10% SDS-PAGE gels were run and transferred to nitrocellulose-- one gel contained 5 l of eluted protein per lane, and the other gel contained 40 l/lane. The blot that had the 5- l samples was reacted with anti-HA (described above), followed by anti rabbit Ig-conjugated HRP, and detection by ECL. The blot containing 40- l samples was incubated with HRP-conjugated anti-MBP (1:5000; New England Biolabs, Beverly, MA) and detected by ECL.-->id=773, page=1, size=8, fam=Times, col=#231f20, type=parrnote, textLines=317--->[]--->parr	To confirm an intera>>>and detected by ECL.
48	Expression Pattern of scpl-1b Promoter-->id=7, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=436--->[]--->parr	Expression Pattern o>>> of scpl-1b Promoter
49	A 7.5-kb genomic segment that includes the initiator ATG, 5 -untranslated region and putative promoter sequence upstream of scpl-1b was produced by PCR and cloned into pPD95.77 (a gift from A. Fire, Stanford University, Stanford, CA), by using the SphI and BamHI sites. The primers used were SCPL-1b-promo-F and SCPL-1b-promo-R. The promoter-gfp expression plasmid was injected into wild-type strain N2 worms, and one transgenic line was recovered. Green fluorescent protein (GFP) fluorescence images of adults were obtained as described in Qadota et al., 2007.-->id=773, page=1, size=8, fam=Times, col=#231f20, type=parrnote, textLines=317--->[]--->parr	A 7.5-kb genomic seg>>>Qadota et al., 2007.
50	SCPL-1 Antibodies-->id=7, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=436--->[]--->parr	SCPL-1 Antibodies>>>SCPL-1 Antibodies
51	For production of anti-SCPL-1b and anti-SCPL-1a, MBP fusion of residues 1-302 of SCPL-1b and MBP fusion of residues 1-156 of SCPL-1a were used as immunogens. For affinity purification of anti-SCPL-1b and anti-SCPL-1a, GST fusion of residues 1-146 of SCPL-1b and GST fusion of residues 1-156 of SCPL-1a were used, respectively. Affinity purification was carried out as described in Mercer et al. (2006).-->id=773, page=1, size=8, fam=Times, col=#231f20, type=parrnote, textLines=317--->[]--->parr	For production of an>>>ercer et al. (2006).
52	Western Blot and Immunofluorescence Microscopy-->id=7, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=436--->[]--->parr	Western Blot and Imm>>>orescence Microscopy
53	Procedures for preparing worm protein lysates and Western blots were described previously (Mercer et al., 2006). The following worm strains were used to make lysates: wild type (N2), scpl-1(ok1080) and scpl-1(gk283). The blot also contained lysates from yeast expressing myc tagged SCPL-1a and -1b. Yeast lysates were prepared by suspending a yeast pellet froma2ml overnight culture in Laemmli buffer and heating at 95o for 5 min. Affinity-purified anti-SCPL-1a was used at 1:200 dilution, and affinity purified anti-SCPL-1b was used at 1:200 dilution. Wild type adult worms were immunostained after fixation by the method described by Nonet et al. (1993). Anti-SCPL-1b was used at 1:100 dilution, and anti-UNC-89 (MH42) was used at 1:200 dilution. Secondary antibodies and confocal microscopy were as described in Qadota et al. (2007).-->id=773, page=1, size=8, fam=Times, col=#231f20, type=parrnote, textLines=317--->[]--->parr	Procedures for prepa>>>adota et al. (2007).
54	SCPL-1 Phosphatase Activity Assays-->id=7, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=436--->[]--->parr	SCPL-1 Phosphatase A>>>tase Activity Assays
55	Hydrolysis of phosphate from p-nitrophenyl phosphate (pNPP) was assayed in a 1-ml reaction mixture containing 50 mM Tris acetate, pH 5.0, 10 mM MgCl2,20mM pNPP, and MBP or MBP-SCPL-1b (typically 10 g) as described in Zheng et al. (2005). The amount of protein, the pH, and inclusion of different divalent cations was as described in Zheng et al. (2005), and the use of various phosphatase inhibitors was as described in Kamenski et al. (2004).-->id=773, page=1, size=8, fam=Times, col=#231f20, type=parrnote, textLines=317--->[]--->parr	Hydrolysis of phosph>>>enski et al. (2004).
56	Egg-Laying Assays-->id=7, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=436--->[]--->parr	Egg-Laying Assays>>>Egg-Laying Assays
57	Examination of drug response to egg laying was performed as described in Trent et al. (1983). Briefly, 96-well plates with 100 l of M9 containing 5 mg/ml serotonin or 0.75 mg/ml imipramine in each well were prepared. Single adult worms were soaked in each well for 1 h (serotonin) or 1.5 h (imipramine) at room temperature. After incubation, the numbers of laid eggs were counted.-->id=773, page=1, size=8, fam=Times, col=#231f20, type=parrnote, textLines=317--->[]--->parr	Examination of drug >>>d eggs were counted.
58	RESULTS inhibitory domain. After screening 8.1 106 colonies, 42 positives were confirmed after retransformation, and all of them represented portions of a single gene, B0379.4. As predicted on WormBase and confirmed by sequencing cDNAs, this gene encodes two transcripts, B0379.4a and B0379.4b, by alternative splicing in the 5 half of the gene. Our prey clones included representatives of both splice isoforms. The proteins encoded by B0379.4a and .4b are 345 and 491 residues, respectively. The only recognizable domain found in these proteins is a protein phosphatase domain ( 170 ­200 aa) in the C-terminal half of each isoform (Figures 1C and 3A). For reasons explained below, we have renamed B0379.4 as SCPL-1.-->id=7, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=436--->[]--->parr	RESULTS inhibitory d>>>d B0379.4 as SCPL-1.
59	With the goal of identifying binding partners or possible substrates for the protein kinase domains of UNC-89, a portion of the protein containing PK2 was used to screen a yeast two-hybrid library. The bait contained the domains Ig-Fn3-PK2 (Figure 1A, 15/14), including the putative auto--->id=7, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=436--->[]--->parr	With the goal of ide>>>g the putative auto-
60	Figure 3. The SCPL family of proteins in C. elegans and demonstration that UNC-89 PK2 region specifically interacts with SCPL-1. (A) Schematic representation of domain organization of proteins containing CTD type (or FCPH) phosphatase domains. C. elegans has five genes that encode proteins with FCPH domains: one domain called CeFCP-1 is more closely related to FCP proteins in that it has both phosphatase and BRCT domains. The others, including SCPL-1, which interacts with UNC-89, are more closely related to small CTD phosphatases (called SCPs), and they are designated SCP-L. The percentages indicate the percentage of identical amino acids in the 200-residue phosphatase domains, compared with the phosphatase in CeB0379.4a (SCPL-1). (B) By yeast two-hybrid assays, the UNC-89 PK2 region interacts with SCPL-1, but not SCPL-2, -3, or -4. The comparable regions of twitchin and TTN-1 fail to interact with any of the SCPL proteins. , growth and ,no growth on Ade plates.-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->capfig	Figure 3. The SCPL f>>>rowth on Ade plates.
61	Novel Phosphatase Binds Giant Protein Kinase-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->note	Novel Phosphatase Bi>>>Giant Protein Kinase
62	Vol. 19, June 2008-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->note	Vol. 19, June 2008>>>Vol. 19, June 2008
63	2427-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->note	2427>>>2427
64	To determine which portions of the UNC-89 bait are minimally required to interact with SCPL-1, deletion derivatives of the segment Ig-Fn3-PK2 were tested by two-hybrid against SCPL-1a and -1b full-length prey. As shown in Figure 1A, all the domains are required for interaction, including the C-terminal autoinhibitory region. Because the pattern of domains, Ig-Fn3-kinase, is conserved among the giant protein kinases, we tested the comparable regions from the two other giant kinases in worm muscle, twitchin and TTN-1. As can be seen in Figure 1A, these giant kinases failed to interact with SCPL-1a or -1b.-->id=7, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=436--->[]--->parr	To determine which p>>>with SCPL-1a or -1b.
65	Given that UNC-89 has a second protein kinase domain, PK1, which is predicted to be catalytically silent (Small et al., 2004), we wondered whether it, too, might interact with SCPL-1. Therefore, fragment Fn3-Ig-PK1 was used as bait to check for interaction with SCPL-1 by two-hybrid analysis. As indicated in Figure 1B, interaction was found, and all the domains, Fn3, Ig, and PK1 need to be present to obtain interaction of the PK1 region with either SCPL-1a or -1b. To determine which portion of SCPL-1a or -1b is required for interaction with either PK1 or PK2 full-length regions, deletion derivatives of SCPL-1a and -1b were tested by twohybrid analysis. As shown in Figure 1C, the minimal region required is the phosphatase domain.-->id=7, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=436--->[]--->parr	Given that UNC-89 ha>>> phosphatase domain.
66	To provide additional evidence that SCPL-1 interacts with the PK2 and PK1 regions of UNC-89, further experiments were performed. Although each isoform of SCPL-1 could be expressed in bacteria as MBP or GST fusions, segments of UNC-89, either Ig-Fn3-PK2, or Fn3-Ig-PK1 as GST or Histagged fusions, could not be expressed. In fact, the PK1 or PK2 proteins seemed to significantly reduce the growth rate of E. coli. Therefore, in the same yeast cell, myc-tagged SCPL-1b and individual HA-tagged derivatives of the PK2 region corresponding to those depicted in Figure 1A were expressed. Total protein lysates were prepared and incubated with agarose beads coated with antibodies to the HA tag, washed, and eluted, and portions were run on two gels and blotted. One blot was reacted with anti-HA to detect the presence of the PK2 derivatives. As shown in Figure 2A, appropriately sized proteins were detected from each PK2 derivative. The second blot was reacted with anti-myc to detect the presence of any SCPL-1b that might have been brought down with the PK2 protein. As shown in Figure 2A (bottom), only derivative 15/14 (Ig-Fn3-PK2-autoinhibitory region) was coimmunoprecipitated, consistent with the domain mapping two-hybrid experiment shown in Figure 1A. A variation of this experiment was conducted in which yeast was used to express the PK2 or PK1 segments, and E. coli was used to express SCPL-1. Protein extracts were prepared from yeast expressing either HA-PK2 15/14 (Figure 1A) or HA-PK1 AB (Figure 1B), and these UNC-89 segments were immunoprecipitated with anti-HA antibody-conjugated agarose beads. After washing, these beads were incubated with purified, bacterially expressed MBP or MBPSCPL-1a (Figure 2C), washed, and the proteins were eluted. Portions of each sample were run on two gels and blotted. One blot was reacted with anti-HA to detect the presence of the yeast expressed protein, the other blot was reacted with anti-MBP to detect possible binding with MBP or MBPSCPL-1a. As shown in Figure 2B, this method demonstrates interaction between MBP-SCPL-1a and either Ig-Fn3-PK2, or Fn3-Ig-PK1. Using MBP-SCPL-1b, we obtained similar results (data not shown).-->id=7, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=436--->[]--->parr	To provide additiona>>>ts (data not shown).
67	Figure 4. Expression pattern of the SCPL-1b promoter and characterization of antibodies to SCPL-1. (A) SCPL-1b is expressed in the same muscle cells as UNC-89. The exon/intron structure of the SCPL-1b gene is shown, as predicted on WormBase. 7.5 kb of DNA sequence upstream of the initiator methionine together with codons for a few amino acids of the first exon of SCPL-1b were fused in-frame with GFP, and used to create transgenic animals, and the sites of GFP expression were recorded. This promoter is expressed in pharyngeal (left), vulval (center), and body wall muscle (right). (B) Anti-SCPL-1b and anti-SCPL-1a antibodies specifically recognize SCPL-1b and SCPL-1a, respectively. Above is shown immunogens used to generate rabbit antibodies that were later affinity purified using the same regions. Below are Western blots reacted with the designated antibodies. In each case, the left-most three lanes are worm extracts, and the right-most lanes are yeast extracts. WT, wild type; scpl-1(ok1080) and scpl-1(gk283) are intragenic deletions of the scpl-1 gene. Vector, Myc-SCPL-1a, and Myc-SCPL-1b are yeast harboring either the empty vector or myc-tagged SCPL-1 isoforms. (C) Anti-SCPL-1 localize to M-lines and I-bands in body wall muscle. Anti-SCPL-1b and anti-UNC-89 were coincubated with wild-type worms, and the muscle was imaged by immunofluorescence microscopy. The images show a portion of one body wall muscle cell. Weak labeling of the M-line and a portion of the I-band is seen. UNC-89 is a marker of the M-line. Bar, 10 m.-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->capfig	Figure 4. Expression>>>e M-line. Bar, 10 m.
68	H. Qadota et al.-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->note	H. Qadota et al.>>>H. Qadota et al.
69	Molecular Biology of the Cell-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->note	Molecular Biology of>>> Biology of the Cell
70	2428-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->note	2428>>>2428
71	Application of BLAST and Pfam reveals that the phosphatase domain of B0379.4 (SCPL-1) is most closely related to the protein phosphatase domains of the CTD phosphatase family. The founding member of this family is FCP1 (TFIIFassociating component of CTD phosphatase), which is conserved from yeast to humans, and it is known to dephosphorylate the C-terminal domain (CTD) of the largest subunit of RNA polymerase II. This phosphatase family has no sequence similarity to other phosphatases, except for the motif DXDX(T/V) that is common to both phosphotransferases and phosphohydrolases (Collet et al., 1998). In addition to a phosphatase domain, FCP proteins have a Brca1 C-terminal (BRCT) domain. Other members of this family are generally smaller proteins that lack the BRCT domain, and they are called small CTD phosphatases (SCPs). The function of the SCPs is still being established (see Discussion). Our analysis has revealed that C. elegans has five genes that encode proteins with FCP1 homology (FCPH) or CTDtype phosphatase domains (Figure 3A). One of them is more closely related to FCP (in sequence and the presence of both phosphatase and BRCT domains); thus, it is called CeFCP-1. The others, including B0379.4 (SCPL-1), are more closely related to SCPs; therefore, we call these proteins SCP-like (SCPL). It seems noteworthy that the phosphatase domain of worm SCPL-1 is more closely related to the phosphatase domains of human SCP1, SCP2, and SCP3 (64 ­ 67% identical amino acids over a span of 200 amino acids), than it is to the other SCPs in C. elegans (ranging from 23 to 38% identity). It is also clear that the phosphatase domain of SCPL-1 is least similar to the phosphatase domains from worm, yeast, and human FCPs (16 ­19% identity).-->id=7, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=436--->[]--->parr	Application of BLAST>>> (16 ­19% identity).
72	To determine whether the PK2 and PK1 regions of UNC-89 interact specifically with SCPL-1, prey plasmids were generated specifying the complete coding sequences for nematode SCPL-2, SCPL-3, and SCPL-4, and they were tested by two-hybrid for interaction. As shown in Figure 3B, when the Ig-Fn3-PK2 segment was tested against all the SCPL proteins, interaction was found only with SCPL-1. In addition, as shown in Figure 3B, when the comparable kinase-containing regions of twitchin and TTN-1 were tested, they failed to interact with any of the SCPL proteins.-->id=7, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=436--->[]--->parr	To determine whether>>>f the SCPL proteins.
73	Because the two-hybrid library contains cDNAs made from mRNA prepared from whole worms (including all tissues and cell types), it was important to determine whether scpl-1 is expressed in muscle. To do this, we fused in-frame with GFP, 7.5 kb of DNA sequence upstream of the predicted initiator methionine together with codons for a few amino acids of the first exon of scpl-1b. This DNA was used to create transgenic animals, and the sites of GFP expression were recorded. As shown in Figure 4A, scpl-1b is expressed in pharyngeal, vulval, and body wall muscle, the same muscle cells that express unc-89 (Small et al., 2004). This result bolsters the likelihood that SCPL-1 and UNC-89 interact in vivo.-->id=7, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=436--->[]--->parr	Because the two-hybr>>>89 interact in vivo.
74	To determine where the SCPL-1 proteins are expressed and localized, we generated affinity-purified rabbit antibodies that specifically recognize SCPL-1a or SCPL-1b. As shown in Figure 4B, by Western blot, anti-SCPL-1b recognizes yeast-expressed myc-tagged SCPL-1b, but not SCPL1a, and anti-SCPL-1a recognizes yeast-expressed myctagged SCPL-1a but not SCPL-1b. These antibodies recognize the expected sized proteins from C. elegans. AntiSCPL-1b reacts with an 70-kDa protein from wild-type worms likely to be SCPL-1b, and with truncated proteins from the intragenic deletion mutants, ok1080 and gk283. Similarly, anti-SCPL-1a reacts with an 50-kDa protein from wild-type worms likely to be SCPL-1a, and this protein is missing from the intragenic deletion mutants.-->id=7, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=436--->[]--->parr	To determine where t>>>ic deletion mutants.
75	Table 1. Loss of function of scpl-1 results in a defect in egg laying-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->captable	Table 1. Loss of fun>>>defect in egg laying
76	Strain-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->celltab	Strain>>>Strain
77	RNAi-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->celltab	RNAi>>>RNAi
78	Line-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->celltab	Line>>>Line
79	Serotonin-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->celltab	Serotonin>>>Serotonin
80	Imipramine-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->celltab	Imipramine>>>Imipramine
81	( )( )-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->celltab	( )( )>>>( )( )
82	( )( )-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->celltab	( )( )>>>( )( )
83	N2-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->celltab	N2>>>N2
84	scpl-1a full-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->celltab	scpl-1a full>>>scpl-1a full
85	a-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->celltab	a>>>a
86	5-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->celltab	5>>>5
87	3-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->celltab	3>>>3
88	2-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->celltab	2>>>2
89	0-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->celltab	0>>>0
90	8-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->celltab	8>>>8
91	2-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->celltab	2>>>2
92	0-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->celltab	0>>>0
93	0-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->celltab	0>>>0
94	b7-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->celltab	b7>>>b7
95	1-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->celltab	1>>>1
96	0-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->celltab	0>>>0
97	1-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->celltab	1>>>1
98	9-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->celltab	9>>>9
99	0-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->celltab	0>>>0
100	1-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->celltab	1>>>1
101	0-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->celltab	0>>>0
102	N2-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->celltab	N2>>>N2
103	scpl-1b full-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->celltab	scpl-1b full>>>scpl-1b full
104	a-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->celltab	a>>>a
105	7-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->celltab	7>>>7
106	1-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->celltab	1>>>1
107	1-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->celltab	1>>>1
108	0-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->celltab	0>>>0
109	9-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->celltab	9>>>9
110	1-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->celltab	1>>>1
111	0-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->celltab	0>>>0
112	0-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->celltab	0>>>0
113	b8-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->celltab	b8>>>b8
114	1-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->celltab	1>>>1
115	1-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->celltab	1>>>1
116	0-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->celltab	0>>>0
117	10-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->celltab	10>>>10
118	0-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->celltab	0>>>0
119	0-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->celltab	0>>>0
120	0-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->celltab	0>>>0
121	N2-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->celltab	N2>>>N2
122	empty vector-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->celltab	empty vector>>>empty vector
123	a-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->celltab	a>>>a
124	10-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->celltab	10>>>10
125	0-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->celltab	0>>>0
126	0-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->celltab	0>>>0
127	0-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->celltab	0>>>0
128	10-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->celltab	10>>>10
129	0-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->celltab	0>>>0
130	0-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->celltab	0>>>0
131	0-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->celltab	0>>>0
132	b9-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->celltab	b9>>>b9
133	1-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->celltab	1>>>1
134	0-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->celltab	0>>>0
135	0-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->celltab	0>>>0
136	8-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->celltab	8>>>8
137	2-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->celltab	2>>>2
138	0-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->celltab	0>>>0
139	0-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->celltab	0>>>0
140	unc-89 (tm752)-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->celltab	unc-89 (tm752)>>>unc-89 (tm752)
141	scpl-1b full-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->celltab	scpl-1b full>>>scpl-1b full
142	a-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->celltab	a>>>a
143	7-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->celltab	7>>>7
144	2-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->celltab	2>>>2
145	1-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->celltab	1>>>1
146	0-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->celltab	0>>>0
147	10-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->celltab	10>>>10
148	0-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->celltab	0>>>0
149	0-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->celltab	0>>>0
150	0-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->celltab	0>>>0
151	b9-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->celltab	b9>>>b9
152	0-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->celltab	0>>>0
153	1-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->celltab	1>>>1
154	0-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->celltab	0>>>0
155	10-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->celltab	10>>>10
156	0-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->celltab	0>>>0
157	0-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->celltab	0>>>0
158	0-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->celltab	0>>>0
159	unc-89 (tm752)-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->celltab	unc-89 (tm752)>>>unc-89 (tm752)
160	empty vector-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->celltab	empty vector>>>empty vector
161	a-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->celltab	a>>>a
162	9-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->celltab	9>>>9
163	1-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->celltab	1>>>1
164	0-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->celltab	0>>>0
165	0-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->celltab	0>>>0
166	9-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->celltab	9>>>9
167	1-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->celltab	1>>>1
168	0-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->celltab	0>>>0
169	0-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->celltab	0>>>0
170	b-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->celltab	b>>>b
171	10-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->celltab	10>>>10
172	0-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->celltab	0>>>0
173	0-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->celltab	0>>>0
174	0-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->celltab	0>>>0
175	10-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->celltab	10>>>10
176	0-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->celltab	0>>>0
177	0-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->celltab	0>>>0
178	0-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->celltab	0>>>0
179	unc-89 (su75)-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->celltab	unc-89 (su75)>>>unc-89 (su75)
180	scpl-1b full-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->celltab	scpl-1b full>>>scpl-1b full
181	a-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->celltab	a>>>a
182	8-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->celltab	8>>>8
183	1-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->celltab	1>>>1
184	0-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->celltab	0>>>0
185	1-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->celltab	1>>>1
186	9-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->celltab	9>>>9
187	1-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->celltab	1>>>1
188	0-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->celltab	0>>>0
189	0-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->celltab	0>>>0
190	b7-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->celltab	b7>>>b7
191	2-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->celltab	2>>>2
192	1-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->celltab	1>>>1
193	0-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->celltab	0>>>0
194	10-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->celltab	10>>>10
195	0-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->celltab	0>>>0
196	0-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->celltab	0>>>0
197	0-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->celltab	0>>>0
198	unc-89 (su75)-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->celltab	unc-89 (su75)>>>unc-89 (su75)
199	empty vector-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->celltab	empty vector>>>empty vector
200	a-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->celltab	a>>>a
201	8-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->celltab	8>>>8
202	2-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->celltab	2>>>2
203	0-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->celltab	0>>>0
204	0-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->celltab	0>>>0
205	10-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->celltab	10>>>10
206	0-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->celltab	0>>>0
207	0-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->celltab	0>>>0
208	0-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->celltab	0>>>0
209	b9-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->celltab	b9>>>b9
210	1-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->celltab	1>>>1
211	0-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->celltab	0>>>0
212	0-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->celltab	0>>>0
213	10-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->celltab	10>>>10
214	0-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->celltab	0>>>0
215	0-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->celltab	0>>>0
216	0-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->celltab	0>>>0
217	, more than 8 eggs; ( ), 4 ­7 eggs; ( ), 1­3 eggs; and , 0 eggs.-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->parr	, more than 8 eggs; >>> eggs; and , 0 eggs.
218	Novel Phosphatase Binds Giant Protein Kinase-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->note	Novel Phosphatase Bi>>>Giant Protein Kinase
219	Vol. 19, June 2008-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->note	Vol. 19, June 2008>>>Vol. 19, June 2008
220	2429-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->note	2429>>>2429
221	Anti-SCPL-1b and anti-UNC-89 were coincubated with wild-type worms, and the muscle was imaged by immunofluorescence microscopy. The image presented in Figure 4C shows a portion of one body wall muscle cell. Labeling of the M-line (marked by UNC-89) and a portion of the I-band is seen. In experiments not shown, the I-band location was confirmed by use of known I-band or dense body markers. Similar results were obtained using anti-SCPL-1a (data not shown). Thus, the partial colocalization of SCPL-1 with UNC-89 at the M-line adds further credibility to a functional interaction between SCPL-1 and UNC-89.-->id=7, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=436--->[]--->parr	Anti-SCPL-1b and ant>>>n SCPL-1 and UNC-89.
222	Genome-wide RNAi screening (on WormBase) has reported that scpl-1(RNAi) results in a reduction of egg laying or Egl phenotype. We had a difficult time discerning this subtle effect. Therefore, we chose to assay the response to drugs that induce egg laying as described in Trent et al. (1983) and shown in Table 1. Compared with wild type, scpl-1(RNAi) shows a weak response to serotonin, suggesting a defect in function of egg laying muscles. unc-89 mutants showed normal responses to serotonin, unc-89; scpl1(RNAi) double showed the same defect as scpl-1(RNAi), suggesting that scpl-1 is epistatic to unc-89. Thus, scpl-1 may function downstream of unc-89 in egg laying muscles.-->id=7, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=436--->[]--->parr	Genome-wide RNAi scr>>> egg laying muscles.
223	To determine whether SCPL-1 has phosphatase catalytic activity, bacterially expressed MBP fusion proteins of SCPL-1a and SCPL-1b (Figure 2C) were tested in vitro for ability to remove the phosphate from the nonspecific substrate p-nitrophenyl phosphate. Results are shown in Figure 5A for SCPL-1b; similar results were obtained for SCPL-1a; MBP itself showed no activity. As shown in Figure 5A, the amount of product generated was proportional to the amount of added protein. The enzymatic properties are very similar to those of previously characterized FCPs and SCPs (Kamenski et al. (2004); Zheng et al. (2005)), including a pH optimum of 5.0, a preference for Mg 2 as divalent cation, and unusual response to small molecule inhibitors. Figure 5D shows that SCPL-1 is not inhibited by the typical phosphatase inhibitors NaF or Na3VO4, but it is inhibited by muscle, SCPL-1 is at least partially colocalized to the Mlines, where it may interact with UNC-89 in situ. Although knockdown of scpl-1 mRNA by RNAi has no obvious phenotype in the structure or function of body wall muscle, it does show a mild defect in egg-laying muscles, as manifest by an abnormal response to serotonin-induced egg laying. unc-89 mutants showed a normal response to serotonin, whereas the unc-89;scpl-1 (RNAi) doubles show the same deficit as spcl-1(RNAi), indicating that scpl-1 is epistatic to unc-89. Therefore, scpl-1 functions downstream of unc-89 in egg-laying muscles. Finally, we have shown that the phosphatase domain of SCPL-1 is an active phosphatase, because phosphatase activity can be demonstrated in vitro with a model substrate. Moreover, because the phosphatase activity is optimal in acidic pH conditions, this might have functional implications. For example, in mammalian muscle, repeated or excessive muscle activity leads to acidosis, and this may stimulate SCPL-1 activity. Alternatively, the phosphatase might be recruited to a different cellular compartment that is acidic (e.g., peroxisomes or autophagosomes). What might be the biochemical consequences of this interaction? There are at least five possibilities: 1) The UNC-89 putatively active kinase PK2 phosphorylates SCPL-1 to affect its activity. This idea is consistent with SCPL-1 functioning downstream of UNC-89, as suggested by the epistasis results. However, protein kinase assays using yeast-expressed HA-tagged PK2 and bacterially expressed MBP-SCPL-1b were negative (data not shown). 2) The SCPL-1 phosphatase dephosphorylates UNC-89 active kinase PK2 to affect its activity. Precedence for the regulatory affect of phosphorylation on a kinase domain in a giant protein kinase is the tyrosine phosphorylation within the kinase domain of mammalian titin that is one step required in embryonic muscle to relieve autoinhibition (Mayans et al., 1998). 3) Each enzyme, UNC-89 kinase and SCPL-1 phosphatase, has the same substrate, and the two proteins act antagonistically. Vertebrate titin kinase (Grater et al., 2005; Lange et al., 2005), and perhaps the other titin-like kinases such as twitchin, TTN-1, and UNC-89, may be activated (from their autoinhibited states) by small mechanical forces that occur with each contraction/ relaxation cycle. Thus, it could be imagined that contraction activates UNC-89 PK2 to phosphorylate the substrate, and during relaxation when UNC-89 PK2 has returned to its autoinhibited state, SCPL-1 phosphatase takes over and dephosphorylates the substrate. 4) The interaction of UNC-89 kinase regions and SCPL-1 forms a structure that recognizes a substrate that neither of the separate enzymes can recognize alone. 5) The interaction of UNC-89 kinase regions with SCPL-1 recruits SCPL-1 to the M-line so that SCPL-1 can dephoshorylate a substrate located there. This scenario does not require that the UNC-89 kinase domains have phosphotransferase activities, but instead act as protein­protein interaction modules. This model is consistent with the finding that both the predicted active PK2 and inactive PK1 bind to SCPL-1. This model of PK1 or PK2 functioning to target SCPL-1 to the M-line is further supported by our finding that prior incubation of PK1 or PK2 with SCPL-1 under conditions that are favorable for binding does not affect the subsequent phosphatase activity of SCPL-1 (data not shown). Further experiments will be required to sort out these different scenarios.-->id=7, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=436--->[]--->parr	To determine whether>>>different scenarios.
224	BeF3 and AlF4.-->id=3661, page=4, size=6, fam=Times, col=#231f20, type=note, textLines=1--->[]--->note	BeF3 and AlF4.>>>BeF3 and AlF4.
225	DISCUSSION-->id=7, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=436--->[]--->parr	DISCUSSION>>>DISCUSSION
226	We have shown that both the putatively catalytically active (PK2) and catalytically inactive (PK1) kinase domains of UNC-89 interact with a novel protein phosphatase, SCPL-1. For UNC-89, this requires the upstream Ig and Fn3 domains, as well as the autoinhibitory region at least in PK2. For SCPL-1, only the phosphatase domain itself is required for interaction with UNC-89. There are two indications of the specificity of this interaction: 1) SCPL-1 does not interact with similar regions of the two other giant titin-like kinases in the worm, twitchin and TTN-1. 2) The UNC-89 PK2 region does not interact with three other related proteins in C. elegans: SCPL-2, -3, and -4, which SAGE data available on WormBase suggest express in body wall muscle. The plausibility of the UNC-89 ­SCPL-1 interaction is indicated by finding the following: 1) The scpl-1b promoter is expressed in the same sets of muscles (body wall, pharyngeal, and vulval) as unc-89. 2) By antibody staining of body wall striated-->id=7, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=436--->[]--->parr	We have shown that b>>>f body wall striated
227	Figure 5. Biochemical properties of SCPL-1. SCPL-1b was expressed as an MBP fusion protein in E. coli, and they are shown to have phosphatase activity toward a model substrate, p-nitrophenyl phosphate (A). Enzymatic properties were very similar to previously characterized FCPs and SCPs, including a pH optimum of 5.0 (B), preference for Mg 2 (C), and unusual response to smallmolecule inhibitors (not inhibited by NaF or Na3VO4 but inhibited by BeF3 and AlF4; shown in D).-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->capfig	Figure 5. Biochemica>>>d AlF4; shown in D).
228	H. Qadota et al.-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->note	H. Qadota et al.>>>H. Qadota et al.
229	Molecular Biology of the Cell-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->note	Molecular Biology of>>> Biology of the Cell
230	2430-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=293--->[]--->note	2430>>>2430
231	Our studies have pointed to a new function for the CTD phosphatases, that is, in muscle giant protein signaling. The founding member of the CTD phosphatase family is FCP, found in all eukaryotes, and it is known to dephosphorylate serines in the CTD of the largest subunit of RNA polymerase II. This activity of FCP is required for RNA polymerase recycling and global transcription (Yeo et al., 2003). FCP proteins contain both a phosphatase domain (FCPH domain) and a BRCT domain. Higher eukaryotes also contain small CTD phosphatases, proteins possessing the CTD phosphatase domain, but lacking the BRCT domain (Yeo et al., 2003). In vitro, SCPs can dephosphorylate the CTD of RNA polymerase II, but a definitive role as general regulators of transcription has not been demonstrated. So far, there is evidence that SCPs have more specialized functions in transcriptional regulation. For example, SCP1-3 silence neuronspecific gene expression in non-neuronal cells by being recruited by a silencer factor complex (Yeo et al., 2005). Recently, in mammalian cells and Xenopus embryos, SCP1-3, but not FCP1, have been shown to dephosphorylate Smad transcription factors and by doing so, regulate transforming growth factor- and BMP signaling (Knockaert et al., 2006; Sapkota et al., 2006; Wrighton et al., 2006). Our finding that one of four SCPs in C. elegans specifically interacts with the kinase domains of the giant protein UNC-89 demonstrates yet another function for an SCP protein, a function probably not related to transcriptional regulation. In addition, until our findings, all SCPs have been reported to be nuclear (e.g., Yeo et al., 2003 for SCP1), whereas worm SCPL-1 is located in myofibrils. It is possible that the kinase domains of obscurin (UNC-89 in vertebrates) similarly interact with an SCP protein. It is possible that the true vertebrate orthologue for nematode SCPL-1 has not been identified; for example, although searches of vertebrate proteomes have not revealed an SCP protein with sequence homology outside the phosphatase domain, it is possible that there is a functional homologue of worm SCPL-1 that has an upstream region that forms a similar structure, function, or both. Nevertheless, given the high degree of sequence identity between the 200-residue phosphatase domain of worm SCPL-1 to human SCP1, SCP2, and SCP3 (67, 64, and 64%, respectively), even if these phosphatases target different substrate proteins, the recognition sequences that immediately surround the phophorylated serines/threonines might be very similar.-->id=7, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=436--->[]--->parr	Our studies have poi>>>ght be very similar.
232	ACKNOWLEDGMENTS Collet, J. F., Stroobant, V., Pirard, M., Delpierre, G., and Van Schaftingen, E. (1998). A new class of phosphotransferases phosphorylated on an aspartate residue in an amino-terminal DXDX(T/V) motif. J. Biol. Chem. 273, 14107­ 14112.-->id=7, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=436--->[]--->parr	ACKNOWLEDGMENTS Coll>>>. 273, 14107­ 14112.
233	We thank Andy Fire for the promoter-less GFP vector, Robert Barstead for the random primed nematode cDNA library, and Kozo Kaibuchi (Nagoya University) for vectors pMAL-KK1 and pKK51. Worm strains were provided by the Caenorhabditis Genetics Center, which is supported by the National Center for Research Resources of the National Institutes of Health. Support for this work was provided by National Institute of Arthritis & Musculoskeletal & Skin Diseases/National Institutes of Health grant AR-051466.-->id=773, page=1, size=8, fam=Times, col=#231f20, type=parrnote, textLines=317--->[]--->parr	We thank Andy Fire f>>>lth grant AR-051466.
234	REFERENCES-->id=7, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=436--->[]--->parr	REFERENCES>>>REFERENCES
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