G1 cyclin/cyclin-dependent kinase-coordinated phosphorylation of endogenous pocket proteins differentially regulates their interactions with E2F4 and E2F1 and gene expression

Mitogenic stimulation leads to activation of G(1) cyclin-dependent kinases (CDKs), which phosphorylate pocket proteins and trigger progression through the G(0)/G(1) and G(1)/S transitions of the cell cycle. However, the individual role of G(1) cyclin-CDK complexes in the coordinated regulation of pocket proteins and their interaction with E2F family members is not fully understood. Here we report that individually or in concert cyclin D1-CDK and cyclin E-CDK complexes induce distinct and coordinated phosphorylation of endogenous pocket proteins, which also has distinct consequences in the regulation of pocket protein interactions with E2F4 and the expression of p107 and E2F1, both E2F-regulated genes. The up-regulation of these two proteins and the release of p130 and pRB from E2F4 complexes allows formation of E2F1 complexes not only with pRB but also with p130 and p107 as well as the formation of p107-E2F4 complexes. The formation of these complexes occurs in the presence of active cyclin D1-CDK and cyclin E-CDK complexes, indicating that whereas phosphorylation plays a role in the abrogation of certain pocket protein/E2F interactions, these same activities induce the formation of other complexes in the context of a cell expressing endogenous levels of pocket and E2F proteins. Of note, phosphorylated p130 "form 3," which does not interact with E2F4, readily interacts with E2F1. Our data also demonstrate that ectopic overexpression of either cyclin is sufficient to induce mitogen-independent growth in human T98G and Rat-1 cells, although the effects of cyclin D1 require downstream activation of cyclin E-CDK2 activity. Interestingly, in T98G cells, cyclin D1 induces cell cycle progression more potently than cyclin E. This suggests that cyclin D1 activates pathways independently of cyclin E that ensure timely progression through the cell cycle.     

PP2A holoenzymes negatively and positively regulate cell cycle progression by dephosphorylating pocket proteins and multiple CDK substrates

Cell cycle progression is negatively regulated by the retinoblastoma family of pocket proteins and CDK inhibitors (CKIs). In contrast, CDKs promote progression through multiple phases of the cell cycle. One prominent way by which CDKs promote cell cycle progression is by inactivation of pocket proteins via hyperphosphorylation. Reactivation of pocket proteins to halt cell cycle progression requires dephosphorylation of multiple CDK-phosphorylated sites and is accomplished by PP2A and PP1 serine/threonine protein phosphatases. The same phosphatases are also implicated in dephosphorylation of multiple CDK substrates as cells exit mitosis and reenter the G1 phase of the cell cycle. This review is primarily focused on the role of PP2A and PP1 in the activation of pocket proteins during the cell cycle and in response to signaling cues that trigger cell cycle exit. Other functions of PP2A during the cell cycle will be discussed in brief, as comprehensive reviews on this topic have been published recently (De Wulf et al., 2009; Wurzenberger and Gerlich, 2011).     

LIN-9 Phosphorylation on Threonine-96 Is Required for Transcriptional Activation of LIN-9 Target Genes and Promotes Cell Cycle Progression

Cell cycle transitions are governed by the timely expression of cyclins, the activating subunits of Cyclin-dependent kinases (Cdks), which are responsible for the inactivation of the pocket proteins. Overexpression of cyclins promotes cell proliferation and cancer. Therefore, it is important to understand the mechanisms by which cyclins regulate the expression of cell cycle promoting genes including subsequent cyclins. LIN-9 and the pocket proteins p107 and p130 are members of the DREAM complex that in G0 represses cell cycle genes. Interestingly, little is know about the regulation and function of LIN-9 after phosphorylation of p107,p130 by Cyclin D/Cdk4 disassembles the DREAM complex in early G1. In this report, we demonstrate that cyclin E1/Cdk3 phosphorylates LIN-9 on Thr-96. Mutating Thr-96 to alanine inhibits activation of cyclins A2 and B1 promoters, whereas a phosphomimetic Asp mutant strongly activates their promoters and triggers accelerated entry into G2/M phase in 293T cells. Taken together, our data suggest a novel role for cyclin E1 beyond G1/S and into S/G2 phase, most likely by inducing the expression of subsequent cyclins A2 and B1 through LIN-9.     

Overlapping roles of pocket proteins in the myocardium are unmasked by germ line deletion of p130 plus heart-specific deletion of Rb

The pocket protein family of tumor suppressors, and Rb specifically, have been implicated as controlling terminal differentiation in many tissues, including the heart. To establish the biological functions of Rb in the heart and overcome the early lethality caused by germ line deletion of Rb, we used a Cre/loxP system to create conditional, heart-specific Rb-deficient mice. Mice that are deficient in Rb exclusively in cardiac myocytes (CRbL/L) are born with the expected Mendelian distribution, and the adult mice displayed no change in heart size, myocyte cell cycle distribution, myocyte apoptosis, or mechanical function. Since both Rb and p130 are expressed in the adult myocardium, we created double-knockout mice (CRbL/L p130-/-) to determine it these proteins have a shared role in regulating cardiac myocyte cell cycle progression. Adult CRbL/L p130-/- mice demonstrated a threefold increase in the heart weight-to-body weight ratio and showed increased numbers of bromodeoxyuridine- and phosphorylated histone H3-positive nuclei, consistent with persistent myocyte cycling. Likewise, the combined deletion of Rb plus p130 up-regulated myocardial expression of Myc, E2F-1, and G1 cyclin-dependent kinase activities, synergistically. Thus, Rb and p130 have overlapping functional roles in vivo to suppress cell cycle activators, including Myc, and maintain quiescence in postnatal cardiac muscle.     

Inhibition of mitogenesis in Balb/c-3T3 cells by Trichostatin A. Multiple alterations in the induction and activation of cyclin-cyclin-dependent kinase complexes

Trichostatin A (TSA), a global repressor of histone deacetylase activity, inhibits the proliferation of a number of cell types. However, the identification of the mechanisms underlying TSA-mediated growth arrests has remained elusive. In order to resolve in more detail the cellular process modulated during the growth inhibition induced by TSA, we studied the effect of the drug on G(0)/G(1) traverse in mitogen-stimulated quiescent Balb/c-3T3 cells. Cyclin D1 and retinoblastoma proteins were induced following the mitogenic stimulation of both control and TSA-treated cells, and cyclin D1 formed complexes with CDK4 under both conditions. However, cyclin D1-associated kinase was not increased in growth-arrested cells. The lack of cyclin D-associated kinase was paralleled by an accumulation of RB in a hypophosphorylated form, as would be expected. In contrast, p130 became partially phosphorylated, accompanied by a marked increase in p130-dependent E2F DNA binding activity and a partial release of free E2F-4. Despite the presence of E2F complexes not bound to pocket proteins, late G(1) E2F-dependent gene expression was not observed. The lack of cyclin D1-associated kinase in TSA-treated cultures was potentially due to high levels of the cyclin-dependent inhibitor p27(kip1). However, the modulation of p27(kip1) levels by the deacetylase inhibitor cannot be responsible for the induction of the cell cycle arrest, since the growth of murine embryo fibroblasts deficient in both p27(kip1) and p21(cip1) was also inhibited by TSA. These data support a model in which TSA inhibits very early cell cycle traverse, which, in turn, leads to a decrease in cyclin D1-associated kinase activation and a repression of late cell cycle-dependent events. Alterations in early G(0)/G(1) gene expression accompany the TSA-mediated growth arrest.     

E1A blocks hyperphosphorylation of p130 and p107 without affecting the phosphorylation status of the retinoblastoma protein

The phosphorylation status of the pRB family of growth suppressor proteins is regulated in a cell cycle entry-, progression-, and exit-dependent manner in normal cells. We have shown previously that p130, a member of this family, exhibits patterns of phosphorylated forms associated with various cell growth and differentiation stages. However, human 293 cells, which are transformed cells that express the adenoviral oncoproteins E1A and E1B, exhibit an abnormal pattern of p130 phosphorylated forms. Here we report that, unlike pRB, the phosphorylation status of both p130 and p107 is not modulated during the cell cycle in 293 cells as it is in other cells. Conditional overexpression of individual G(1)/S cyclins in 293 cells does not alter the phosphorylation status of p130, suggesting that the expression of E1A and/or E1B blocks hyperphosphorylation of p130. In agreement with these observations, transient cotransfection of vectors expressing E1A 12S, but not E1B, in combination with pocket proteins into U-2 OS cells blocks hyperphosphorylation of both p130 and p107. However, the phosphorylation status of pRB is not altered by cotransfection of E1A 12S vectors. Moreover, MC3T3-E1 preosteoblasts stably expressing E1A 12S also exhibit a block in hyperphosphorylation of endogenous p130 and p107. Direct binding of E1A to p130 and p107 is not required for the phosphorylation block since E1A 12S mutants defective in binding to the pRB family also block hyperphosphorylation of p130 and p107. Our data reported here identify a novel function of E1A, which affects p130 and p107 but does not affect pRB. Since E1A does not bind the hyperphosphorylated forms of p130, this function of E1A might prevent the existence of "free" hyperphosphorylated p130, which could act as a CDK inhibitor.     

Pocket Protein p130/Rb2 Is Required for Efficient Herpes Simplex Virus Type 1 Gene Expression and Viral Replication

We have reported previously that herpes simplex virus type 1 (HSV-1) infection disrupts normal progression of the mammalian cell cycle, causing cells to enter a G(1)-like state. Infected cells were characterized by a decline in cyclin-dependent kinase 2 (CDK2) activities, loss of hyperphosphorylated retinoblastoma protein (pRb), accumulation of E2F-pocket protein complexes, and failure to initiate cellular DNA replication. In the present study, we investigated the role of the pocket proteins pRb, p107, and p130 in HSV-1-dependent cell cycle inhibition and cyclin kinase regulation by infecting murine 3T3 cells derived from wild-type (WT) mouse embryos or embryos with deletions of pRb (pRb(-/-)), p107 (p107(-/-)), p130 (p130(-/-)), or both p130 and p107 (p130(-/-)/p107(-/-)). With respect to CDK2 inhibition, viral protein accumulation, viral DNA replication, and progeny virus yield, WT, pRb(-/-), and p107(-/-) cells were essentially identical. In contrast, after infection of p130(-/-) cells, we observed no inhibition of CDK2 activity, a 5- to 6-h delay in accumulation of viral proteins, an impaired ability to form viral DNA replication compartments, and reduced viral DNA synthesis. As a result, progeny virus yield was reduced 2 logs compared to that in WT cells. Notably, p130(-/-)/p107(-/-) double-knockout cells had a virus replication phenotype intermediate between those of the p107(-/-) and p130(-/-) cells. We conclude from these studies that p130 is a key factor in regulating aspects of cell cycle progression, as well as the timely expression of viral genes and replication of viral DNA.     

G1 arrest and differentiation can occur independently of Rb family function

The ability of progenitor cells to exit the cell cycle is essential for proper embryonic development and homeostasis, but the mechanisms governing cell cycle exit are still not fully understood. Here, we tested the requirement for the retinoblastoma (Rb) protein and its family members p107 and p130 in G0/G1 arrest and differentiation in mammalian cells. We found that Rb family triple knockout (TKO) mouse embryos survive until days 9-11 of gestation. Strikingly, some TKO cells, including in epithelial and neural lineages, are able to exit the cell cycle in G0/G1 and differentiate in teratomas and in culture. This ability of TKO cells to arrest in G0/G1 is associated with the repression of key E2F target genes. Thus, G1 arrest is not always dependent on Rb family members, which illustrates the robustness of cell cycle regulatory networks during differentiation and allows for the identification of candidate pathways to inhibit the expansion of cancer cells with mutations in the Rb pathway.     

Overexpression of cyclin D1 is associated with elevated levels of MAP kinases, Akt and Pak1 during diethylnitrosamine-induced progressive liver carcinogenesis

Hepatocellular carcinoma (HCC) is a multi-factorial and multi-step process. However, the molecular mechanisms, which play a pivotal role during progressive development of HCC, are not known. Accordingly Sprague-Dawley rats were administered diethylnitrosamine (DEN) for one to three months in order to understand the molecular alterations during progressive development of liver tumor. In this study involvement of G1/S regulatory proteins, MAP kinases and cell survival factors were analyzed using RT-PCR, western blotting and in vitro kinase assays. The data showed overexpression of cyclin D1 and increased expression and activation of ERK1/2, p38 kinase and JNK1/2 with progression of tumor suggesting that MAP kinases play an important role during tumorigenesis. These molecular alterations were supported by Akt upregulation and increase in the levels of inactive GSK3beta with progression of liver tumor. Further, p21-actived kinase1 (Pak1) was found to be upregulated with tumor progression, which is a novel observation during progressive liver carcinogenesis. These results indicate that elevated levels of all the three MAP kinases (ERK1/2, p38 and JNK1/2), Akt/GSK3beta and Pak1 are associated with cyclin D1 upregulation, which helps in the disruption of the G1/S regulatory point of the cell cycle and leads to abnormal cell proliferation during progressive hepatocarcinogenesis.     

GSK3 inhibitors stabilize Wee1 and reduce cerebellar granule cell progenitor proliferation

Ubiquitin mediated proteolysis is required for transition from one cell cycle phase to another. For instance, the mitosis inhibitor Wee1 is targeted for degradation during S phase and G2 to allow mitotic entry. Wee1 is an essential tyrosine kinase required for the G2/M transition and S-phase progression. Although several studies have concentrated on Wee1 regulation during mitosis, few have elucidated its degradation during interphase. Our prior studies have demonstrated that Wee1 is degraded via CK1δ dependent phosphorylation during the S and G2/M phases of the cell cycle. Here we demonstrate that GSK3β may work in concert with CK1δ to induce Wee1 destruction during interphase. We generated small molecules that specifically stabilized Wee1. We profiled these compounds against 296 kinases and found that they inhibit GSK3α and GSK3β, suggesting that Wee1 may be targeted for proteolysis by GSK3. Consistent with this notion, known GSK3 inhibitors stabilized Wee1 and GSK3β depletion reduced Wee1 turnover. Given Wee1's central role in cell cycle progression, we predicted that GSK3 inhibitors should limit cell proliferation. Indeed, we demonstrate that GSK3 inhibitors potently inhibited proliferation of the most abundant cell in the mammalian brain, the cerebellar granule cell progenitor (GCP). These studies identify a previously unappreciated role for GSK3β mediated regulation of Wee1 during the cell cycle and in neurogenesis. Furthermore, they suggest that pharmacological inhibition of Wee1 may be therapeutically attractive in some cancers where GSK-3β or Wee1 are dysregulated.     

C-terminal phosphorylation controls the stability and function of p27kip1

Entry of cells into the cell division cycle requires the coordinated activation of cyclin-dependent kinases (cdks) and the deactivation of cyclin kinase inhibitors. Degradation of p27kip1 is known to be a central component of this process as it allows controlled activation of cdk2-associated kinase activity. Turnover of p27 at the G1/S transition is regulated through phosphorylation at T187 and subsequent SCF(skp2)-dependent ubiquitylation. However, detailed analysis of this process revealed the existence of additional pathways that regulate the abundance of the protein in early G1 and as cells exit quiescence. Here, we report on a molecular mechanism that regulates p27 stability by phosphorylation at T198. Phosphorylation of p27 at T198 prevents ubiquitin-dependent degradation of free p27. T198 phosphorylation also controls progression through the G1 phase of the cell cycle by regulating the association of p27 with cyclin-cdk complexes. Our results unveil the molecular composition of a pathway, which regulates the abundance and activity of p27kip1 during early G1. They also explain how the T187- and the T198-dependent turnover systems synergize to allow cell cycle progression in G1.     

Retinoblastoma tumor suppressor protein-dependent methylation of histone H3 lysine 27 is associated with irreversible cell cycle exit

The retinoblastoma tumor suppressor protein (pRb) is involved in mitotic exit, promoting the arrest of myoblasts, and myogenic differentiation. However, it is unclear how permanent cell cycle exit is maintained in differentiated muscle. Using RNA interference, expression profiling, and chromatin immunoprecipitations, we show that pRb is essential for cell cycle exit and the differentiation of myoblasts and is also uniquely required to maintain this arrest in myotubes. Remarkably, we also uncover a function for the pRb-related proteins p107 and p130 as enforcers of a G2/M phase checkpoint that prevents progression into mitosis in cells that have lost pRb. We further demonstrate that pRb effects permanent cell cycle exit in part by maintaining trimethylation of histone H3 lysine 27 (H3K27) on cell cycle genes. H3K27 trimethylation silences other genes, including Cyclin D1, in a pRb-independent but polycomb-dependent manner. Thus, our data distinguish two distinct chromatin-based regulatory mechanisms that lead to terminal differentiation.     

Protein kinase C signaling mediates a program of cell cycle withdrawal in the intestinal epithelium

Members of the protein kinase C (PKC) family of signal transduction molecules have been widely implicated in regulation of cell growth and differentiation, although the underlying molecular mechanisms involved remain poorly defined. Using combined in vitro and in vivo intestinal epithelial model systems, we demonstrate that PKC signaling can trigger a coordinated program of molecular events leading to cell cycle withdrawal into G(0). PKC activation in the IEC-18 intestinal crypt cell line resulted in rapid downregulation of D-type cyclins and differential induction of p21(waf1/cip1) and p27(kip1), thus targeting all of the major G(1)/S cyclin-dependent kinase complexes. These events were associated with coordinated alterations in expression and phosphorylation of the pocket proteins p107, pRb, and p130 that drive cells to exit the cell cycle into G(0) as indicated by concomitant downregulation of the DNA licensing factor cdc6. Manipulation of PKC isozyme levels in IEC-18 cells demonstrated that PKCalpha alone can trigger hallmark events of cell cycle withdrawal in intestinal epithelial cells. Notably, analysis of the developmental control of cell cycle regulatory molecules along the crypt-villus axis revealed that PKCalpha activation is appropriately positioned within intestinal crypts to trigger this program of cell cycle exit-specific events in situ. Together, these data point to PKCalpha as a key regulator of cell cycle withdrawal in the intestinal epithelium.