Protein 6 by a Calcium-binding Regulatory Subunit
tion. Phosphorylation of Cdc6 by cyclin-dependent kinases
inhibits ubiquitination of Cdc6 by APC/C
PR70 member of the PPP2R3 family of regulatory subunits tar-
gets protein phosphatase 2A (PP2A) to Cdc6. Interaction with
Cdc6 is mediated by residues within the C terminus of PR70,
whereas interaction with PP2A requires N-terminal sequences
conserved within the PPP2R3 family. Two functional EF-hand
calcium-binding motifs mediate a calcium-enhanced interac-
tion of PR70 with PP2A. Calcium has no effect on the interac-
tion of PR70 with Cdc6 but enhances the association of PP2A
with Cdc6 through its effects on PR70. Knockdown of PR70 by
RNA interference results in an accumulation of endogenous and
expressed Cdc6 protein that is dependent on the cyclin-depend-
ent protein kinase phosphorylation sites on Cdc6. Knockdown
of PR70 also causes G
cate that PP2A can be targeted in a calcium-regulated manner to
Cdc6 via the PR70 subunit, where it plays a role in regulating
protein phosphorylation and stability.
ing mitosis. A crucial step in regulating DNA replication is the
assembly of pre-replicative complexes at origins of replication
(1). Coordination of DNA replication with the cell cycle is
achieved through a periodic accumulation and destruction of
proteins involved in formation of pre-RCs
anaphase promoting complex/cyclosome (2). The mammalian
Cdc6 protein is required for DNA replication and acts in con-
junction with the Cdt1 protein to recruit the mini-chromosome
maintenance complex into pre-RCs (1, 3, 4). Mammalian cells
have multiple mechanisms to ensure that pre-RCs only assem-
ble during late M and G
protein kinases. Cdc6 is phosphorylated at canonical CDK sites,
including serines 54, 74, and 106 of human Cdc6 (5, 6). Exper-
iments with exogenously expressed protein have shown that
phosphorylation can regulate the nuclear localization of Cdc6
(5, 7–9). However, other studies have shown that a subpopula-
tion of endogenous Cdc6 remains in the nucleus, bound to
chromatin, throughout the cell cycle (10 –12). Phosphorylation
of Cdc6 also plays an important role in regulating the stability of
Cdc6. The N-terminal domain of Cdc6 contains RXXL (D box)
and KEN (KEN box) destruction motifs, which are binding sites
for the form of the APC/C containing the cdh1-targeting sub-
unit (13). Cdc6 is polyubiquitinated and targeted for degrada-
tion by APC/C
tein from degradation by blocking recognition by cdh1 result-
ing in stabilization of Cdc6 during a window of time that allows
formation of pre-RCs during G
showing that the cell cycle arrest caused by DNA damage is due
to dephosphorylation and degradation of Cdc6 (16).
phosphatases, dephosphorylation of Cdc6 can also control for-
mation of pre-RCs. Much less is known about mechanisms that
regulate Cdc6 dephosphorylation. A previous study identified a
fragment of PR70 as a member of the PPP2R3 family of PP2A
regulatory subunits that interacted with Cdc6 and implicated
PP2A in regulating Cdc6 phosphorylation (17). The major
forms of PP2A contain a dimeric core complex composed of a
scaffold (A) and a catalytic subunit (C). The AC core dimer
associates with regulatory subunits that form heterotrimeric
holoenzymes and target the catalytic subunit to specific phos-
phoprotein substrates (18 –20). In this study, the mechanism
and functional consequences of targeting of PP2A to Cdc6 by
PR70 were investigated. The results show that PR70 interacts
with PP2A and Cdc6 through distinct regions of the protein,
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Dallas, TX 75390-9041. Tel.: 214-645-6152; Fax: 214-645-6151; E-mail:
marc.mumby@utsouthwestern.edu.
siRNA, small interfering RNA; Cdk, cyclin-dependent kinase; E3, ubiquitin-
protein isopeptide ligase; PBS, phosphate-buffered saline; IP, immunopre-
cipitation; GST, glutathione S-transferase; aa, amino acid(s); HA, hemagglu-
tinin; CMV, cytomegalovirus; Cdc6, cell division control protein 6.