Cdc6 Protein Activates p27KIP1-bound Cdk2 Protein Only after the Bound p27 Protein Undergoes C-terminal Phosphorylation*

Background: Cdk2 activity essential for chromosome replication is regulated by p27KIP1 the Cdk inhibitor. Results: Cdc6 the AAA+ ATPase, known to assemble pre-replicative complexes and activate p21CIP1-bound Cdk2, also activates p27-bound Cdk2 but only after the p27 undergoes C-terminal phosphorylation. Conclusion: An entirely new mechanism for regulating Cdk2 activity is discovered. Significance: This makes key progress in understanding how the G1-S transition is controlled. In mammalian cells Cdk2 activity during the G1-S transition is mainly controlled by p27KIP1. Although the amount and subcellular localization of p27 influence Cdk2 activity, how Cdk2 activity is regulated during this phase transition still remains virtually unknown. Here we report an entirely new mechanism for this regulation. Cdc6 the AAA+ ATPase, known to assemble prereplicative complexes on chromosomal replication origins and activate p21CIP1-bound Cdk2, also activated p27-bound Cdk2 in its ATPase and cyclin binding motif-dependent manner but only after the p27 bound to the Cdk2 was phosphorylated at the C terminus. ROCK, which mediates a signal for cell anchorage to the extracellular matrix and activates the mTORC1 cascade as well as controls cytoskeleton assembly, was partly responsible for C-terminal phosphorylation of the p27. In vitro reconstitution demonstrated ROCK (Rho-associated kinase)-mediated phosphorylation of Cdk2-bound p27 at the C terminus and subsequent activation of the Cdk2 by Cdc6.

The onset of S phase is prepared in advance by the assembly of prereplication complexes that takes place in late M and G 1 phases (1,2). Cdc6 the AAAϩ ATPase, aided by Cdt1, assembles prereplicative complexes by loading the minichromosome maintenance helicase complex on the origin recognition complex (ORC) 2 -bound origins of replication. After prereplicative complexes are assembled, several factors, some of which require activation by Cdk2, which is negatively regulated by association with Cdk inhibitors such as p27 and p21, are further loaded on the origins (3). Minichromosome maintenance heli-case is then activated by Cdc7, and finally, DNA polymerases are recruited to those origins to initiate DNA replication.
Cdc6 is known to possess two biological functions for the onset and progression of S phase. Besides assembling prereplicative complexes, it activates p21-bound Cdk2 and thereby governs utilization of p21-dependent DNA damage checkpoint (4,5). Cdc6 contains a cyclin binding motif and an ATPase domain, and these two functional domains are essential to activate p21-bound Cdk2. During the G 1 -S transition, Cdk2 activity is regulated mainly by p27, which undergoes modification by phosphorylation at Ser 10 , Thr 187 , and Thr 197 in rodent fibroblasts (6,7). Phosphorylation of this inhibitor at Ser 10 and Thr 197 occurs early in growth-stimulated cells and facilitates its own stabilization and nuclear exclusion, whereas Cdk2-mediated Thr 187 phosphorylation promotes its own degradation by proteasomes (8).
The cells constituting solid organs of animals require an anchorage to the extracellular matrix for their proliferation, and without such an anchorage they arrest in G 1 and eventually die of apoptosis known as anoikis (9,10). The G 1 arrest is caused at least in part by inactivation of Cdk4/Cdk6 and Cdk2 with repression of cyclins A, D1, D3, and induction of p27 (11,12). Inactivation of Cdk4/Cdk6 results in activation of retinoblastoma protein (Rb) and its cognates, which in turn inactivates the E2F transcriptional factors to shut down a subset of genes essential or important for S phase onset, such as Cdc6, cyclin A, and E2F1 (13). Furthermore, Cdc6 protein is rapidly eliminated by proteasomal degradation executed mainly by the APC/ C CDH1 ubiquitin ligase (14).
The evolutionarily conserved Tsc1/Tsc2-Rheb-mTORC1 pathway mediates growth and metabolic signals to control cell proliferation (15,16). Growth factor-activated AKT/PKB stimulates the Rheb small G protein by inactivating the Tsc1/Tsc2 GTPase activating protein complex. Stimulated Rheb activates mTORC1 to phosphorylate S6K1 and 4EBP to enhance translation. In addition, this cascade conveys a cellular anchorage signal to control the G 1 -S transition. ROCK (Rho-associated kinase) activated by an anchorage signal originated from RhoA and integrins up-regulates the mTORC1 pathway by directly phosphorylating Tsc2 at Thr 1203 (17). Consequently, when cells are deprived of anchorage, not only Cdk4/Cdk6 and Cdk2 but also mTORC1 undergo inactivation. Forced activation of mTORC1, however, restores only Cdk4/Cdk6 activity despite marked up-regulation of both cyclin A and D-type cyclins and additional enforced Cdc6 expression. By contrast, expression of both a constitutively active ROCK and Cdc6 stimulates not only Cdk4/Cdk6 but also Cdk2 (17). Although ROCK may participate therein, how anchorage signals regulate Cdk2 activity is unknown.
During a search for a minimum combination of G 1 cell cycle factors the manipulation of which invokes anchorage-independent proliferation of rodent fibroblasts, we came across finding an entirely new mechanism for regulating Cdk2 activity that involves ROCK-mediated C-terminal phosphorylation of Cdk2-bound p27 and subsequent activation of the Cdk2 by Cdc6.
In Vitro Cdk2 Reactivation Assay-Proliferating REF cells were cultured in methylcellulose medium (MC) for 12 h, lysed with lysis buffer (14), and immunoprecipitated for Cdk2 with the agarose beads-conjugated anti-Cdk2 antibody. The Cdk2bound agarose beads were then incubated at 30 ºC in 20 l of 50 mM Tris-HCl (pH 7.5) buffer containing 30 mM MgCl 2 and 10 mM ATP for 30 min with the addition of 1 l of either Escherichia coli-expressed active ROCK1 (17) or its control prepara-tion and then for another 30 min with the addition of 1 l of either recombinant Cdc6/Cdc6 WB /Cdc6 Cy or its control preparation. The bead-bound Cdk2 was determined for its amount and activity as well as coprecipitated p27, its Thr 197 phosphorylated form, and Cdc6 as described (4). Recombinant Cdc6, Cdc6 WB , and Cdc6 Cy were synthesized in reticulocyte lysates and affinity-purified as described (4).
The cDNA encoding rat Cdc6 tagged with 3 ϫ FLAG and 6 ϫ His at its C terminus was inserted into the pFASTBAC plasmid (Invitrogen) and then converted to a recombinant baculovirus (constructed by Q. Kan). The recombinant Cdc6 protein expressed in SF9 cells was double affinity-purified with nickelnitrilotriacetic acid beads and anti-FLAG M2 gels. A phosphomimetic mutant p27 S10D N-terminal-tagged with His 6 and an empty vector control was expressed in E. coli, purified with nickel-nitrilotriacetic acid beads, and used for inactivation of baculovirus-expressed affinity-purified Cdk2-cyclin A complexes (46% purity) (Upstate Biotechnology) in a buffer containing 50 mM Tris-HCl (pH 7.5) and 10 mM MgCl 2 . The inactivated Cdk2 was immunoprecipitated with anti-Cdk2 antibody-conjugated beads and used for subsequent reactivation assay as above.

Cdk2 Remains Activated in REF-overexpressing Cdk6 and
Cyclin D3 Despite Anchorage Deprivation-To achieve the initial goal, we began to examine the potentially anchorage-independent cell cycle-promoting effects of Cdk6-cyclin D3 complexes (K6D3) that are refractory to CDK inhibitors (18,19). Both original REF and REF-K6D3 cells were deprived of anchorage by culturing in MC for 48 h with every 12-h sampling and analyzed for major G 1 cell cycle factors by immunoblotting (Fig. 1A). Confirming our previous results, in REF, expression of Cdc6, G 1 cyclins, and E2F-1 diminished or disappeared with mTORC1 inactivation as indicated by a loss of both S6K1 protein and its Thr 389 phosphorylation. In addition, Rb quickly lost Cdk4/Cdk6-specific Ser 780 and Cdk2-specific Ser 807/811 phosphorylation, consistent with inactivation of these kinases as shown by the parallel in vitro kinase assays (Fig. 1B).
On the other hand, in anchorage-deprived REF-K6D3, G 1 cyclins and E2F1 remained expressed at least for 48 h, but Cdc6 disappeared gradually. Due to the overexpressed K6D3, Rb phosphorylation at Ser 780 persisted for 48 h, albeit significantly reduced perhaps, partly caused by destabilization of Cdk6 protein. Both S6K1 and its Thr 389 phosphorylation markedly diminished but, unlike in REF, persisted at a low level, indicating that mTORC1 was not completely inactivated in this cell despite anchorage loss. Highly interestingly, Rb continued to be phosphorylated at Ser 807/811 for 48 h. We speculated that this phosphorylation might be attributable to the overexpressed K6D3 because there was a report showing that Cdk6 bound to a viral cyclin is refractory to p27 KIP1 like K6D3 and has a Cdk2like activity (20). But this speculation was wrong. In in vitro kinase assays, Cdk6 showed no ability to phosphorylate Rb Ser 807/811 despite its robust activity toward Rb Ser 780 (Fig. 1B). Instead, Cdk2 was found highly active, accounting for the in vivo Rb Ser 807/811 phosphorylation.
Consistent with the continued Cdk6 activation, mRNAs for E2F-regulated genes, such as Cdc6, cyclin A, and E2F1, were up-regulated in REF-K6D3 (Fig. 1C). Thus, in this cell overexpressed Cdk6 was active without anchorage as initially intended. But unexpectedly, Cdk2 was also active albeit transiently, as opposed to its rapid inactivation in anchorage-deprived REF.
Close Association of p27 Thr 197 Phosphorylation with Cdk2 Activation-The factors that would affect Cdk2 activity in G 1 -S are the partner cyclins, p27 and p21, and its own modification by activating Thr 160 phosphorylation and checkpoint-associated inhibitory Tyr 15 phosphorylation (21). As already noted Cdk2 was quickly inactivated upon anchorage deprivation in REF despite continued expression of cyclin E (Fig. 1A) and even in mTORC1-active REFs that express both cyclins E and A (14). These observations indicate that the availability of the partner cyclins is not the determinant of Cdk2 activity during anchorage deprivation.
Consequently we next focused on Cdk2 phosphorylation at Thr 160 and Tyr 15 and examined its causal relation to Cdk2 activity. Rapidly proliferating REF and REF-K6D3 were cultured in MC as in Fig. 1A and analyzed for the levels of Cdk2, its phosphorylation at Thr 160 and Tyr 15 , and others as well as Cdk2 activity ( Fig. 2A). In REF, Cdk2 remained Thr 160 -phosphorylated at least for 12 h during MC culture, whereas Cdk2 activity vanished within 12 h. In REF-K6D3, this phosphorylation persisted for 48 h, whereas Cdk2 activity almost disappeared at 36 h. In addition, throughout this experiment Tyr 15 phosphorylation of Cdk2 could not be detected. Thus there was no direct correlation between the loss of Cdk2 activity and phosphorylation at these sites, although Thr 160 phosphorylation is absolutely essential for Cdk2 activity. The level of p21 was similar between the two during anchorage deprivation ( Fig. 2A).
On the other hand, despite the continued Cdk2 activity, p27 expression was constitutively elevated 2-3-fold in  with no significant fluctuations during MC culture. Because modification of p27 by phosphorylation controls its subcellular localization and ultimate fate, we next examined the levels of p27 phosphorylated at the three sites, Ser 10 , Thr 187 , and Thr 197 , in REF and REF-K6D3 during MC culture. Ser 10 phosphorylation was elevated in REF-K6D3 proportional to the p27 amount. Interestingly, there was a great difference in the levels of both Thr 187 and Thr 197 phosphorylation between the two. In REF, in which Cdk2 was quickly inactivated, phosphorylation at both sites fell sharply to an undetectable level, whereas in REF-K6D3, where Cdk2 was active for 36 h, phosphorylation at these sites persisted for 48 h with a gradual reduction. Because Thr 187 is the site phosphorylated by Cdk2, we attributed the Thr 187 phosphorylation to the activated Cdk2. Consequently, we speculated that the C-terminal phosphorylation of p27 might be causally related to the Cdk2 activation. This speculation is consistent with the previous report showing that overexpression of a C-terminally unphosphorylatable p27 mutant retards both activation of Cdk2 and S phase entry (22).   (14). B, Cdk2 activation is prolonged in anchorage-deprived REF-K6D3. Cdk2 and Cdk6 in A were assayed for their amounts and activities as described (14). C, E2F-dependent genes driving S phase onset are up-regulated in anchorage-deprived REF-K6D3. RNA was prepared from the cells harvested in A, and cyclin A, E2F1, and Cdc6 mRNAs were quantified by reverse transcription-coupled real time PCR as described (14). The data shown are averaged values with S.D obtained from three independently isolated samples.  (24), and Rsk1 (25). In addition to these kinases, we included ROCK in the candidates for in-depth examination not only because this kinase minimum target sequence (RRX(S/T) or RX(S/T)) perfectly matches with the evolutionarily conserved C-terminal sequence of p27 (RRQT) but also because unlike forced mTORC1 activation, expression of a constitutively active ROCK1 activated Cdk2 in the absence of anchorage albeit weakly (17).
To narrow down candidates, we first examined the expression levels of these four kinases and the presence or absence of the physical or biological representations of their activation state in anchorage-deprived REF and REF-K6D3. Both cells were incubated in MC and analyzed for the levels of the four kinases and additionally, Ser 473 -phosphorylated AKT and Thr 573 -phosphorylated Rsk1 because these phosphorylated forms are absolutely essential for their activity (26), whereas ROCK activity was monitored by detecting Thr 508 phosphorylation of LIMK1, a specific ROCK substrate (27). AKT remained expressed but was inactive in REF-K6D3 because its Ser 473 phosphorylation disappeared quickly in this cell, like in REF (Fig. 3A). On the other hand, Pim-1 protein was up-regulated, but Pim-3 was unaltered, whereas Pim-2 was not expressed. Rsk1 was expressed and active in both cells as indicated by continued phosphorylation at the Thr 573 . Finally, ROCK1 and ROCK2 were expressed in both cells, but at least either one was active in REF-K6D3 as Thr 508 phosphorylation of LIMK1 persisted in these cells, albeit as a gradual loss. Thus, both Pim and ROCK appeared to nicely fit the aimed kinase.
We, therefore, examined the effects of specific inhibitors to these two kinases. An Rsk1 inhibitor was also included in the experiment as a provisional negative control. The inhibitors used were GW 5074 for Pim-1 and Pim-3, Y27632 for ROCK1 and ROCK2, and BI-D1870 for Rsk1 (28). Proliferating REF-K6D3 cells were cultured in MC for 12 h first, then for 24 h with or without the addition of the inhibitors and analyzed for the levels of p27 and its Thr 197 phosphorylation (Fig. 3B, upper  panel). Without these additions, the levels of p27 Thr 197 phosphorylation were similar for 24 h. Of the three, the ROCK and Pim inhibitors significantly lowered p27 Thr 197 phosphorylation at 24 h. In this experiment, LIMK1 Thr 508 phosphorylation completely vanished on treatment with the ROCK inhibitor as expected but was also markedly reduced by treatment with the Pim inhibitor. On the other hand, the Rsk1 inhibitor had no inhibitory effect.
To confirm the inhibitory effect and examine a potential synergism between the ROCK and Pim inhibitors, a similar inhibition experiment was carried out for REF-K6D3 proliferating in anchorage-furnished dishes. In this experiment inhibitors were added at 0 h, and cells were incubated for 24 h with every 12 h harvests (Fig. 3B, lower panel). Both inhibitors markedly reduced Thr 197 phosphorylation at 12 and 24 h when used separately. But the simultaneous use of both inhibitors reduced Thr 197 phosphorylation further. These results indicate that both Rock and Pim are mainly responsible for C-terminal phosphorylation of p27 in REF-K6D3 regardless of the presence or absence of anchorage.
Expression of Active ROCK1 Leads to Continued p27 Thr 197 Phosphorylation during Anchorage Deprivation-Because Pim was already known to phosphorylate p27 at Thr 197 , we decided to focus on ROCK and sought to investigate a possible physical interaction between this kinase and p27. REF cells expressing wild-type or a constitutively active ROCK1 (REF-RK and REF-aRK) were constructed as previously (17) and examined for phosphorylation of both p27 Thr 197 and LIMK1 Thr 508 and others (Fig. 3C). Unlike in REF-RK, in which ROCK1 was quickly inactivated, p27 Thr 197 continued to be phosphorylated in REF-aRK, albeit at a lowered level, consistent with the inhibitor data.
ROCK1 Phosphorylates p27 at Thr 197 in Vitro-Consequently we examined whether or not active ROCK1 can directly phosphorylate p27 Thr 197 in vitro. Both E. coli-expressed affinity-purified p27, and similarly, E. coli-expressed affinity-purified active ROCK1 (aRK) were incubated in an ATP-containing reaction mixture in the presence or absence of Y27632 and analyzed for p27 Thr 197 phosphorylation by immunoblotting (Fig. 3D). Active ROCK1 but not the empty vector lysate phos-phorylated p27 Thr 197 when the inhibitor was absent. Thus ROCK1 could physically interact with p27 and phosphorylate its C terminus.
Cdc6 Facilitates Cdk2 Activation in Anchorage-deprived REF-K6D3 Cells-Although essential, p27 Thr 197 phosphorylation was not sufficient for Cdk2 activation in REF-K6D3 because Cdk2 lost activity at 48 h despite the presence of both p27 Thr 197 and Cdk2 Thr 160 phosphorylation (Fig. 2). This indicates that an additional factor(s) is likely to be needed for the Cdk2 activation. Cdc6 is known to activate p21-bound Cdk2 (4,5). Moreover, the loss of Cdk2 activity roughly coincided with the disappearance of Cdc6 protein (Figs. 1 and 2). We, therefore, speculated that Cdc6 might be the additional factor and examined whether or not siRNA-mediated knockdown or antipodal overexpression of Cdc6 would influence the prolonged Cdk2 activation. A duplex siRNA designed for  were constructed with the same doxycycline-repressible system as in Fig. 2 and analyzed similarly (Fig. 4B). When wild-type Cdc6 was induced, Cdk2 remained active throughout the MC culture, confirming the Fig. 4A results. But when the WB or Cy mutant was induced, Cdk2 lost activity within 36 h. Loss of Cdk2 activity was more evident with the WB mutant. By contrast, without induction, Cdk2 activity was indistinguishable among them and similar to that in original REF-K6D3 cells. Thus both the ATPase domain and the cyclin binding motif were required for Cdc6 to induce the prolonged activation of Cdk2.
ROCK-dependent Activation of p27-Bound Cdk2 by Cdc6-Finally, to establish that Cdc6 activates p27-bound Cdk2 in its ATPase-and Cy-dependent manner only after the bound p27 is C-terminal-phosphorylated, we performed in vitro reactivation assays with E. coli-expressed affinity-purified aRK and in vitro reticulocyte lysate-synthesized affinity-purified Cdc6. First, inactive Cdk2 was immunoprecipitated from 12-h MC-cultured REF with anti-Cdk2 antibody-conjugated agarose beads. In this cell, Cdk2 was inactivated completely, and Thr 197 phosphorylation of p27 disappeared also nearly completely, yet Thr 160 phosphorylation of Cdk2 essential for its activity was retained ( Fig. 2A). The immunoprecipitated Cdk2 was then incubated in an ATP-containing reaction mixture added with either aRK or Cdc6 or with aRK first and Cdc6 next and split into halves. One-half was used to determine the amounts of Cdk2, p27, aRK, and Cdc6 in the reaction mixture. From the other half, the bead-bound Cdk2 was recovered by a brief centrifugation, incubated in the Cdk2 assay mixture, and determined for Cdk2 activity and the amounts of Cdk2, coprecipitated 27, its Thr 197 phosphorylation, and Cdc6 (Fig. 5A). In parallel, Cdk2 immunoprecipitated from logarithmically proliferating REF cells was similarly incubated with E. coli and reticulocyte control preparations (C1 and C2) and assayed for its activity as a positive control. When the inactive Cdk2 was incubated with active ROCK1, p27 bound to the Cdk2 was phosphorylated at Thr 197 , but the Cdk2 was only marginally activated. When the Cdk2 was incubated with Cdc6, it was activated marginally too. By contrast, when incubated with ROCK1 first and Cdc6 next, the Cdk2 was activated nearly as high as the fully active positive control Cdk2. The activated Cdk2 was associated with Cdc6, a slightly less amount of p27, and a much less amount of its C-terminal-phosphorylated form. On the other hand, the active Cdk2 recovered from proliferating cells did not contain any significant amounts of Cdc6 or C-terminal-phosphorylated p27, perhaps because of their rapid removal from the Cdk2 in proliferating cells. Instead it was associated with C-terminal-nonphosphorylated p27. Consistent with the in vivo results, the WB or Cy-motif mutant Cdc6 failed to reactivate the Cdk2 in the same in vitro assay although they bound to the Cdk2 similarly (Fig. 5B). Furthermore, consistently the WB mutant even suppressed the slight reactivation induced upon incubation with active ROCK1 perhaps by a residual amount of endogenous Cdc6 associated with the inactive Cdk2.
As for activation of p21-bound Cdk2, no other cellular protein may be required for this activation because the same reactivation occurred with baculovirus-expressed highly purified recombinant Cdk2-cyclin A complexes inactivated by bacterially expressed p27 S10D , bacterially expressed active ROCK1, and baculovirus-expressed double affinity-purified Cdc6 (Fig.  5C). In this experiment, a phospho-mimetic S10D substitution mutant p27 S10D was used to reconstitute inactive Cdk2-p27 complexes because p27 in REF was phosphorylated at Ser 10 regardless of the presence or absence of anchorage (Fig. 2). These results demonstrate that ROCK1 phosphorylates the C terminus of Cdk2-bound p27 and that Cdc6 can activate the Cdk2 in its ATPase-and Cy-dependent manner only after the bound p27 is C-terminal-phosphorylated. Combined Overexpression of Cdk6, Cyclin D3, Cdc6, and Active Rheb Induces Anchorage-independent Proliferation of Rodent Embryonic Fibroblasts-As expected from the anchorage-independent Cdk2 activation by combined overexpression of Cdk6, cyclin D3, and Cdc6 as described above, REF-K6D3-Cdc6 and the same cell but with additional overexpression of active Rheb to enhance mRNA translation (REF-K6D3-Cdc6-aRheb) proliferated in soft agar and formed colonies. Remarkably, the active Rheb overexpressor proliferated in soft agar as rapidly as HeLa, a fully developed human cancer cell line (Fig.  6A). In this cell line all the G 1 cell cycle factors examined continued to be expressed with activated mTORC1 despite the absence of anchorage at least for 48 h (Fig. 6B). Mouse embryonic fibroblasts overexpressing the same combination of the factors formed smaller colonies infrequently mingled with large ones (supplemental Fig. S2). Thus, overexpression of four G 1 cell cycle or related factors can induce sustainable anchorageindependent proliferation of otherwise absolutely anchoragedependent rodent embryonic fibroblasts.

DISCUSSION
Cdc6 is the bifunctional AAAϩ ATPase initially discovered to assemble prereplicative complexes on ORC-bound replication origins and later to activate p21-bound Cdk2. The most striking finding in this study is that Cdc6 can activate also p27bound Cdk2 but only after the bound p27 acquires C-terminal phosphorylation. Despite this difference, both the ATPase domain and the Cy motif are required for this ATPase to activate p27-bound Cdk2, just like p21-bound Cdk2. This implies that the basic mechanism for the activation is perhaps similar between the two, but understanding the reason for the requirement of C-terminal phosphorylation of the bound p27 awaits three-dimensional structural analysis of the tetrameric complex. From the regulatory point of view, this Cdc6-driven mechanism provides cells with a highly effective tool to overcome  . Active ROCK1 phosphorylates the C terminus of Cdk2-bound p27 and Cdc6 activates the Cdk2 in its ATPase-and Cy-dependent manner only after bound p27 is C-terminal-phosphorylated. A, agarose bead-bound inactive Cdk2 immunoprecipitated (IP) from 12-h anchorage-deprived REF cells was incubated first with either E. coli-expressed aRK or its negative control preparation (C1) and then with either reticulocyte-synthesized Cdc6 or its negative control preparation (C2). After incubation, a half of the reaction mixture was denatured and analyzed for the amounts of Cdk2, Cdc6, p27, and its Thr 197 phosphorylated form. From the other half, bead-bound Cdk2 was collected and analyzed for its activity and coprecipitated p27-, Cdc6-, and Thr 197 -phosphorylated p27. B, the immunoprecipitated Cdk2 as above was incubated first with aRK and then with in vitro synthesized recombinant Cdc6, Cdc6 WB , or Cdc6 Cy and analyzed as in A. C, baculovirus-produced and highly purified Cdk2-cyclin A complexes were incubated with E. coli-expressed p27 S10D or an empty vector counterpart (C1) and immunoprecipitated with anti-Cdk2 antibody-conjugated beads. The bead-bound Cdk2 was then incubated with aRK or C1 and finally with baculovirus-produced double affinity-purified Cdc6 or its negative control preparation (C3) and analyzed as in A.
inhibition of Cdk2 by high levels of p27 that have accumulated during G 0 or G 1 arrest invoked by growth factor withdrawal or anchorage deprivation.
It is to our surprise that p27 Thr 197 phosphorylation was exerted not only by Pim but also ROCK, which mediates anchorage signals to control cytoskeleton as well as activate mTORC1, because this kinase has never been implicated to phosphorylate p27. As already noted, we previously observed that unlike mTORC1 activation, expression of an active ROCK1 together with Cdc6 restored both Cdk4 and Cdk2 activities and induced anchorage-independent proliferation albeit weakly (17). In light of the current finding, the mechanistic basis for the Cdk2 activation by expression of active ROCK1 and Cdc6 is now understood.
Although ROCK1 can phosphorylate the C terminus of free p27 as demonstrated in Fig. 3D, it appears that this kinase more efficiently phosphorylates the p27 molecule that properly binds and thereby inactivates Cdk2. Notably, upon Cdk2 activation by Cdc6, the amount of the Thr 197 -phosphorylated form of bound p27 markedly diminished, whereas the total amount of bound p27 decreased only marginally at most in the in vitro reactivation assay (Fig. 5A). This observation implies the following scenario. As well documented, multiple molecules of p27 or p21 bind one molecule of Cdk2 (29), but only one molecule of these inhibitors properly binds Cdk2 and causes its inactivation. The properly bound p27 molecule is preferentially phosphorylated by ROCK and removed by Cdc6 with full activation of the Cdk2. This scenario also well explains the strange association of C-terminal-nonphosphorylated p27 with the active Cdk2 in proliferating cells (Fig. 5A) as well as only the marginal reduction in the amount of Cdk2-associated p21 despite full reactivation of the Cdk2 by Cdc6 in vitro (4).
Finally, we would like to briefly comment on the induction of anchorage-independent proliferation by manipulation of G 1 cell cycle factors or their related. As shown in Fig. 6, combined overexpression of Cdk6, cyclin D3, Cdc6, and active Rheb was sufficient to induce proliferation of rat embryonic fibroblasts in soft agar as rapidly as HeLa, the fully developed human cancer cells. There are numerous reports documenting that many of these factors are highly expressed in various cancer cells (30 -34). In light of our finding, overexpression of these factors might in part account for their anchorage-independent proliferation capability.