with Cdc6.
ing PP2A activity. Experiments with N-terminal truncation
mutants showed that PR70 can associate with Cdc6 independ-
ently of the A- and C-subunits. This observation contrasts with
the prevailing view of PP2A in which the regulatory subunits
have been thought to be constitutively associated with the core
dimer (18 –20). The importance of the existence of PP2A in
heterotrimeric forms is supported by data showing that some
PP2A regulatory subunits are only stable when incorporated
into holoenzymes (29 –31). Overexpression of the PPP2R2 fam-
ily member, B
zymes (31). In contrast, several lines of evidence indicate that
members of the PPP2R3/PR72 family are stable regardless of
whether or not they are incorporated into holoenzymes. As
shown here, mutants of PR70 that cannot bind to PP2A accu-
mulate to the same levels as the wild-type protein. The apparent
stability of PR70 is also independent of interaction with Cdc6,
because a mutant (
PR72 subunit that block interaction with the AC core dimer
have no effect on the levels of expressed protein (27). In addi-
tion, the PR59 subunit is not degraded following loss of the
A-subunit (31). Thus, in contrast to the PPP2R2/B and
PPP2R5/B56 families of PP2A regulatory subunits, members of
the PPP2R3/PR72 family are stable proteins whose levels and
functional interactions with substrates and other proteins may
be independent of the core dimer of PP2A.
the levels of Cdc6. Excess free PR70 would associate with Cdc6
and displace endogenous PR70-AC holoenzyme. Loss of the
active AC core dimer from Cdc6 would inhibit dephosphoryl-
ation leading to decreased ubiquitination by APC/C
pressed PR70 is supported by observations that the effects on
Cdc6 levels are not dependent on interaction of PR70 with the
AC core dimer (e.g. the
increase Cdc6 levels appear to be dependent on intact phospho-
rylation sites, because the levels of co-expressed DDD and AAA
mutants of Cdc6 were not significantly affected. Forced over-
expression of the related PR72 subunit has also been reported
to act in a dominant-negative manner. Expression of either
wild-type PR72 or an EF-hand 2 mutant, which cannot bind the
AC core dimer, both cause G
important role for PR70 in progression of cells through into S
phase. Similarly, overexpression of a fragment that contains the
complete R3 domain and the C terminus of PR70 (termed PR48
or
stability of Cdc6 enhance formation of pre-replicative com-
plexes (15). The increase in pre-replicative complex formation
would be expected to enhance entry into S phase. Consistent
with this idea, expression of exogenous wild-type Cdc6 leads to
accelerated entry into S phase (14). However, exogenous
expression of a non-phosphorylatable (AAA) mutant of Cdc6
(5) or an N-terminally truncated version of Cdc6, missing the
CDK phosphorylation sites and destruction motifs recognized
by APC/C
ation is required for stabilization of Cdc6 and assembly of pre-
replicative complexes during G
replication. Knockdown or overexpression of PR70 might
inhibit this step and retard entry into S phase. Although the
ability of PR70 knockdown to cause G
plays other roles during G
PP2A-mediated dephosphorylation or actions that are inde-
pendent of PP2A.
PPP2R3/PR72 family in mediating calcium-regulated dephos-
phorylation. All four members of this family contain conserved
EF-hand sequence motifs (supplemental Fig. S1
PR70, calcium binding to the second EF-hand enhances inter-
action with the A-subunit (27). PR72 has been shown to medi-
ate calcium-dependent dephosphorylation of threonine-75 in
the dopamine- and cAMP-regulated phosphoprotein of 32 kDa
(DARPP-32). This report showed that, in addition to the role of
EF-hand 2 in interaction with the AC core dimer, calcium bind-
ing to EF-hand 1 increased the phosphatase activity of the
PR72-holoenzyme toward DARPP-32 (32). Both PR72 and its
alternative splice variant (PR130) have been reported to inter-
act with the mammalian Naked cuticle protein and regulate
Wnt signaling (33, 34). Calcium may therefore influence Wnt
signaling through recruitment and/or regulation of PP2A asso-
ciated with Naked cuticle. The other member of the PPP2R3
family, PR59, targets PP2A to the retinoblastoma-related p107
protein (35) and may provide a mechanism for calcium regula-
tion of the cell cycle functions of p107.
bridging the two proteins. The N-terminal domain of PR70,
which is not conserved with other members of the PPP2R3
family, is not required for interaction with either PP2A or Cdc6.
Deletion of the C-terminal region, including the PR70-unique
sequence and a portion of the conserved R3 domain, had no
effect on interaction with the PP2A core dimer but severely
inhibited binding to Cdc6. Conversely, deletion of N-terminal
sequences within the conserved R3 domain blocked binding to
the A-subunit but had no effect on interaction with Cdc6. The
N-terminal region required for interaction with the A-subunit
contains an FYF amino acid motif that plays a role in the inter-
action and is conserved between members of the PPP2R3 fam-