Mammalian Cell Cycles without Cyclin E-CDK2

The family of mammalian E-type cyclins is composed of two proteins, termed cyclin E1 and E2. These two cyclins are widely expressed in proliferating cells. E-cyclins bind and activate cyclin dependent kinase CDK2. Cyclin E-CDK2 complexes were believed to play critical function in driving cell cycle progression of normal, nontransformed cells and of cancer cells. Several recent reports challenge this notion.     

Mammalian cell cycles without cyclin E-CDK2

The family of mammalian E-type cyclins is composed of two proteins, termed cyclin E1 and E2. These two cyclins are widely expressed in proliferating cells. E-cyclins bind and activate cyclin dependent kinase CDK2. Cyclin E-CDK2 complexes were believed to play critical function in driving cell cycle progression of normal, nontransformed cells and of cancer cells. Several recent reports challenge this notion.     

Distinct roles for cyclins E and A during DNA replication complex assembly and activation

Initiation of DNA replication is regulated by cyclin-dependent protein kinase 2 (Cdk2) in association with two different regulatory subunits, cyclin A and cyclin E (reviewed in ref. 1). But why two different cyclins are required and why their order of activation is tightly regulated are unknown. Using a cell-free system for initiation of DNA replication that is based on G1 nuclei, G1 cytosol and recombinant proteins, we find that cyclins E and A have specialized roles during the transition from G0 to S phase. Cyclin E stimulates replication complex assembly by cooperating with Cdc6, to make G1 nuclei competent to replicate in vitro. Cyclin A has two separable functions: it activates DNA synthesis by replication complexes that are already assembled, and it inhibits the assembly of new complexes. Thus, cyclin E opens a 'window of opportunity' for replication complex assembly that is closed by cyclin A. The dual functions of cyclin A ensure that the assembly phase (G1) ends before DNA synthesis (S) begins, thereby preventing re-initiation until the next cell cycle.     

Ubiquitination of free cyclin D1 is independent of phosphorylation on threonine 286

Cyclin D1 binds and regulates the activity of cyclin-dependent kinases (CDKs) 4 and 6. Phosphorylation of the retinoblastoma protein by cyclin D1.CDK4/6 complexes during the G(1) phase of the cell cycle promotes entry into S phase. Cyclin D1 protein is ubiquitinated and degraded by the 26 S proteasome. Previous studies have demonstrated that cyclin D1 ubiquitination is dependent on its phosphorylation by glycogen synthase kinase 3beta (GSK-3beta) on threonine 286 and that this phosphorylation event is greatly enhanced by binding to CDK4 (Diehl, J. A., Cheng, M. G., Roussel, M. F., and Sherr, C. J. (1998) Genes Dev. 12, 3499-3511). We now report an additional pathway for the ubiquitination of free cyclin D1 (unbound to CDKs). We show that, when unbound to CDK4, a cyclin D1-T286A mutant is ubiquitinated. Further, we show that a mutant of cyclin D1 that cannot bind to CDK4 (cyclin D1-KE) is also ubiquitinated in vivo. Our results demonstrate that free cyclin D1 is ubiquitinated independently of its phosphorylation on threonine 286 by GSK-3beta, suggesting that, as has been shown for cyclin E, distinct pathways of ubiquitination lead to the degradation of free and CDK-bound cyclin D1. The pathway responsible for ubiquitination of free cyclin D1 may be important in limiting the effects of cyclin D1 overexpression in a variety of cancers.     

Cyclin T1 expression is regulated by multiple signaling pathways and mechanisms during activation of human peripheral blood lymphocytes

Stimulation of primary human T lymphocytes results in up-regulation of cyclin T1 expression, which correlates with phosphorylation of the C-terminal domain of RNA polymerase II (RNAP II). Up-regulation of cyclin T1 and concomitant stabilization of cyclin-dependent kinase 9 (CDK9) may facilitate productive replication of HIV in activated T cells. We report that treatment of PBLs with two mitogens, PHA and PMA, results in accumulation of cyclin T1 via distinct mechanisms. PHA induces accumulation of cyclin T1 mRNA and protein, which results from cyclin T1 mRNA stabilization, without significant change in cyclin T1 promoter activity. Cyclin T1 mRNA stabilization requires the activation of both calcineurin and JNK because inhibition of either precludes cyclin T1 accumulation. In contrast, PMA induces cyclin T1 protein up-regulation by stabilizing cyclin T1 protein, apparently independently of the proteasome and without accumulation of cyclin T1 mRNA. This process is dependent on Ca2+-independent protein kinase C activity but does not require ERK1/2 activation. We also found that PHA and anti-CD3 Abs induce the expression of both the cyclin/CDK complexes involved in RNAP II C-terminal domain phosphorylation and the G1-S cyclins controlling cell cycle progression. In contrast, PMA alone is a poor inducer of the expression of G1-S cyclins but often as potent as PHA in inducing RNAP II cyclin/CDK complexes. These findings suggest coordination in the expression and activation of RNAP II kinases by pathways that independently stimulate gene expression but are insufficient to induce S phase entry in primary T cells.     

Kinase-independent function of cyclin E

E-type cyclins are thought to drive cell-cycle progression by activating cyclin-dependent kinases, primarily CDK2. We previously found that cyclin E-null cells failed to incorporate MCM helicase into DNA prereplication complex during G(0) --> S phase progression. We now report that a kinase-deficient cyclin E mutant can partially restore MCM loading and S phase entry in cyclin E-null cells. We found that cyclin E is loaded onto chromatin during G(0) --> S progression. In the absence of cyclin E, CDT1 is normally loaded onto chromatin, whereas MCM is not, indicating that cyclin E acts between CDT1 and MCM loading. We observed a physical association of cyclin E with CDT1 and with MCMs. We propose that cyclin E facilitates MCM loading in a kinase-independent fashion, through physical interaction with CDT1 and MCM. Our work indicates that-in addition to their function as CDK activators-E cyclins play kinase-independent functions in cell-cycle progression.     

Cyclin A-CDK activity during G1 phase impairs MCM chromatin loading and inhibits DNA synthesis in mammalian cells

Progression through the mammalian cell division cycle is regulated by the sequential activation of cyclin-dependent kinases, CDKs, at specific phases of the cell cycle. Cyclin A-CDK2 and cyclin A-CDK1 phosphorylate nuclear substrates during S and G2 phases, respectfully. However, the DNA helicase complex, MCM2-7, is loaded onto the origin of replications in G1, prior to the normally scheduled induction of cyclin A. It has previously been shown that cyclin A-CDKs phosphorylate MCM2 and MCM4 in vitro, thereby diminishing helicase activity. Thus, in this study we hypothesize that, in vivo, cyclin A-CDK activity during G1 would result in an inhibition of progression into the S phase. To test this, we establish an in vivo method of inducing cyclin A-CDK activity in G1 phase and observe that activation of cyclin A-CDK, but not cyclin E-CDK complexes, inhibit DNA synthesis without affecting other G1 events such as cyclin D synthesis, E2F activation and cdc6 loading onto chromatin. We further report that the mechanism of this S phase inhibition occurs, at least in part, through impaired loading of MCM onto chromatin, presumably due to decreased levels of cdt1 and premature phosphorylation of MCM by cyclin A-CDK. In addition to providing in vivo confirmation of in vitro predictions regarding cyclin A-CDK phosphorylation of the MCM complex, our results provide insight into the cellular effects of unscheduled cyclin A-CDK activity in mammalian cells.     

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.     

Human cyclin F.

Cyclins are important regulators of cell cycle transitions through their ability to bind and activate cyclin-dependent protein kinases. In mammals several classes of cyclins exist which are thought to co-ordinate the timing of different events necessary for cell cycle progression. Here we describe the identification of a novel human cyclin, cyclin F, isolated as a suppressor of the G1/S deficiency of a Saccharomyces cerevisiae cdc4 mutant. Cyclin F is the largest cyclin, with a molecular weight of 87 kDa, and migrates as a 100-110 kDa protein. It contains an extensive PEST-rich C-terminus and a cyclin box region that is most closely related to cyclins A and B. Cyclin F mRNA is ubiquitiously expressed in human tissues. It fluctuates dramatically through the cell cycle, peaking in G2 like cyclin A and decreasing prior to decline of cyclin B mRNA. Cyclin F protein accumulates in interphase and is destroyed at mitosis at a time distinct from cyclin B. Cyclin F shows regulated subcellular localization, being localized in the nucleus in most cells, with a significant percentage of cells displaying only perinuclear staining. Overexpression of cyclin F, or a mutant lacking the PEST region, in human cells resulted in a significant increase in the G2 population, implicating cyclin F in the regulation of cell cycle transitions. The ubiquitous expression and phylogentic conservation of cyclin F suggests that it is likely to coordinate essential cell cycle events distinct from those regulated by other cyclins.     

Phosphorylation of mammalian CDC6 by cyclin A/CDK2 regulates its subcellular localization.

Cyclin-dependent kinases (CDKs) are essential for regulating key transitions in the cell cycle, including initiation of DNA replication, mitosis and prevention of re-replication. Here we demonstrate that mammalian CDC6, an essential regulator of initiation of DNA replication, is phosphorylated by CDKs. CDC6 interacts specifically with the active Cyclin A/CDK2 complex in vitro and in vivo, but not with Cyclin E or Cyclin B kinase complexes. The cyclin binding domain of CDC6 was mapped to an N-terminal Cy-motif that is similar to the cyclin binding regions in p21(WAF1/SDI1) and E2F-1. The in vivo phosphorylation of CDC6 was dependent on three N-terminal CDK consensus sites, and the phosphorylation of these sites was shown to regulate the subcellular localization of CDC6. Consistent with this notion, we found that the subcellular localization of CDC6 is cell cycle regulated. In G1, CDC6 is nuclear and it relocalizes to the cytoplasm when Cyclin A/CDK2 is activated. In agreement with CDC6 phosphorylation being specifically mediated by Cyclin A/CDK2, we show that ectopic expression of Cyclin A, but not of Cyclin E, leads to rapid relocalization of CDC6 from the nucleus to the cytoplasm. Based on our data we suggest that the phosphorylation of CDC6 by Cyclin A/CDK2 is a negative regulatory event that could be implicated in preventing re-replication during S phase and G2.     

Novel INK4 proteins, p19 and p18, are specific inhibitors of the cyclin D-dependent kinases CDK4 and CDK6.

Cyclin D-dependent kinases act as mitogen-responsive, rate-limiting controllers of G1 phase progression in mammalian cells. Two novel members of the mouse INK4 gene family, p19 and p18, that specifically inhibit the kinase activities of CDK4 and CDK6, but do not affect those of cyclin E-CDK2, cyclin A-CDK2, or cyclin B-CDC2, were isolated. Like the previously described human INK4 polypeptides, p16INK4a/MTS1 and p15INK4b/MTS2, mouse p19 and p18 are primarily composed of tandemly repeated ankyrin motifs, each ca. 32 amino acids in length, p19 and p18 bind directly to CDK4 and CDK6, whether untethered or in complexes with D cyclins, and can inhibit the activity of cyclin D-bound cyclin-dependent kinases (CDKs). Although neither protein interacts with D cyclins or displaces them from preassembled cyclin D-CDK complexes in vitro, both form complexes with CDKs at the expense of cyclins in vivo, suggesting that they may also interfere with cyclin-CDK assembly. In proliferating macrophages, p19 mRNA and protein are periodically expressed with a nadir in G1 phase and maximal synthesis during S phase, consistent with the possibility that INK4 proteins limit the activities of CDKs once cells exit G1 phase. However, introduction of a vector encoding p19 into mouse NIH 3T3 cells leads to constitutive p19 synthesis, inhibits cyclin D1-CDK4 activity in vivo, and induces G1 phase arrest.     

Human cyclin B3. mRNA expression during the cell cycle and identification of three novel nonclassical nuclear localization signals

Cyclins form complexes with cyclin-dependent kinases. By controlling activity of the enzymes, cyclins regulate progression through the cell cycle. A- and B-type cyclins were discovered due to their distinct appearance in S and G(2) phases and their rapid proteolytic destruction during mitosis. Transition from G(2) to mitosis is basically controlled by B-type cyclins. In mammals, two cyclin B proteins are well characterized, cyclin B1 and cyclin B2. Recently, a human cyclin B3 gene was described. In contrast to the expression pattern of other B-type cyclins, we find cyclin B3 mRNA expressed not only in S and G(2)/M cells but also in G(0) and G(1). Human cyclin B3 is expressed in different variants. We show that one isoform remains in the cytoplasm, whereas the other variant is translocated to the nucleus. Transport to the nucleus is dependent on three autonomous nonclassical nuclear localization signals that where previously not implicated in nuclear translocation. It had been shown that cyclin B3 coimmunoprecipitates with cdk2; but this complex does not exhibit any kinase activity. Furthermore, a degradation-resistant version of cyclin B3 can arrest cells in G(1) and G(2). Taken together with the finding that cyclin B3 mRNA is not only expressed in G(2)/M but is also detected in significant amounts in resting cells and in G(1) cells. This may suggest a dominant-negative function of human cyclin B3 in competition with activating cyclins in G(0) and the G(1) phase of the cell cycle.     

Ultraviolet light induces the sustained unscheduled expression of cyclin E in the absence of functional p53

Cell cycle progression is regulated through changes in the activity of cyclin-dependent kinases that are, in turn, regulated by the expression of their respective cyclin partners. In primary cells, cyclin E expression increases through the G1 phase of the cell cycle and peaks near the G1/S boundary. The unscheduled expression of cyclin E in primary human fibroblasts leads to chromosomal instability that is greatly increased by loss of the p53 tumour suppressor. Intriguingly, ultraviolet light (UV), the most prevalent environmental carcinogen, is similarly known to induce chromosomal instability more dramatically in the absence of p53. Here we report that UV light transiently increased the expression of cyclin E in normal human fibroblasts. Strikingly, cyclin E levels remained elevated for an extended period of time in the absence of functional p53. UV-induced cyclin E expression was not restricted to the G1/S boundary but remained elevated throughout S phase and this correlated with a massive accumulation of p53-deficient fibroblasts in this phase of the cell cycle. Forced expression of cyclin E alone was insufficient to cause a similar S phase arrest but forced expression of cyclin E led to an increase in the proportion of UV-irradiated cells in S phase. The present work suggests that p53 affects S phase progression following UV exposure by preventing the sustained unscheduled expression of cyclin E and that this may limit the clastogenic and carcinogenic effects of UV light.     

Adeno-associated virus type 2 increases proteosome-dependent degradation of p21WAF1 in a human papillomavirus type 31b-positive cervical carcinoma line

Adeno-associated virus type 2 (AAV2) seropositivity is negatively correlated with the development of human papillomavirus (HPV)-associated cervical cancer. We have begun analysis of the molecular mechanisms underlying AAV2-mediated onco-suppression through cell cycle regulation in HPV-infected keratinocytes isolated from a low-grade cervical lesion. AAV2 superinfection of HPV type 31b (HPV31b)-positive cells at early times postinfection resulted in degradation of the cyclin-dependent kinase (CDK) inhibitor p21(WAF1) protein in a proteosome-dependent manner. Downstream consequences of lowering p21(WAF1) levels included a proportional loss of cyclin E/CDK2 complexes bound to p21(WAF1). The loss of stable p21(WAF1)/cyclin E/CDK2 complexes coincided with an increase in CDK2-associated kinase activity and cyclin E levels. Both events have the potential to enhance the G(1)/S transition point mediated by active cyclin E/CDK2 complexes. Concurrently, cyclin A and E2F levels were decreased, conditions reminiscent of delayed entrance into the S phase of the cell cycle. On the other hand, infection of primary human foreskin keratinocytes with AAV2 resulted in upregulation of p21(WAF1) protein levels, reminiscent of a block in G(1) phase progression. We propose that by down regulating p21(WAF1), AAV2 initiates cell cycle activities leading to enhanced G(1)/S phase-like conditions which may be favorable for AAV2-specific functions and may lead to downstream interference with HPV-associated cervical cancer progression.