0	Reduced phosphatase activity of SHP-2 in LEOPARD, otherFeat=[]-->, belongsTo=title
1	syndrome: Consequences for PI3K binding on Gab1, otherFeat=[]-->, belongsTo=title
2	Nadine Hannaa,1, Alexandra Montagnerb,1, Wen Hwa Leec, Maria Mitevad, Michel Vidald,, otherFeat=[]-->, belongsTo=parrnote
3	Michel Vidaud a,Be?atrice Parfaita,*,1, Patrick Raynalb,*,1, otherFeat=[]-->, belongsTo=parrnote
4	a INSERM U745, Faculte? des Sciences Pharmaceutiques et Biologiques, Universite? Rene? Descartes 4 avenue de l'Observatoire,, otherFeat=[]-->, belongsTo=parrnote
5	75270 Paris Cedex 06, France, otherFeat=[]-->, belongsTo=parrnote
6	b INSERM U563, De?partement Lipoprote?ines et Me?diateurs Lipidiques, Ho^pital Purpan, BP 3028, 31024 Toulouse Cedex 03, France, otherFeat=[]-->, belongsTo=parrnote
7	c Structural Genomics Consortium, University of Oxford, Botnar Research Center, OX 3 7LD Oxford, United Kingdom, otherFeat=[]-->, belongsTo=parrnote
8	d INSERM U648, UFR Biome?dicale, Universite? Rene? Descartes 45 rue des Saints Pe`res, 75006 Paris, France, otherFeat=[]-->, belongsTo=parrnote
9	Received 14 February 2006; revised 10 March 2006; accepted 31 March 2006, otherFeat=[]-->, belongsTo=parrnote
10	Available online 12 April 2006, otherFeat=[]-->, belongsTo=parrnote
11	Edited by Francesc Posas, otherFeat=[]-->, belongsTo=parrnote
12	Abstract LEOPARD (LS) and Noonan (NS) are overlapping, otherFeat=[]-->, belongsTo=parr
13	syndromes associated with distinct mutations of SHP-2., otherFeat=[]-->, belongsTo=parr
14	Whereas NS mutations enhance SHP-2 catalytic activity, we, otherFeat=[]-->, belongsTo=parr
15	show that the activity of three representative LS mutants is unde-, otherFeat=[]-->, belongsTo=parr
16	tectable when assayed using a standard protein tyrosine phospha-, otherFeat=[]-->, belongsTo=parr
17	tase (PTP) substrate. A different assay using a specific SHP-2, otherFeat=[]-->, belongsTo=parr
18	substrate confirms their decreased PTP activity, but also reveals, otherFeat=[]-->, belongsTo=parr
19	a significant activity of the T468M mutant. In transfected cells, otherFeat=[]-->, belongsTo=parr
20	stimulated with epidermal growth factor, the least active LS mu-, otherFeat=[]-->, belongsTo=parr
21	tants promote Gab1/PI3K binding, validating our in vitro data., otherFeat=[]-->, belongsTo=parr
22	LS mutants thus display a reduced PTP activity both in vitro, otherFeat=[]-->, belongsTo=parr
23	and in transfected cells., otherFeat=[]-->, belongsTo=parr
24	? 2006 Federation of European Biochemical Societies. Published, otherFeat=[]-->, belongsTo=parr
25	by Elsevier B.V. All rights reserved., otherFeat=[]-->, belongsTo=parr
26	Keywords: LEOPARD syndrome; PTPN11; SHP-2, otherFeat=[]-->, belongsTo=parr
27	phosphatase activity; Gab1; PI3K, otherFeat=[]-->, belongsTo=parr
28	1. Introduction, otherFeat=[]-->, belongsTo=parr
29	LEOPARD syndrome (multiple lentigines, electrocardio-, otherFeat=[]-->, belongsTo=parr
30	graphic conduction abnormalities, ocular hypertelorism,, otherFeat=[]-->, belongsTo=parr
31	pulmonary stenosis, abnormal genitalia, retardation of growth,, otherFeat=[]-->, belongsTo=parr
32	sensorineural deafness; LS; MIM#151100) is a rare genetic dis-, otherFeat=[]-->, belongsTo=parr
33	order characterized by multiple lentigines and other cutaneous, otherFeat=[]-->, belongsTo=parr
34	abnormalities such as ``cafe? au lait'' spots, dysmorphic facies,, otherFeat=[]-->, belongsTo=parr
35	cardiac defects, growth retardation and increased risk of, otherFeat=[]-->, belongsTo=parr
36	malignancy. Several clinical manifestations overlap those of, otherFeat=[]-->, belongsTo=parr
37	Noonan syndrome (NS; MIM#163950), a common genetic, otherFeat=[]-->, belongsTo=parr
38	disease also typified by facial dysmorphy, heart defects and, otherFeat=[]-->, belongsTo=parr
39	growth retardation. However ``cafe? au lait'' spots are rare, otherFeat=[]-->, belongsTo=parr
40	and multiple lentigines are typically absent in NS., otherFeat=[]-->, belongsTo=parr
41	Recently, germline missense mutations in the PTPN11 gene, otherFeat=[]-->, belongsTo=parr
42	were identified in both syndromes and are now causally linked, otherFeat=[]-->, belongsTo=parr
43	to the diseases  [1,2] . Somatic PTPN11 mutations are also, otherFeat=[]-->, belongsTo=parr
44	responsible for juvenile myelomonocytic leukemia  [3]. PTPN11, otherFeat=[]-->, belongsTo=parr
45	encodes SHP-2, a widely expressed protein tyrosine phosphatase, otherFeat=[]-->, belongsTo=parr
46	(PTP) involved in intracellular signalling downstream of several, otherFeat=[]-->, belongsTo=parr
47	growth factors, cytokines, and hormone receptors  [4] . The struc-, otherFeat=[]-->, belongsTo=parr
48	ture of SHP-2 consists of two Src-homology 2 (SH2) domains, otherFeat=[]-->, belongsTo=parr
49	(N-SH2 and C-SH2), a single PTP domain and a C-terminal, otherFeat=[]-->, belongsTo=parr
50	hydrophilic tail. SHP-2 displays a low basal catalytic activity, otherFeat=[]-->, belongsTo=parr
51	due to close interactions between the N-SH2 and PTP domains, otherFeat=[]-->, belongsTo=parr
52	that keeps the phosphatase in an autoinhibited closed conforma-, otherFeat=[]-->, belongsTo=parr
53	tion. Its catalytic activation thus requires to open the molecule, otherFeat=[]-->, belongsTo=parr
54	by releasing these interactions, which occurs when its SH2 do-, otherFeat=[]-->, belongsTo=parr
55	mains bind to specific phosphotyrosine motifs on SHP-2 up-, otherFeat=[]-->, belongsTo=parr
56	stream signalling partners. These partners include the insulin, otherFeat=[]-->, belongsTo=parr
57	receptor substrate 1 (IRS-1) and the Grb2-associated binder 1,, otherFeat=[]-->, belongsTo=parr
58	Gab1. Both are adapter proteins rapidly phosphorylated in re-, otherFeat=[]-->, belongsTo=parr
59	sponse to the stimulation of insulin or epidermal growth factor, otherFeat=[]-->, belongsTo=parr
60	(EGF) receptors, respectively. Downstream of Gab1 or IRS-1,, otherFeat=[]-->, belongsTo=parr
61	SHP-2 promotes the activation of the Ras/mitogen-activated, otherFeat=[]-->, belongsTo=parr
62	pathway, through mechanisms that are still under investigation, otherFeat=[]-->, belongsTo=parr
63	[4,5]. Besides this signal-enhancing function, SHP-2 can play a, otherFeat=[]-->, belongsTo=parr
64	modulatory role in the activation of phosphoinositide 3-kinase, otherFeat=[]-->, belongsTo=parr
65	(PI3K), at least downstream of the EGF receptor. Indeed, this, otherFeat=[]-->, belongsTo=parr
66	receptor recruits PI3K by phosphorylating Gab1 on binding, otherFeat=[]-->, belongsTo=parr
67	sites for p85, the regulatory subunit of PI3K, and it was recently, otherFeat=[]-->, belongsTo=parr
68	shown that these sites are dephosphorylated by SHP-2 during, otherFeat=[]-->, belongsTo=parr
69	EGF stimulation  [6] ., otherFeat=[]-->, belongsTo=parr
70	In NS, most of SHP-2 mutations described so far affect res-, otherFeat=[]-->, belongsTo=parr
71	idues located in or close to the interaction surface between the, otherFeat=[]-->, belongsTo=parr
72	N-SH2 and the PTP domains  [7]. This is thought to disrupt the, otherFeat=[]-->, belongsTo=parr
73	autoinhibited closed conformation of SHP-2, resulting in an, otherFeat=[]-->, belongsTo=parr
74	increased basal PTP activity of most NS-associated SHP-2 mu-, otherFeat=[]-->, belongsTo=parr
75	tants observed in vitro  [8,9] . Thus the basis of NS pathogenesis, otherFeat=[]-->, belongsTo=parr
76	is certainly due to activating, ``gain-of-function'' mutations of, otherFeat=[]-->, belongsTo=parr
77	SHP-2, even though the SHP-2-dependent mechanisms altered, otherFeat=[]-->, belongsTo=parr
78	by its NS-mutant derivatives remain to be identified. In LS,, otherFeat=[]-->, belongsTo=parr
79	only nine mutations were reported to date, affecting seven dif-, otherFeat=[]-->, belongsTo=parr
80	ferent amino acids  [7]. These residues are all located in the PTP, otherFeat=[]-->, belongsTo=parr
81	domain, and, even if no functional data were reported yet,, otherFeat=[]-->, belongsTo=parr
82	these mutations are predicted to be also ``gain-of-function'',, otherFeat=[]-->, belongsTo=parr
83	due to the clinical overlap between NS and LS., otherFeat=[]-->, belongsTo=parr
84	This work aimed to define the effect of LS mutations on SHP-2, otherFeat=[]-->, belongsTo=parr
85	phosphatase activity. Using molecular modelling, all LS muta-, otherFeat=[]-->, belongsTo=parr
86	tions were strikingly located in the catalytic cleft of SHP-2. We, otherFeat=[]-->, belongsTo=parr
87	Abbreviations: EGF, epidermal growth factor; IRS-1, insulin receptor, otherFeat=[]-->, belongsTo=parrnote
88	substrate 1; LS, LEOPARD syndrome; NS, Noonan syndrome; PTP,, otherFeat=[]-->, belongsTo=parrnote
89	protein tyrosine phosphatase; WT, wild type, otherFeat=[]-->, belongsTo=parrnote
90	*Corresponding authors. Fax: +33 1 4407 1754 (B. Parfait),, otherFeat=[]-->, belongsTo=parrnote
91	+33 5 6177 9401 (P. Raynal)., otherFeat=[]-->, belongsTo=parrnote
92	E-mail addresses:  beatrice.parfait@univ-paris5.fr (B. Parfait),, otherFeat=[]-->, belongsTo=parrnote
93	raynal@toulouse.inserm.fr (P. Raynal)., otherFeat=[]-->, belongsTo=parrnote
94	1 Equal contribution., otherFeat=[]-->, belongsTo=parrnote
95	0014-5793/$32.00 ? 2006 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved., otherFeat=[]-->, belongsTo=nota_cab_pie
96	doi:10.1016/j.febslet.2006.03.088, otherFeat=[]-->, belongsTo=nota_cab_pie
97	FEBS Letters 580 (2006) 2477?2482, otherFeat=[]-->, belongsTo=nota_cab_pie
98	then explored, both in vitro and in intact cells, the effect of three, otherFeat=[]-->, belongsTo=parr
99	representative LS mutations on SHP-2 catalytic activity., otherFeat=[]-->, belongsTo=parr
100	2. Materials and methods, otherFeat=[]-->, belongsTo=parr
101	2.1. Structural analysis of LS-associated SHP-2 mutant residues, otherFeat=[]-->, belongsTo=parrnote
102	The structure of wild-type (WT) SHP-2  [10] (PDB code 2shp) was, otherFeat=[]-->, belongsTo=parrnote
103	loaded into the program ICM (Molsoft LLC,  www.molsoft.com ), otherFeat=[]-->, belongsTo=parrnote
104	and the LS-associated residues were mapped and visually inspected., otherFeat=[]-->, belongsTo=parrnote
105	2.2. Phosphopeptide synthesis, otherFeat=[]-->, belongsTo=parrnote
106	The substrate phosphopeptide Gab1-pY589 (DSEEN-pY-, otherFeat=[]-->, belongsTo=parrnote
107	VPMNPNL) and the activating peptide IRS1-pY1172 (SLN-pY-, otherFeat=[]-->, belongsTo=parrnote
108	IDLDLVK)  [8] were synthesized using small-scale Fmoc chemistry., otherFeat=[]-->, belongsTo=parrnote
109	2.3. PTPN11 constructs, otherFeat=[]-->, belongsTo=parrnote
110	The human WT PTPN11 cDNA and the mutants D61Y and C459G, otherFeat=[]-->, belongsTo=parrnote
111	subcloned into the pcDNA6/V5-HisA vector were kindly provided by, otherFeat=[]-->, belongsTo=parrnote
112	Bruce D. Gelb. The NS-associated D61del mutation  [11] and the LS-, otherFeat=[]-->, belongsTo=parrnote
113	associated mutations resulting in Y279C, T468M, and Q510P were, otherFeat=[]-->, belongsTo=parrnote
114	introduced using site-directed mutagenesis (QuickChange, Stratagene), otherFeat=[]-->, belongsTo=parrnote
115	and verified by sequencing., otherFeat=[]-->, belongsTo=parrnote
116	2.4. Cell lines, transfections and stimulation, otherFeat=[]-->, belongsTo=parrnote
117	Vero cells were grown, transiently transfected and stimulated with, otherFeat=[]-->, belongsTo=parrnote
118	30 ng/ml EGF as described  [5]., otherFeat=[]-->, belongsTo=parrnote
119	2.5. Immune complex PTP assays, otherFeat=[]-->, belongsTo=parrnote
120	Transfected cells were harvested in lysis buffer: 25 mM HEPES, pH, otherFeat=[]-->, belongsTo=parrnote
121	7.4, 150 mM NaCl, 2 mM EDTA, 0.5% Triton X-100 and Protease, otherFeat=[]-->, belongsTo=parrnote
122	Inhibitor Cocktail (Sigma). SHP-2 was immunoprecipitated with, otherFeat=[]-->, belongsTo=parrnote
123	anti-V5 antibody (Invitrogen) then washed in lysis buffer and in PTP, otherFeat=[]-->, belongsTo=parrnote
124	buffer: 20 mM HEPES, pH 7.4, EDTA 1 mM, 5% glycerol, and, otherFeat=[]-->, belongsTo=parrnote
125	1 mM DTT. Immunocomplexes were resuspended in 50 ll of PTP buf-, otherFeat=[]-->, belongsTo=parrnote
126	fer supplemented with 250 lM of Src-pY529 phosphopeptide, otherFeat=[]-->, belongsTo=parrnote
127	(TSTEPQ-pY-QPGENL, Upstate Biotech) or 100 lM of Gab1-, otherFeat=[]-->, belongsTo=parrnote
128	pY589 phosphopeptide alone or with 10 lM of the activating peptide, otherFeat=[]-->, belongsTo=parrnote
129	IRS1-pY1172, and incubated for 30 min at 37 ?C. To assess free phos-, otherFeat=[]-->, belongsTo=parrnote
130	phate release, supernatants were then placed in 96-well plates with, otherFeat=[]-->, belongsTo=parrnote
131	Malachite Green solution (Upstate Biotech) and incubated for, otherFeat=[]-->, belongsTo=parrnote
132	15 min. Absorbances (630 nm) were compared to a phosphate stan-, otherFeat=[]-->, belongsTo=parrnote
133	dard curve  [3,12]., otherFeat=[]-->, belongsTo=parrnote
134	2.6. GST-p85 affinity precipitation assay, otherFeat=[]-->, belongsTo=parrnote
135	This was performed as described  [5]., otherFeat=[]-->, belongsTo=parrnote
136	3. Results and discussion, otherFeat=[]-->, belongsTo=parr
137	3.1. LS-associated SHP-2 mutated residues are clustered in the, otherFeat=[]-->, belongsTo=parr
138	PTP catalytic cleft, otherFeat=[]-->, belongsTo=parr
139	The nine missense mutations reported to date in LS affect se-, otherFeat=[]-->, belongsTo=parr
140	ven different amino acids localized exclusively in the SHP-2, otherFeat=[]-->, belongsTo=parr
141	PTP domain (  Fig. 1A). To gain insight on LS mutations effects, otherFeat=[]-->, belongsTo=parr
142	on SHP-2 function, we performed a structural model analysis, otherFeat=[]-->, belongsTo=parr
143	of the residues which are found mutated in LS with respect to, otherFeat=[]-->, belongsTo=parr
144	their positioning in the SHP-2 molecule  [10].  Fig. 1B show that, otherFeat=[]-->, belongsTo=parr
145	the seven residues are highly clustered in the catalytic core of, otherFeat=[]-->, belongsTo=parr
146	the PTP domain, suggesting that LS mutations might alter, otherFeat=[]-->, belongsTo=parr
147	SHP-2 catalytic properties, rather than modify its activation, otherFeat=[]-->, belongsTo=parr
148	state as in the case of NS-associated mutations., otherFeat=[]-->, belongsTo=parr
149	3.2. PTP activity of LS mutants is abolished when assayed using, otherFeat=[]-->, belongsTo=parr
150	a standard PTP substrate, otherFeat=[]-->, belongsTo=parr
151	To study the effect of LS mutations on SHP-2 catalytic activ-, otherFeat=[]-->, belongsTo=parr
152	ity, we introduced by site-directed mutagenesis the mutations, otherFeat=[]-->, belongsTo=parr
153	giving rise to the two common Y279C and T468M and the rare, otherFeat=[]-->, belongsTo=parr
154	Q510P single amino acid changes in a plasmid encoding hu-, otherFeat=[]-->, belongsTo=parr
155	man WT-SHP-2. After transfection of these constructs in, otherFeat=[]-->, belongsTo=parr
156	mammalian cells, we measured in vitro the catalytic activity, otherFeat=[]-->, belongsTo=parr
157	of each mutant isolated by immunoprecipitation. Vero cells, otherFeat=[]-->, belongsTo=parr
158	were chosen for their low basal level of SHP-2-dependent sig-, otherFeat=[]-->, belongsTo=parr
159	nalling pathways once incubated overnight in serum-free med-, otherFeat=[]-->, belongsTo=parr
160	ium  [5,13]. Following immunoprecipitation, the PTP activity, otherFeat=[]-->, belongsTo=parr
161	of the different constructs was assayed using as substrate a syn-, otherFeat=[]-->, belongsTo=parr
162	thetic phosphopeptide encompassing Y529 of Src, a standard, otherFeat=[]-->, belongsTo=parr
163	in vitro substrate of PTP  [3,9]. To test these mutants under, otherFeat=[]-->, belongsTo=parr
164	stimulation by an upstream SHP-2 activator, the immunocom-, otherFeat=[]-->, belongsTo=parr
165	plexes were also incubated with 10 lM of a phosphopeptide, otherFeat=[]-->, belongsTo=parr
166	containing the SHP-2-binding site of IRS-1 (IRS1-pY1172), otherFeat=[]-->, belongsTo=parr
167	[8] . As shown in  Fig. 2 , addition of IRS1-pY1172 phosphopep-, otherFeat=[]-->, belongsTo=parr
168	tide resulted in a 2?3-fold increase of WT-SHP-2 catalytic, otherFeat=[]-->, belongsTo=parr
169	activity, attesting that these experimental conditions allow to, otherFeat=[]-->, belongsTo=parr
170	detect SHP-2 activation. As additional controls, we verified, otherFeat=[]-->, belongsTo=parr
171	that the well-characterized catalytically dead C459G construct, otherFeat=[]-->, belongsTo=parr
172	displayed no PTP activity, and that the leukemia-associated, otherFeat=[]-->, belongsTo=parr
173	D61Y mutant and of the NS-associated D61del mutant have, otherFeat=[]-->, belongsTo=parr
174	an increased PTP activity compared to WT-SHP-2. These, otherFeat=[]-->, belongsTo=parr
175	two mutants are not significantly overstimulated by the, otherFeat=[]-->, belongsTo=parr
176	IRS1-pY1172 peptide in these conditions, but this is in agree-, otherFeat=[]-->, belongsTo=parr
177	ment with a previous report  [8] . In addition, when the immu-, otherFeat=[]-->, belongsTo=parr
178	nocomplexes were incubated with the activating peptide, otherFeat=[]-->, belongsTo=parr
179	alone, the release of phosphate was undetectable for any of, otherFeat=[]-->, belongsTo=parr
180	SHP-2 construct (data not shown), indicating that this phos-, otherFeat=[]-->, belongsTo=parr
181	phopeptide was not significantly dephosphorylated by SHP-2, otherFeat=[]-->, belongsTo=parr
182	when used in these conditions appropriate to observe its acti-, otherFeat=[]-->, belongsTo=parr
183	vating effect. This series of controls validated the methodology, otherFeat=[]-->, belongsTo=parr
184	used in this study., otherFeat=[]-->, belongsTo=parr
185	More interestingly, when considering the three LS-associ-, otherFeat=[]-->, belongsTo=parr
186	ated SHP-2 mutants,  Fig. 2 shows that their basal catalytic, otherFeat=[]-->, belongsTo=parr
187	activity is similar to that of the catalytically dead C459G con-, otherFeat=[]-->, belongsTo=parr
188	struct. In the presence of the activating peptide, a slight but, otherFeat=[]-->, belongsTo=parr
189	significant increase of PTP activity was detected in the case, otherFeat=[]-->, belongsTo=parr
190	of Y279C and T468M mutants, but this activity remains at, otherFeat=[]-->, belongsTo=parr
191	the most around 10% of that of WT-SHP-2. Taken together,, otherFeat=[]-->, belongsTo=parr
192	these results suggest that LS mutations lead to the loss of, otherFeat=[]-->, belongsTo=parr
193	SHP-2 catalytic activity, probably by disruption of the integ-, otherFeat=[]-->, belongsTo=parr
194	rity of SHP-2 catalytic cleft., otherFeat=[]-->, belongsTo=parr
195	3.3. PTP assay of LS mutants using a specific SHP-2 substrate, otherFeat=[]-->, belongsTo=parr
196	confirms their decreased activity but reveals a significant, otherFeat=[]-->, belongsTo=parr
197	activity of T468M mutant, otherFeat=[]-->, belongsTo=parr
198	The above results were obtained with a standard PTP sub-, otherFeat=[]-->, belongsTo=parr
199	strate, which is not considered as an undeniable SHP-2 sub-, otherFeat=[]-->, belongsTo=parr
200	strate in vivo. We thus determined if LS mutations had the, otherFeat=[]-->, belongsTo=parr
201	same effect on SHP-2 catalytic activity when assayed on a, otherFeat=[]-->, belongsTo=parr
202	validated SHP-2 substrate. To this aim, we designed a phos-, otherFeat=[]-->, belongsTo=parr
203	phopeptide encompassing Y589 of Gab1 (Gab1-pY589 phos-, otherFeat=[]-->, belongsTo=parr
204	phopeptide), which corresponds to a PI3K-binding site and, otherFeat=[]-->, belongsTo=parr
205	is known to be dephosphorylated by SHP-2 in cells treated, otherFeat=[]-->, belongsTo=parr
206	with EGF  [6]. As shown in  Fig. 3 , the basal PTP activity of, otherFeat=[]-->, belongsTo=parr
207	the three LS mutants assayed with this peptide is still severely, otherFeat=[]-->, belongsTo=parr
208	reduced in comparison with the WT protein. However, in the, otherFeat=[]-->, belongsTo=parr
209	case of the T468M mutant, its basal activity is around 30%, otherFeat=[]-->, belongsTo=parr
210	of that of WT-SHP-2. Under stimulation with IRS1-pY1172,, otherFeat=[]-->, belongsTo=parr
211	the Q510P mutant still displayed a negligible PTP activity. In, otherFeat=[]-->, belongsTo=parr
212	contrast, the two other mutants seem to display a significant, otherFeat=[]-->, belongsTo=parr
213	2478, otherFeat=[]-->, belongsTo=nota_cab_pie
214	N. Hanna et al. / FEBS Letters 580 (2006) 2477?2482, otherFeat=[]-->, belongsTo=nota_cab_pie
215	Fig. 1. LS-associated SHP-2 mutated residues are clustered in the PTP domain of the protein. (A) Schematic representation of SHP-2 showing the, otherFeat=[]-->, belongsTo=fig_caption
216	distribution of mutations identified in LS (above). Position of hyperactive and inactive SHP-2 mutants studied in this report are also indicated, otherFeat=[]-->, belongsTo=fig_caption
217	(below). (B) Left: ribbon representation of SHP-2 structure in its autoinhibited closed conformation  [10] . The N- and C-terminal SH2 domains are, otherFeat=[]-->, belongsTo=fig_caption
218	coloured in yellow and green, respectively, the catalytic domain in blue. The red square depicts the closeup region. Right: Close-up of the active site, otherFeat=[]-->, belongsTo=fig_caption
219	and location of LS-associated SHP-2 mutated residues. The two most frequently altered residues Y279 and T468 are represented as orange sticks,, otherFeat=[]-->, belongsTo=fig_caption
220	while other mutations are shown as magenta sticks. Position of D61 (on the inhibitory loop of N-SH2 domain protruding into the catalytic site), otherFeat=[]-->, belongsTo=fig_caption
221	responsible for autoinhibition of PTP activity is highlighted in red., otherFeat=[]-->, belongsTo=fig_caption
222	Fig. 2. PTP activity of LS mutants is abolished when assayed using a standard PTP substrate. Immune complex PTP assay against 250 lM Src-, otherFeat=[]-->, belongsTo=fig_caption
223	pY529 with or without 10 lM activating peptide IRS1-pY1172 carried out on serum-starved Vero cells transfected with the indicated SHP-2, otherFeat=[]-->, belongsTo=fig_caption
224	construct. Bottom: representative anti-V5 immunoblots of immunoprecipitates. NT, experiments performed from non-transfected cells. Data, otherFeat=[]-->, belongsTo=fig_caption
225	represent means ? S.D. from three independent experiments. Significant differences in PTP activity between basal and activated conditions are, otherFeat=[]-->, belongsTo=fig_caption
226	indicated (* P < 0.05, *** P < 0.001)., otherFeat=[]-->, belongsTo=fig_caption
227	N. Hanna et al. / FEBS Letters 580 (2006) 2477?2482, otherFeat=[]-->, belongsTo=nota_cab_pie
228	2479, otherFeat=[]-->, belongsTo=nota_cab_pie
229	PTP activity, notably the T468M that reaches 50% of WT, otherFeat=[]-->, belongsTo=parr
230	SHP-2. These results thus confirm the  Fig. 2 data showing that, otherFeat=[]-->, belongsTo=parr
231	LS mutations, in contrast with NS mutations, lead to a de-, otherFeat=[]-->, belongsTo=parr
232	crease in SHP-2 catalytic activity. However, these experiments, otherFeat=[]-->, belongsTo=parr
233	also indicate that different LS mutations produce distinct levels, otherFeat=[]-->, belongsTo=parr
234	of alteration of SHP-2 activity, which was not detected using a, otherFeat=[]-->, belongsTo=parr
235	standard PTP substrate. This suggest that at least one LS, otherFeat=[]-->, belongsTo=parr
236	mutation influences SHP-2 substrate recognition rather than, otherFeat=[]-->, belongsTo=parr
237	suppresses its catalytic activity., otherFeat=[]-->, belongsTo=parr
238	3.4. In situ confirmation of in vitro assays: the least active LS, otherFeat=[]-->, belongsTo=parr
239	mutants promote Gab1/PI3K binding in EGF-stimulated, otherFeat=[]-->, belongsTo=parr
240	cells, otherFeat=[]-->, belongsTo=parr
241	We next determined whether these in vitro features of LS, otherFeat=[]-->, belongsTo=parr
242	mutants could be confirmed in transfected cells, by testing, otherFeat=[]-->, belongsTo=parr
243	the effects of these mutants on the dephosphorylation of a, otherFeat=[]-->, belongsTo=parr
244	cellular substrate of SHP-2. Because SHP-2 can dephosphor-, otherFeat=[]-->, belongsTo=parr
245	ylate Gab1 on its PI3K-binding sites in response to EGF  [6],, otherFeat=[]-->, belongsTo=parr
246	we designed an experiment to monitor these sites phosphor-, otherFeat=[]-->, belongsTo=parr
247	ylation. Vero cells were chosen again, since they can be effi-, otherFeat=[]-->, belongsTo=parr
248	ciently stimulated by EGF, leading to the phosphorylation, otherFeat=[]-->, belongsTo=parr
249	of Gab1 and the downstream activation of SHP-2 and, otherFeat=[]-->, belongsTo=parr
250	PI3K  [5,13]. Following stimulation with EGF, cells were, otherFeat=[]-->, belongsTo=parr
251	incubated with a GST-p85 fusion protein, which allows to, otherFeat=[]-->, belongsTo=parr
252	precipitate Gab1 only if phosphorylated on PI3K binding, otherFeat=[]-->, belongsTo=parr
253	sites  [5,13]. As positive control, in cells transfected with cat-, otherFeat=[]-->, belongsTo=parr
254	alytically dead C459G SHP-2, GST-p85 precipitates an high-, otherFeat=[]-->, belongsTo=parr
255	er amount of Gab1 in comparison with WT SHP-2,, otherFeat=[]-->, belongsTo=parr
256	whereas, as negative control, the hyperactive D61del mutant, otherFeat=[]-->, belongsTo=parr
257	prevents Gab1 precipitation (  Fig. 4A?C, compare lanes, otherFeat=[]-->, belongsTo=parr
258	``C459G'' and ``D61del'' with ``WT''). Thus Gab1 amount, otherFeat=[]-->, belongsTo=parr
259	precipitated with GST-p85 appears inversely proportional, otherFeat=[]-->, belongsTo=parr
260	to SHP-2 catalytic activity, showing that the Gab1/PI3K, otherFeat=[]-->, belongsTo=parr
261	interaction can be taken as marker of the cellular catalytic, otherFeat=[]-->, belongsTo=parr
262	activity of SHP-2., otherFeat=[]-->, belongsTo=parr
263	Once validated, this assay was applied to cells expressing LS, otherFeat=[]-->, belongsTo=parr
264	mutants. The results show that the Y279C and Q510P mutants, otherFeat=[]-->, belongsTo=parr
265	enhance, compare to the WT-SHP-2, Gab1 recovery in GST-, otherFeat=[]-->, belongsTo=parr
266	p85 pulldowns, measured either after 5 or 10 min of EGF stim-, otherFeat=[]-->, belongsTo=parr
267	ulation  (Fig. 4A and B). A quantitative analysis of data from, otherFeat=[]-->, belongsTo=parr
268	different experiments indicates that these mutants produce an, otherFeat=[]-->, belongsTo=parr
269	effect similar to that of catalytically dead SHP-2 C459G, otherFeat=[]-->, belongsTo=parr
270	(Fig. 4 D). In contrast, the T468M mutant did not enhance, otherFeat=[]-->, belongsTo=parr
271	Gab1 precipitation by GST-p85 in comparison with WT-, otherFeat=[]-->, belongsTo=parr
272	SHP-2, implying that this mutant species retains some of its, otherFeat=[]-->, belongsTo=parr
273	phosphatase activity towards Gab1 PI3K-binding sites, otherFeat=[]-->, belongsTo=parr
274	(  Fig. 4 C and D). We conclude that the LS mutants Y279C, otherFeat=[]-->, belongsTo=parr
275	and Q510P behave in situ like catalytically inactive mutants,, otherFeat=[]-->, belongsTo=parr
276	which confirms our in vitro data showing that these mutants, otherFeat=[]-->, belongsTo=parr
277	display the lowest PTP activity. In addition, the fact that the, otherFeat=[]-->, belongsTo=parr
278	T468M mutant does not promote Gab1/PI3K interaction, otherFeat=[]-->, belongsTo=parr
279	more than the WT protein also confirms the in vitro results, otherFeat=[]-->, belongsTo=parr
280	since this mutation was found to reduce, but not to abolish,, otherFeat=[]-->, belongsTo=parr
281	its catalytic activity on Gab1 phosphopeptide. It is thus likely, otherFeat=[]-->, belongsTo=parr
282	that this mutation does alter the catalytic efficiency of the, otherFeat=[]-->, belongsTo=parr
283	T468M mutant, but its residual activity appears sufficient to, otherFeat=[]-->, belongsTo=parr
284	dephosphorylate Gab1 in EGF-stimulated cells., otherFeat=[]-->, belongsTo=parr
285	The apparent lack of hyperactivity of D61 mutants in vitro, otherFeat=[]-->, belongsTo=parr
286	on Gab1-pY589 substrate is probably due to the use of a con-, otherFeat=[]-->, belongsTo=parr
287	centration of Gab1-pY589 lower than that of Src-pY529 phos-, otherFeat=[]-->, belongsTo=parr
288	phopeptide, according to previous reports that utilized these, otherFeat=[]-->, belongsTo=parr
289	substrates  [3,12] , which likely results in a more rapid substrate, otherFeat=[]-->, belongsTo=parr
290	depletion of Gab1-pY589. In support of this view, the experi-, otherFeat=[]-->, belongsTo=parr
291	ments in intact cells (  Fig. 4 ) show that the D61del mutant dis-, otherFeat=[]-->, belongsTo=parr
292	plays hyperactivity toward Gab1 PI3K-binding sites, which, otherFeat=[]-->, belongsTo=parr
293	include Y589., otherFeat=[]-->, belongsTo=parr
294	During the preparation of this manuscript, it was reported, otherFeat=[]-->, belongsTo=parr
295	that LS-associated SHP-2 mutants, including Y279C and, otherFeat=[]-->, belongsTo=parr
296	T468M, have lost their catalytic activity when assayed on stan-, otherFeat=[]-->, belongsTo=parr
297	dard PTP substrates  [14] . The data obtained from our experi-, otherFeat=[]-->, belongsTo=parr
298	ments performed in identical conditions confirm these, otherFeat=[]-->, belongsTo=parr
299	observations. However, we measured for the first time the, otherFeat=[]-->, belongsTo=parr
300	PTP activity of LS mutants using a specific SHP-2 substrate., otherFeat=[]-->, belongsTo=parr
301	At least in the case of the T468M mutant, we were able to de-, otherFeat=[]-->, belongsTo=parr
302	tect a significant PTP activity, an observation which was vali-, otherFeat=[]-->, belongsTo=parr
303	dated ex vivo by monitoring Gab1/PI3K interaction. This, otherFeat=[]-->, belongsTo=parr
304	reveals the importance of substrate choice for in vitro measure-, otherFeat=[]-->, belongsTo=parr
305	ments of SHP-2 catalytic activity., otherFeat=[]-->, belongsTo=parr
306	In conclusion, in contrast to NS, LS-associated SHP-2 mu-, otherFeat=[]-->, belongsTo=parr
307	tants display a reduced PTP activity. How these apparently, otherFeat=[]-->, belongsTo=parr
308	opposite behaviours of SHP-2 mutants lead to clinically over-, otherFeat=[]-->, belongsTo=parr
309	lapped syndromes still remain to be explained. However, the, otherFeat=[]-->, belongsTo=parr
310	observation that one mutation (T468M) might possibly alter, otherFeat=[]-->, belongsTo=parr
311	the substrate specificity of SHP-2 suggests that an heterogene-, otherFeat=[]-->, belongsTo=parr
312	ity of biochemical features exists between the different mu-, otherFeat=[]-->, belongsTo=parr
313	tants, which may well be the key to the understanding of the, otherFeat=[]-->, belongsTo=parr
314	variable occurrence of the different symptoms associated to, otherFeat=[]-->, belongsTo=parr
315	each of these syndromes., otherFeat=[]-->, belongsTo=parr
316	Fig. 3. PTP assays of LS mutants using a specific SHP-2 substrate. Immune complex PTP assays against 100 lM Gab1-pY589 with or without, otherFeat=[]-->, belongsTo=fig_caption
317	activating peptide IRS1-pY1172 carried out on serum-starved Vero cell transfected with the indicated SHP-2 construct. Bottom: representative anti-, otherFeat=[]-->, belongsTo=fig_caption
318	V5 immunoblot of immunoprecipitates. NT, experiments performed from non-transfected cells. Data represent means ? S.D. from three independent, otherFeat=[]-->, belongsTo=fig_caption
319	experiments. *, different from condition without IRS1-pY1172, P < 0.05., otherFeat=[]-->, belongsTo=fig_caption
320	2480, otherFeat=[]-->, belongsTo=nota_cab_pie
321	N. Hanna et al. / FEBS Letters 580 (2006) 2477?2482, otherFeat=[]-->, belongsTo=nota_cab_pie
322	Acknowledgements: We thank Bruce D. Gelb (Mount Sinai School of, otherFeat=[]-->, belongsTo=parrnote
323	Medicine, New York) for providing PTPN11 constructs and Armelle, otherFeat=[]-->, belongsTo=parrnote
324	Yart for valuable comments., otherFeat=[]-->, belongsTo=parrnote
325	References, otherFeat=[]-->, belongsTo=parr
326	[1] Digilio, M.C., Conti, E., Sarkozy, A., Mingarelli, R., Dottorini,, otherFeat=[]-->, belongsTo=parrnote
327	T., Marino, B., Pizzuti, A. and Dallapiccola, B. (2002) Grouping, otherFeat=[]-->, belongsTo=parrnote
328	of multiple-lentigines/LEOPARD and Noonan syndromes on the, otherFeat=[]-->, belongsTo=parrnote
329	PTPN11 gene. Am. J. Hum. Genet. 71, 389?394., otherFeat=[]-->, belongsTo=parrnote
330	[2] Tartaglia, M. et al. (2001) Mutations in PTPN11, encoding the, otherFeat=[]-->, belongsTo=parrnote
331	protein tyrosine phosphatase SHP-2, cause Noonan syndrome., otherFeat=[]-->, belongsTo=parrnote
332	Nat. Genet. 29, 465?468., otherFeat=[]-->, belongsTo=parrnote
333	[3] Tartaglia, M., Niemeyer, C.M., Fragale, A., Song, X.,, otherFeat=[]-->, belongsTo=parrnote
334	Buechner, J., Jung, A., Hahlen, K., Hasle, H., Licht, J.D., otherFeat=[]-->, belongsTo=parrnote
335	and Gelb, B.D. (2003) Somatic mutations in PTPN11 in, otherFeat=[]-->, belongsTo=parrnote
336	juvenile myelomonocytic leukemia, myelodysplastic syn-, otherFeat=[]-->, belongsTo=parrnote
337	dromes and acute myeloid leukemia. Nat. Genet. 34,, otherFeat=[]-->, belongsTo=parrnote
338	148?150., otherFeat=[]-->, belongsTo=parrnote
339	[4] Neel, B.G., Gu, H. and Pao, L. (2003) The `Shp'ing news: SH2, otherFeat=[]-->, belongsTo=parrnote
340	domain-containing tyrosine phosphatases in cell signaling. Trends, otherFeat=[]-->, belongsTo=parrnote
341	Biochem. Sci. 28, 284?293., otherFeat=[]-->, belongsTo=parrnote
342	Fig. 4. Inactive LS mutants promote Gab1/PI3K binding in EGF-stimulated cells. (A?C) Vero cells cotransfected with Myc-tagged WT Gab1 and, otherFeat=[]-->, belongsTo=fig_caption
343	the indicated V5-tagged SHP-2 construct were stimulated with EGF when indicated. Cleared lysates were then subjected to a p85 affinity, otherFeat=[]-->, belongsTo=fig_caption
344	precipitation assay (``pulldown'') using a GST-p85a fusion protein. The amount of Gab1-Myc precipitated with GST-p85a has analysed by anti-Myc, otherFeat=[]-->, belongsTo=fig_caption
345	immunoblotting (top panels). Corresponding lysates were subjected to anti-Myc (middle panels) and anti-V5 (bottom panels) immunoblotting. (D), otherFeat=[]-->, belongsTo=fig_caption
346	The immunoblots from three independent GST-p85 pulldown experiments were quantified using the software ImageQuant (Molecular Dynamics)., otherFeat=[]-->, belongsTo=fig_caption
347	Data represent means ? S.D. from three independent experiments. ***, different from WT (EGF 5 min), P < 0.001., otherFeat=[]-->, belongsTo=fig_caption
348	N. Hanna et al. / FEBS Letters 580 (2006) 2477?2482, otherFeat=[]-->, belongsTo=nota_cab_pie
349	2481, otherFeat=[]-->, belongsTo=nota_cab_pie
350	[5] Montagner, A., Yart, A., Dance, M., Perret, B., Salles, J.P. and, otherFeat=[]-->, belongsTo=parrnote
351	Raynal, P. (2005) A novel role for Gab1 and SHP2 in epidermal, otherFeat=[]-->, belongsTo=parrnote
352	growth factor-induced Ras activation. J. Biol. Chem. 280, 5350?, otherFeat=[]-->, belongsTo=parrnote
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354	[6] Zhang, S.Q., Tsiaras, W.G., Araki, T., Wen, G., Minichiello,, otherFeat=[]-->, belongsTo=parrnote
355	L., Klein, R. and Neel, B.G. (2002) Receptor-specific regu-, otherFeat=[]-->, belongsTo=parrnote
356	lation of phosphatidylinositol 30-kinase activation by the, otherFeat=[]-->, belongsTo=parrnote
357	protein tyrosine phosphatase Shp2. Mol. Cell. Biol. 22,, otherFeat=[]-->, belongsTo=parrnote
358	4062?4072., otherFeat=[]-->, belongsTo=parrnote
359	[7] Tartaglia, M. et al. (2006) Diversity and functional consequences, otherFeat=[]-->, belongsTo=parrnote
360	of germline and somatic PTPN11 mutations in human disease., otherFeat=[]-->, belongsTo=parrnote
361	Am. J. Hum. Genet. 78, 279?290., otherFeat=[]-->, belongsTo=parrnote
362	[8] Keilhack, H., David, F.S., McGregor, M., Cantley, L.C. and, otherFeat=[]-->, belongsTo=parrnote
363	Neel, B.G. (2005) Diverse biochemical properties of Shp2, otherFeat=[]-->, belongsTo=parrnote
364	mutants. Implications for disease phenotypes. J. Biol. Chem., otherFeat=[]-->, belongsTo=parrnote
365	280, 30984?30993., otherFeat=[]-->, belongsTo=parrnote
366	[9] Fragale, A., Tartaglia, M., Wu, J. and Gelb, B.D. (2004) Noonan, otherFeat=[]-->, belongsTo=parrnote
367	syndrome-associated SHP2/PTPN11 mutants cause EGF-depen-, otherFeat=[]-->, belongsTo=parrnote
368	dent prolonged GAB1 binding and sustained ERK2/MAPK1, otherFeat=[]-->, belongsTo=parrnote
369	activation. Hum. Mutat. 23, 267?277., otherFeat=[]-->, belongsTo=parrnote
370	[10] Hof, P., Pluskey, S., Dhe-Paganon, S., Eck, M.J. and Shoelson,, otherFeat=[]-->, belongsTo=parrnote
371	S.E. (1998) Crystal structure of the tyrosine phosphatase SHP-2., otherFeat=[]-->, belongsTo=parrnote
372	Cell 92, 441?450., otherFeat=[]-->, belongsTo=parrnote
373	[11] Lee, W.H., Raas-Rotschild, A., Miteva, M.A., Bolasco, G., Rein,, otherFeat=[]-->, belongsTo=parrnote
374	A., Gillis, D., Vidaud, D., Vidaud, M., Villoutreix, B.O. and, otherFeat=[]-->, belongsTo=parrnote
375	Parfait, B. (2005) Noonan syndrome type I with PTPN11 3 bp, otherFeat=[]-->, belongsTo=parrnote
376	deletion: structure?function implications. Proteins 58, 7?13., otherFeat=[]-->, belongsTo=parrnote
377	[12] Cunnick, J.M., Mei, L., Doupnik, C.A. and Wu, J. (2001), otherFeat=[]-->, belongsTo=parrnote
378	Phosphotyrosines 627 and 659 of Gab1 constitute a bisphosphoryl, otherFeat=[]-->, belongsTo=parrnote
379	tyrosine-based activation motif (BTAM) conferring binding and, otherFeat=[]-->, belongsTo=parrnote
380	activation of SHP2. J. Biol. Chem. 276, 24380?24387., otherFeat=[]-->, belongsTo=parrnote
381	[13] Yart, A., Laffargue, M., Mayeux, P., Chretien, S., Peres, C., Tonks,, otherFeat=[]-->, belongsTo=parrnote
382	N., Roche, S., Payrastre, B., Chap, H. and Raynal, P. (2001) A, otherFeat=[]-->, belongsTo=parrnote
383	critical role for phosphoinositide 3- kinase upstream of Gab1 and, otherFeat=[]-->, belongsTo=parrnote
384	SHP2 in the activation of ras and mitogen-activated protein kinases, otherFeat=[]-->, belongsTo=parrnote
385	by epidermal growth factor. J. Biol. Chem. 276, 8856?8864., otherFeat=[]-->, belongsTo=parrnote
386	[14] Kontaridis, M.I., Swanson, K.D., David, F.S., Barford, D. and, otherFeat=[]-->, belongsTo=parrnote
387	Neel, B.G. (2006) PTPN11 (SHP2) mutations in LEOPARD, otherFeat=[]-->, belongsTo=parrnote
388	syndrome have dominant negative, not activating, effects. J. Biol., otherFeat=[]-->, belongsTo=parrnote
389	Chem. 281, 6785?6792., otherFeat=[]-->, belongsTo=parrnote
390	2482, otherFeat=[]-->, belongsTo=nota_cab_pie
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==============================
0	Reduced phosphatase activity of SHP-2 in LEOPARD-->id=0, page=0, size=21, fam=Times, col=#000000, type=title, textLines=2--->[]--->title	Reduced phosphatase >>> of SHP-2 in LEOPARD
1	syndrome: Consequences for PI3K binding on Gab1-->id=0, page=0, size=21, fam=Times, col=#000000, type=title, textLines=2--->[]--->title	syndrome: Consequenc>>>PI3K binding on Gab1
2	Nadine Hannaa,1, Alexandra Montagnerb,1, Wen Hwa Leec, Maria Mitevad, Michel Vidald, Michel Vidaud a,Be´atrice Parfaita,*,1, Patrick Raynalb,*,1-->id=2, page=0, size=9, fam=Times, col=#000000, type=parrnote, textLines=176--->[]--->note	Nadine Hannaa,1, Ale>>> Patrick Raynalb,*,1
3	a INSERM U745, Faculte´ des Sciences Pharmaceutiques et Biologiques, Universite´ Rene´ Descartes 4 avenue de l'Observatoire, 75270 Paris Cedex 06, France-->id=2, page=0, size=9, fam=Times, col=#000000, type=parrnote, textLines=176--->[]--->note	a INSERM U745, Facul>>>ris Cedex 06, France
4	b INSERM U563, De´partement Lipoprote´ines et Me´diateurs Lipidiques, Ho^pital Purpan, BP 3028, 31024 Toulouse Cedex 03, France-->id=2, page=0, size=9, fam=Times, col=#000000, type=parrnote, textLines=176--->[]--->note	b INSERM U563, De´pa>>>use Cedex 03, France
5	c Structural Genomics Consortium, University of Oxford, Botnar Research Center, OX 3 7LD Oxford, United Kingdom d INSERM U648, UFR Biome´dicale, Universite´ Rene´ Descartes 45 rue des Saints Pe`res, 75006 Paris, France-->id=2, page=0, size=9, fam=Times, col=#000000, type=parrnote, textLines=176--->[]--->note	c Structural Genomic>>> 75006 Paris, France
6	Received 14 February 2006; revised 10 March 2006; accepted 31 March 2006-->id=2, page=0, size=9, fam=Times, col=#000000, type=parrnote, textLines=176--->[]--->note	Received 14 February>>>cepted 31 March 2006
7	Available online 12 April 2006-->id=2, page=0, size=9, fam=Times, col=#000000, type=parrnote, textLines=176--->[]--->note	Available online 12 >>>online 12 April 2006
8	Edited by Francesc Posas-->id=2, page=0, size=9, fam=Times, col=#000000, type=parrnote, textLines=176--->[]--->note	Edited by Francesc P>>>ed by Francesc Posas
9	Abstract LEOPARD (LS) and Noonan (NS) are overlapping syndromes associated with distinct mutations of SHP-2. Whereas NS mutations enhance SHP-2 catalytic activity, we show that the activity of three representative LS mutants is undetectable when assayed using a standard protein tyrosine phosphatase (PTP) substrate. A different assay using a specific SHP-2 substrate confirms their decreased PTP activity, but also reveals a significant activity of the T468M mutant. In transfected cells stimulated with epidermal growth factor, the least active LS mutants promote Gab1/PI3K binding, validating our in vitro data. LS mutants thus display a reduced PTP activity both in vitro and in transfected cells.-->id=4, page=0, size=11, fam=Times, col=#000000, type=parr, textLines=253--->[]--->parr	Abstract LEOPARD (LS>>>n transfected cells.
10	Ó 2006 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.-->id=4, page=0, size=11, fam=Times, col=#000000, type=parr, textLines=253--->[]--->parr	Ó 2006 Federation of>>>All rights reserved.
11	Keywords: LEOPARD syndrome; PTPN11; SHP-2 phosphatase activity; Gab1; PI3K-->id=4, page=0, size=11, fam=Times, col=#000000, type=parr, textLines=253--->[]--->parr	Keywords: LEOPARD sy>>>activity; Gab1; PI3K
12	1. Introduction-->id=4, page=0, size=11, fam=Times, col=#000000, type=parr, textLines=253--->[]--->parr	1. Introduction>>>1. Introduction
13	LEOPARD syndrome (multiple lentigines, electrocardiographic conduction abnormalities, ocular hypertelorism, pulmonary stenosis, abnormal genitalia, retardation of growth, sensorineural deafness; LS; MIM#151100) is a rare genetic disorder characterized by multiple lentigines and other cutaneous abnormalities such as ``cafe´ au lait'' spots, dysmorphic facies, cardiac defects, growth retardation and increased risk of malignancy. Several clinical manifestations overlap those of Noonan syndrome (NS; MIM#163950), a common genetic disease also typified by facial dysmorphy, heart defects and growth retardation. However ``cafe´ au lait'' spots are rare and multiple lentigines are typically absent in NS.-->id=4, page=0, size=11, fam=Times, col=#000000, type=parr, textLines=253--->[]--->parr	LEOPARD syndrome (mu>>>ically absent in NS.
14	Recently, germline missense mutations in the PTPN11 gene were identified in both syndromes and are now causally linked to the diseases  [1,2] . Somatic PTPN11 mutations are also responsible for juvenile myelomonocytic leukemia  [3]. PTPN11 encodes SHP-2, a widely expressed protein tyrosine phosphatase (PTP) involved in intracellular signalling downstream of several growth factors, cytokines, and hormone receptors  [4] . The structure of SHP-2 consists of two Src-homology 2 (SH2) domains (N-SH2 and C-SH2), a single PTP domain and a C-terminal hydrophilic tail. SHP-2 displays a low basal catalytic activity due to close interactions between the N-SH2 and PTP domains that keeps the phosphatase in an autoinhibited closed conformation. Its catalytic activation thus requires to open the molecule by releasing these interactions, which occurs when its SH2 domains bind to specific phosphotyrosine motifs on SHP-2 upstream signalling partners. These partners include the insulin receptor substrate 1 (IRS-1) and the Grb2-associated binder 1, Gab1. Both are adapter proteins rapidly phosphorylated in response to the stimulation of insulin or epidermal growth factor (EGF) receptors, respectively. Downstream of Gab1 or IRS-1, SHP-2 promotes the activation of the Ras/mitogen-activated pathway, through mechanisms that are still under investigation [4,5]. Besides this signal-enhancing function, SHP-2 can play a modulatory role in the activation of phosphoinositide 3-kinase (PI3K), at least downstream of the EGF receptor. Indeed, this receptor recruits PI3K by phosphorylating Gab1 on binding sites for p85, the regulatory subunit of PI3K, and it was recently shown that these sites are dephosphorylated by SHP-2 during EGF stimulation  [6] .-->id=4, page=0, size=11, fam=Times, col=#000000, type=parr, textLines=253--->[]--->parr	Recently, germline m>>>F stimulation  [6] .
15	In NS, most of SHP-2 mutations described so far affect residues located in or close to the interaction surface between the N-SH2 and the PTP domains  [7]. This is thought to disrupt the autoinhibited closed conformation of SHP-2, resulting in an increased basal PTP activity of most NS-associated SHP-2 mutants observed in vitro  [8,9] . Thus the basis of NS pathogenesis is certainly due to activating, ``gain-of-function'' mutations of SHP-2, even though the SHP-2-dependent mechanisms altered by its NS-mutant derivatives remain to be identified. In LS, only nine mutations were reported to date, affecting seven different amino acids  [7]. These residues are all located in the PTP domain, and, even if no functional data were reported yet, these mutations are predicted to be also ``gain-of-function'', due to the clinical overlap between NS and LS.-->id=4, page=0, size=11, fam=Times, col=#000000, type=parr, textLines=253--->[]--->parr	In NS, most of SHP-2>>>p between NS and LS.
16	This work aimed to define the effect of LS mutations on SHP-2 phosphatase activity. Using molecular modelling, all LS mutations were strikingly located in the catalytic cleft of SHP-2. We then explored, both in vitro and in intact cells, the effect of three representative LS mutations on SHP-2 catalytic activity.-->id=4, page=0, size=11, fam=Times, col=#000000, type=parr, textLines=253--->[]--->parr	This work aimed to d>>> catalytic activity.
17	Abbreviations: EGF, epidermal growth factor; IRS-1, insulin receptor substrate 1; LS, LEOPARD syndrome; NS, Noonan syndrome; PTP, protein tyrosine phosphatase; WT, wild type-->id=2, page=0, size=9, fam=Times, col=#000000, type=parrnote, textLines=176--->[]--->parr	Abbreviations: EGF, >>>atase; WT, wild type
18	*Corresponding authors. Fax: +33 1 4407 1754 (B. Parfait), +33 5 6177 9401 (P. Raynal). E-mail addresses:  beatrice.parfait@univ-paris5.fr (B. Parfait), raynal@toulouse.inserm.fr (P. Raynal).-->id=2, page=0, size=9, fam=Times, col=#000000, type=parrnote, textLines=176--->[]--->parr	*Corresponding autho>>>serm.fr (P. Raynal).
19	1 Equal contribution.-->id=2, page=0, size=9, fam=Times, col=#000000, type=parrnote, textLines=176--->[]--->parr	1 Equal contribution>>> Equal contribution.
20	0014-5793/$32.00 Ó 2006 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.-->id=2, page=0, size=9, fam=Times, col=#000000, type=parrnote, textLines=176--->[]--->note	0014-5793/$32.00 Ó 2>>>All rights reserved.
21	doi:10.1016/j.febslet.2006.03.088-->id=2, page=0, size=9, fam=Times, col=#000000, type=parrnote, textLines=176--->[]--->note	doi:10.1016/j.febsle>>>.febslet.2006.03.088
22	FEBS Letters 580 (2006) 2477­2482-->id=2, page=0, size=9, fam=Times, col=#000000, type=parrnote, textLines=176--->[]--->note	FEBS Letters 580 (20>>>580 (2006) 2477­2482
23	2. Materials and methods-->id=4, page=0, size=11, fam=Times, col=#000000, type=parr, textLines=253--->[]--->parr	2. Materials and met>>>aterials and methods
24	2.1. Structural analysis of LS-associated SHP-2 mutant residues-->id=2, page=0, size=9, fam=Times, col=#000000, type=parrnote, textLines=176--->[]--->parr	2.1. Structural anal>>>HP-2 mutant residues
25	The structure of wild-type (WT) SHP-2  [10] (PDB code 2shp) was loaded into the program ICM (Molsoft LLC,  www.molsoft.com ) and the LS-associated residues were mapped and visually inspected.-->id=2, page=0, size=9, fam=Times, col=#000000, type=parrnote, textLines=176--->[]--->parr	The structure of wil>>> visually inspected.
26	2.2. Phosphopeptide synthesis-->id=2, page=0, size=9, fam=Times, col=#000000, type=parrnote, textLines=176--->[]--->parr	2.2. Phosphopeptide >>>phopeptide synthesis
27	The substrate phosphopeptide Gab1-pY589 (DSEEN-pYVPMNPNL) and the activating peptide IRS1-pY1172 (SLN-pYIDLDLVK)  [8] were synthesized using small-scale Fmoc chemistry.-->id=2, page=0, size=9, fam=Times, col=#000000, type=parrnote, textLines=176--->[]--->parr	The substrate phosph>>>cale Fmoc chemistry.
28	2.3. PTPN11 constructs-->id=2, page=0, size=9, fam=Times, col=#000000, type=parrnote, textLines=176--->[]--->parr	2.3. PTPN11 construc>>>3. PTPN11 constructs
29	The human WT PTPN11 cDNA and the mutants D61Y and C459G subcloned into the pcDNA6/V5-HisA vector were kindly provided by Bruce D. Gelb. The NS-associated D61del mutation  [11] and the LSassociated mutations resulting in Y279C, T468M, and Q510P were introduced using site-directed mutagenesis (QuickChange, Stratagene) and verified by sequencing.-->id=2, page=0, size=9, fam=Times, col=#000000, type=parrnote, textLines=176--->[]--->parr	The human WT PTPN11 >>>ified by sequencing.
30	2.4. Cell lines, transfections and stimulation Vero cells were grown, transiently transfected and stimulated with-->id=2, page=0, size=9, fam=Times, col=#000000, type=parrnote, textLines=176--->[]--->parr	2.4. Cell lines, tra>>> and stimulated with
31	30 ng/ml EGF as described  [5].-->id=2, page=0, size=9, fam=Times, col=#000000, type=parrnote, textLines=176--->[]--->parr	30 ng/ml EGF as desc>>>F as described  [5].
32	2.5. Immune complex PTP assays-->id=2, page=0, size=9, fam=Times, col=#000000, type=parrnote, textLines=176--->[]--->parr	2.5. Immune complex >>>e complex PTP assays
33	Transfected cells were harvested in lysis buffer: 25 mM HEPES, pH 7.4, 150 mM NaCl, 2 mM EDTA, 0.5% Triton X-100 and Protease Inhibitor Cocktail (Sigma). SHP-2 was immunoprecipitated with anti-V5 antibody (Invitrogen) then washed in lysis buffer and in PTP buffer: 20 mM HEPES, pH 7.4, EDTA 1 mM, 5% glycerol, and 1 mM DTT. Immunocomplexes were resuspended in 50 ll of PTP buffer supplemented with 250 lM of Src-pY529 phosphopeptide (TSTEPQ-pY-QPGENL, Upstate Biotech) or 100 lM of Gab1pY589 phosphopeptide alone or with 10 lM of the activating peptide IRS1-pY1172, and incubated for 30 min at 37 °C. To assess free phosphate release, supernatants were then placed in 96-well plates with Malachite Green solution (Upstate Biotech) and incubated for 15 min. Absorbances (630 nm) were compared to a phosphate standard curve  [3,12].-->id=2, page=0, size=9, fam=Times, col=#000000, type=parrnote, textLines=176--->[]--->parr	Transfected cells we>>>ndard curve  [3,12].
34	2.6. GST-p85 affinity precipitation assay This was performed as described  [5].-->id=2, page=0, size=9, fam=Times, col=#000000, type=parrnote, textLines=176--->[]--->parr	2.6. GST-p85 affinit>>>d as described  [5].
35	3. Results and discussion-->id=4, page=0, size=11, fam=Times, col=#000000, type=parr, textLines=253--->[]--->parr	3. Results and discu>>>sults and discussion
36	3.1. LS-associated SHP-2 mutated residues are clustered in the PTP catalytic cleft-->id=4, page=0, size=11, fam=Times, col=#000000, type=parr, textLines=253--->[]--->parr	3.1. LS-associated S>>> PTP catalytic cleft
37	The nine missense mutations reported to date in LS affect seven different amino acids localized exclusively in the SHP-2 PTP domain (  Fig. 1A). To gain insight on LS mutations effects on SHP-2 function, we performed a structural model analysis of the residues which are found mutated in LS with respect to their positioning in the SHP-2 molecule  [10].  Fig. 1B show that the seven residues are highly clustered in the catalytic core of the PTP domain, suggesting that LS mutations might alter SHP-2 catalytic properties, rather than modify its activation state as in the case of NS-associated mutations.-->id=4, page=0, size=11, fam=Times, col=#000000, type=parr, textLines=253--->[]--->parr	The nine missense mu>>>ssociated mutations.
38	3.2. PTP activity of LS mutants is abolished when assayed using a standard PTP substrate-->id=4, page=0, size=11, fam=Times, col=#000000, type=parr, textLines=253--->[]--->parr	3.2. PTP activity of>>>andard PTP substrate
39	To study the effect of LS mutations on SHP-2 catalytic activity, we introduced by site-directed mutagenesis the mutations giving rise to the two common Y279C and T468M and the rare Q510P single amino acid changes in a plasmid encoding human WT-SHP-2. After transfection of these constructs in mammalian cells, we measured in vitro the catalytic activity of each mutant isolated by immunoprecipitation. Vero cells were chosen for their low basal level of SHP-2-dependent signalling pathways once incubated overnight in serum-free medium  [5,13]. Following immunoprecipitation, the PTP activity of the different constructs was assayed using as substrate a synthetic phosphopeptide encompassing Y529 of Src, a standard in vitro substrate of PTP  [3,9]. To test these mutants under stimulation by an upstream SHP-2 activator, the immunocomplexes were also incubated with 10 lM of a phosphopeptide containing the SHP-2-binding site of IRS-1 (IRS1-pY1172) [8] . As shown in  Fig. 2 , addition of IRS1-pY1172 phosphopeptide resulted in a 2­3-fold increase of WT-SHP-2 catalytic activity, attesting that these experimental conditions allow to detect SHP-2 activation. As additional controls, we verified that the well-characterized catalytically dead C459G construct displayed no PTP activity, and that the leukemia-associated D61Y mutant and of the NS-associated D61del mutant have an increased PTP activity compared to WT-SHP-2. These two mutants are not significantly overstimulated by the IRS1-pY1172 peptide in these conditions, but this is in agreement with a previous report  [8] . In addition, when the immunocomplexes were incubated with the activating peptide alone, the release of phosphate was undetectable for any of SHP-2 construct (data not shown), indicating that this phosphopeptide was not significantly dephosphorylated by SHP-2 when used in these conditions appropriate to observe its activating effect. This series of controls validated the methodology used in this study.-->id=4, page=0, size=11, fam=Times, col=#000000, type=parr, textLines=253--->[]--->parr	To study the effect >>> used in this study.
40	More interestingly, when considering the three LS-associated SHP-2 mutants,  Fig. 2 shows that their basal catalytic activity is similar to that of the catalytically dead C459G construct. In the presence of the activating peptide, a slight but significant increase of PTP activity was detected in the case of Y279C and T468M mutants, but this activity remains at the most around 10% of that of WT-SHP-2. Taken together, these results suggest that LS mutations lead to the loss of SHP-2 catalytic activity, probably by disruption of the integrity of SHP-2 catalytic cleft.-->id=4, page=0, size=11, fam=Times, col=#000000, type=parr, textLines=253--->[]--->parr	More interestingly, >>>P-2 catalytic cleft.
41	3.3. PTP assay of LS mutants using a specific SHP-2 substrate confirms their decreased activity but reveals a significant activity of T468M mutant-->id=4, page=0, size=11, fam=Times, col=#000000, type=parr, textLines=253--->[]--->parr	3.3. PTP assay of LS>>>vity of T468M mutant
42	The above results were obtained with a standard PTP substrate, which is not considered as an undeniable SHP-2 substrate in vivo. We thus determined if LS mutations had the same effect on SHP-2 catalytic activity when assayed on a validated SHP-2 substrate. To this aim, we designed a phosphopeptide encompassing Y589 of Gab1 (Gab1-pY589 phosphopeptide), which corresponds to a PI3K-binding site and is known to be dephosphorylated by SHP-2 in cells treated with EGF  [6]. As shown in  Fig. 3 , the basal PTP activity of the three LS mutants assayed with this peptide is still severely reduced in comparison with the WT protein. However, in the case of the T468M mutant, its basal activity is around 30% of that of WT-SHP-2. Under stimulation with IRS1-pY1172, the Q510P mutant still displayed a negligible PTP activity. In contrast, the two other mutants seem to display a significant-->id=4, page=0, size=11, fam=Times, col=#000000, type=parr, textLines=253--->[]--->parr	The above results we>>>isplay a significant
43	2478-->id=2, page=0, size=9, fam=Times, col=#000000, type=parrnote, textLines=176--->[]--->note	2478>>>2478
44	N. Hanna et al. / FEBS Letters 580 (2006) 2477­2482-->id=2, page=0, size=9, fam=Times, col=#000000, type=parrnote, textLines=176--->[]--->note	N. Hanna et al. / FE>>>580 (2006) 2477­2482
45	Fig. 1. LS-associated SHP-2 mutated residues are clustered in the PTP domain of the protein. (A) Schematic representation of SHP-2 showing the distribution of mutations identified in LS (above). Position of hyperactive and inactive SHP-2 mutants studied in this report are also indicated (below). (B) Left: ribbon representation of SHP-2 structure in its autoinhibited closed conformation  [10] . The N- and C-terminal SH2 domains are coloured in yellow and green, respectively, the catalytic domain in blue. The red square depicts the closeup region. Right: Close-up of the active site and location of LS-associated SHP-2 mutated residues. The two most frequently altered residues Y279 and T468 are represented as orange sticks, while other mutations are shown as magenta sticks. Position of D61 (on the inhibitory loop of N-SH2 domain protruding into the catalytic site) responsible for autoinhibition of PTP activity is highlighted in red.-->id=2, page=0, size=9, fam=Times, col=#000000, type=parrnote, textLines=176--->[]--->capfig	Fig. 1. LS-associate>>> highlighted in red.
46	Fig. 2. PTP activity of LS mutants is abolished when assayed using a standard PTP substrate. Immune complex PTP assay against 250 lM SrcpY529 with or without 10 lM activating peptide IRS1-pY1172 carried out on serum-starved Vero cells transfected with the indicated SHP-2 construct. Bottom: representative anti-V5 immunoblots of immunoprecipitates. NT, experiments performed from non-transfected cells. Data represent means ± S.D. from three independent experiments. Significant differences in PTP activity between basal and activated conditions are indicated (* P < 0.05, *** P < 0.001).-->id=2, page=0, size=9, fam=Times, col=#000000, type=parrnote, textLines=176--->[]--->capfig	Fig. 2. PTP activity>>>.05, *** P < 0.001).
47	N. Hanna et al. / FEBS Letters 580 (2006) 2477­2482-->id=2, page=0, size=9, fam=Times, col=#000000, type=parrnote, textLines=176--->[]--->note	N. Hanna et al. / FE>>>580 (2006) 2477­2482
48	2479-->id=2, page=0, size=9, fam=Times, col=#000000, type=parrnote, textLines=176--->[]--->note	2479>>>2479
49	PTP activity, notably the T468M that reaches 50% of WT SHP-2. These results thus confirm the  Fig. 2 data showing that LS mutations, in contrast with NS mutations, lead to a decrease in SHP-2 catalytic activity. However, these experiments also indicate that different LS mutations produce distinct levels of alteration of SHP-2 activity, which was not detected using a standard PTP substrate. This suggest that at least one LS mutation influences SHP-2 substrate recognition rather than suppresses its catalytic activity.-->id=4, page=0, size=11, fam=Times, col=#000000, type=parr, textLines=253--->[]--->parr	PTP activity, notabl>>> catalytic activity.
50	3.4. In situ confirmation of in vitro assays: the least active LS mutants promote Gab1/PI3K binding in EGF-stimulated cells-->id=4, page=0, size=11, fam=Times, col=#000000, type=parr, textLines=253--->[]--->parr	3.4. In situ confirm>>>EGF-stimulated cells
51	We next determined whether these in vitro features of LS mutants could be confirmed in transfected cells, by testing the effects of these mutants on the dephosphorylation of a cellular substrate of SHP-2. Because SHP-2 can dephosphorylate Gab1 on its PI3K-binding sites in response to EGF  [6], we designed an experiment to monitor these sites phosphorylation. Vero cells were chosen again, since they can be efficiently stimulated by EGF, leading to the phosphorylation of Gab1 and the downstream activation of SHP-2 and PI3K  [5,13]. Following stimulation with EGF, cells were incubated with a GST-p85 fusion protein, which allows to precipitate Gab1 only if phosphorylated on PI3K binding sites  [5,13]. As positive control, in cells transfected with catalytically dead C459G SHP-2, GST-p85 precipitates an higher amount of Gab1 in comparison with WT SHP-2, whereas, as negative control, the hyperactive D61del mutant prevents Gab1 precipitation (  Fig. 4A­C, compare lanes ``C459G'' and ``D61del'' with ``WT''). Thus Gab1 amount precipitated with GST-p85 appears inversely proportional to SHP-2 catalytic activity, showing that the Gab1/PI3K interaction can be taken as marker of the cellular catalytic activity of SHP-2.-->id=4, page=0, size=11, fam=Times, col=#000000, type=parr, textLines=253--->[]--->parr	We next determined w>>>c activity of SHP-2.
52	Once validated, this assay was applied to cells expressing LS mutants. The results show that the Y279C and Q510P mutants enhance, compare to the WT-SHP-2, Gab1 recovery in GSTp85 pulldowns, measured either after 5 or 10 min of EGF stimulation  (Fig. 4A and B). A quantitative analysis of data from different experiments indicates that these mutants produce an effect similar to that of catalytically dead SHP-2 C459G (Fig. 4 D). In contrast, the T468M mutant did not enhance Gab1 precipitation by GST-p85 in comparison with WTSHP-2, implying that this mutant species retains some of its phosphatase activity towards Gab1 PI3K-binding sites (  Fig. 4 C and D). We conclude that the LS mutants Y279C and Q510P behave in situ like catalytically inactive mutants, which confirms our in vitro data showing that these mutants display the lowest PTP activity. In addition, the fact that the T468M mutant does not promote Gab1/PI3K interaction more than the WT protein also confirms the in vitro results since this mutation was found to reduce, but not to abolish, its catalytic activity on Gab1 phosphopeptide. It is thus likely that this mutation does alter the catalytic efficiency of the T468M mutant, but its residual activity appears sufficient to dephosphorylate Gab1 in EGF-stimulated cells.-->id=4, page=0, size=11, fam=Times, col=#000000, type=parr, textLines=253--->[]--->parr	Once validated, this>>>GF-stimulated cells.
53	The apparent lack of hyperactivity of D61 mutants in vitro on Gab1-pY589 substrate is probably due to the use of a concentration of Gab1-pY589 lower than that of Src-pY529 phosphopeptide, according to previous reports that utilized these substrates  [3,12] , which likely results in a more rapid substrate depletion of Gab1-pY589. In support of this view, the experiments in intact cells (  Fig. 4 ) show that the D61del mutant displays hyperactivity toward Gab1 PI3K-binding sites, which include Y589.-->id=4, page=0, size=11, fam=Times, col=#000000, type=parr, textLines=253--->[]--->parr	The apparent lack of>>> which include Y589.
54	During the preparation of this manuscript, it was reported that LS-associated SHP-2 mutants, including Y279C and T468M, have lost their catalytic activity when assayed on standard PTP substrates  [14] . The data obtained from our experiments performed in identical conditions confirm these observations. However, we measured for the first time the PTP activity of LS mutants using a specific SHP-2 substrate. At least in the case of the T468M mutant, we were able to detect a significant PTP activity, an observation which was validated ex vivo by monitoring Gab1/PI3K interaction. This reveals the importance of substrate choice for in vitro measurements of SHP-2 catalytic activity.-->id=4, page=0, size=11, fam=Times, col=#000000, type=parr, textLines=253--->[]--->parr	During the preparati>>> catalytic activity.
55	In conclusion, in contrast to NS, LS-associated SHP-2 mutants display a reduced PTP activity. How these apparently opposite behaviours of SHP-2 mutants lead to clinically overlapped syndromes still remain to be explained. However, the observation that one mutation (T468M) might possibly alter the substrate specificity of SHP-2 suggests that an heterogeneity of biochemical features exists between the different mutants, which may well be the key to the understanding of the variable occurrence of the different symptoms associated to each of these syndromes.-->id=4, page=0, size=11, fam=Times, col=#000000, type=parr, textLines=253--->[]--->parr	In conclusion, in co>>> of these syndromes.
56	Fig. 3. PTP assays of LS mutants using a specific SHP-2 substrate. Immune complex PTP assays against 100 lM Gab1-pY589 with or without activating peptide IRS1-pY1172 carried out on serum-starved Vero cell transfected with the indicated SHP-2 construct. Bottom: representative antiV5 immunoblot of immunoprecipitates. NT, experiments performed from non-transfected cells. Data represent means ± S.D. from three independent experiments. *, different from condition without IRS1-pY1172, P < 0.05.-->id=2, page=0, size=9, fam=Times, col=#000000, type=parrnote, textLines=176--->[]--->capfig	Fig. 3. PTP assays o>>>S1-pY1172, P < 0.05.
57	2480-->id=2, page=0, size=9, fam=Times, col=#000000, type=parrnote, textLines=176--->[]--->note	2480>>>2480
58	N. Hanna et al. / FEBS Letters 580 (2006) 2477­2482-->id=2, page=0, size=9, fam=Times, col=#000000, type=parrnote, textLines=176--->[]--->note	N. Hanna et al. / FE>>>580 (2006) 2477­2482
59	Acknowledgements: We thank Bruce D. Gelb (Mount Sinai School of Medicine, New York) for providing PTPN11 constructs and Armelle Yart for valuable comments.-->id=2, page=0, size=9, fam=Times, col=#000000, type=parrnote, textLines=176--->[]--->parr	Acknowledgements: We>>>r valuable comments.
60	References-->id=4, page=0, size=11, fam=Times, col=#000000, type=parr, textLines=253--->[]--->parr	References>>>References
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65	Fig. 4. Inactive LS mutants promote Gab1/PI3K binding in EGF-stimulated cells. (A­C) Vero cells cotransfected with Myc-tagged WT Gab1 and the indicated V5-tagged SHP-2 construct were stimulated with EGF when indicated. Cleared lysates were then subjected to a p85 affinity precipitation assay (``pulldown'') using a GST-p85a fusion protein. The amount of Gab1-Myc precipitated with GST-p85a has analysed by anti-Myc immunoblotting (top panels). Corresponding lysates were subjected to anti-Myc (middle panels) and anti-V5 (bottom panels) immunoblotting. (D) The immunoblots from three independent GST-p85 pulldown experiments were quantified using the software ImageQuant (Molecular Dynamics). Data represent means ± S.D. from three independent experiments. ***, different from WT (EGF 5 min), P < 0.001.-->id=2, page=0, size=9, fam=Times, col=#000000, type=parrnote, textLines=176--->[]--->capfig	Fig. 4. Inactive LS >>>F 5 min), P < 0.001.
66	N. Hanna et al. / FEBS Letters 580 (2006) 2477­2482-->id=2, page=0, size=9, fam=Times, col=#000000, type=parrnote, textLines=176--->[]--->note	N. Hanna et al. / FE>>>580 (2006) 2477­2482
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78	2482-->id=2, page=0, size=9, fam=Times, col=#000000, type=parrnote, textLines=176--->[]--->note	2482>>>2482
79	N. Hanna et al. / FEBS Letters 580 (2006) 2477­2482-->id=2, page=0, size=9, fam=Times, col=#000000, type=parrnote, textLines=176--->[]--->note	N. Hanna et al. / FE>>>580 (2006) 2477­2482