Cdc42Hs, but not Rac1, inhibits serum-stimulated cell cycle progression at G1/S through a mechanism requiring p38/RK.

Antimitogenic stimuli such as environmental or genotoxic stress, transforming growth factor-beta, and the inflammatory cytokines tumor necrosis factor and interleukin-1 activate two extracellular signal-regulated kinase (ERK)-based signaling pathways: the stress-activated protein kinase (SAPK/JNK) pathway and the p38 pathway. Activated p38 phosphorylates transcription factors important in the regulation of cell growth and apoptosis, including activating transcription factor 2 (ATF2), Max, cAMP response element-binding protein-homologous protein/growth arrest DNA damage 153 (CHDP/GADD153). In turn, p38 lies downstream of the Rho family GTPases Cdc42Hs and Rac1, as well as at least three mitogen-activated protein kinase (MAPK)/ERK-kinases (MEKs): MAPK kinases-3, -6, and SAPK/ERK-kinase-1. Although many of the stimuli that activate p38 can also inhibit cell cycle progression, a clear-cut role for the p38 pathway in cell cycle regulation has not been established. Using a quantitative microinjection approach, we show here that Cdc42Hs, but not Rac1 or RhoA, can inhibit cell cycle progression at G1/S through a mechanism requiring activation of p38. These results suggest a novel role for Cdc42Hs in cell cycle inhibition. Furthermore, these results suggest that although both Cdc42Hs and Rac1 can activate p38 in situ, the effects of Cdc42Hs and Rac1 on cell cycle progression are, in fact, quite distinct.

Kip1 degradation via the ubiquitin-proteasome pathway.

The cell cycle has been the object of extensive studies for the past years. A complex network of molecular interactions has been identified. In particular, a class of cell cycle inhibitory proteins has been identified but details of the molecular mechanism of their action have yet to be resolved. These inhibitors regulate the progression through G1 and the G1/S transition via the inhibition of the cyclin-dependent kinase (Cdk) activity. The potential function of these negative regulators as tumor suppressors provides new insights into the link between the cell cycle and oncogenesis. Kip1 is a potent inhibitor of Cdks. In quiescent cells Kip1 accumulates without an increase in mRNA or protein synthesis. We demonstrated that cell cycle regulation of Kip1 levels, both in normal and transformed human cells, occurs via the ubiquitin-proteasome pathway. In a crude in vitro system, Kip1 is ubiquitinated and degraded in an ATP dependent manner and inhibition or depletion of the proteasome blocks Kip1 degradation. Human Ubc2 and Ubc3, the homologs of yeast Rad6 and Cdc34 gene products respectively, are specifically involved in the ubiquitination of Kip1. Compared to proliferating cells, quiescent cells contain a far lower amount of Kip1 ubiquitinating activity. These results represent the first demonstration that the ubiquitin-proteasome pathway plays a role in the regulation of a cell cycle protein in human cells, namely the Cdk inhibitor Kip1. The specific proteolysis of Kip1 may be involved in the pathway of inactivation of Cdks.

Role of the ubiquitin-proteasome pathway in regulating abundance of the cyclin-dependent kinase inhibitor p27.

The p27 mammalian cell cycle protein is an inhibitor of cyclin-dependent kinases. Both in vivo and in vitro, p27 was found to be degraded by the ubiquitin-proteasome pathway. The human ubiquitin-conjugating enzymes Ubc2 and Ubc3 were specifically involved in the ubiquitination of p27. Compared with proliferating cells, quiescent cells exhibited a smaller amount of p27 ubiquitinating activity, which accounted for the marked increase of p27 half-life measured in these cells. Thus, the abundance of p27 in cells is regulated by degradation. The specific proteolysis of p27 may represent a mechanism for regulating the activity of cyclin-dependent kinases.

Rescue of defective mitogenic signaling by D-type cyclins.

Three gene products, including Myc and the D- and E-type G1 cyclins, are rate limiting for G1 progression in mammalian fibroblasts. Quiescent mouse NIH 3T3 fibroblasts engineered to express a mutant colony-stimulating factor (CSF-1) receptor (CSF-1R 809F) fail to synthesize c-myc and cyclin D1 mRNAs upon CSF-1 stimulation and remain arrested in early G1 phase. Ectopic expression of c-myc or either of three D-type cyclin genes, but not cyclin E, resensitized these cells to the mitogenic effects of CSF-1, enabling them to proliferate continuously in liquid culture and to form colonies in agar in response to the growth factor. Rescue by cyclin D1 was enhanced by c-myc but not by cyclin E and was reversed by infecting cyclin D1-reconstituted cells with a retroviral vector encoding catalytically inactive cyclin-dependent kinase 4. Induction of cyclin D1 mRNA by CSF-1 was restored in cells forced to express c-myc, and vice versa, suggesting that expression of the two genes is interdependent. Cells reconstituted with c-myc were prevented from entering S phase when microinjected with a monoclonal antibody to cyclin D1, and conversely, those rescued by cyclin D1 were inhibited from forming CSF-1-dependent colonies when challenged with a dominant-negative c-myc mutant. Cyclin D mutants defective in binding to the retinoblastoma protein were impaired in rescuing mitogenic signaling. Therefore, Myc and D-type cyclins collaborate during the mitogenic response to CSF-1, whereas cyclin E functions in a separate pathway.

Human cyclin E, a nuclear protein essential for the G1-to-S phase transition.

Cyclin E was first identified by screening human cDNA libraries for genes that would complement G1 cyclin mutations in Saccharomyces cerevisiae and has subsequently been found to have specific biochemical and physiological properties that are consistent with it performing a G1 function in mammalian cells. Most significantly, the cyclin E-Cdk2 complex is maximally active at the G1/S transition, and overexpression of cyclin E decreases the time it takes the cell to complete G1 and enter S phase. We have now found that mammalian cells express two forms of cyclin E protein which differ from each other by the presence or absence of a 15-amino-acid amino-terminal domain. These proteins are encoded by alternatively spliced mRNAs and are localized to the nucleus during late G1 and early S phase. Fibroblasts engineered to constitutively overexpress either form of cyclin E showed elevated cyclin E-dependent kinase activity and a shortened G1 phase of the cell cycle. The overexpressed cyclin E protein was detected in the nucleus during all cell cycle phases, including G0. Although the cyclin E protein could be overexpressed in quiescent cells, the cyclin E-Cdk2 complex was inactive. It was not activated until 6 to 8 h after readdition of serum, 4 h earlier than the endogenous cyclin E-Cdk2. This premature activation of cyclin E-Cdk2 was consistent with the extent of G1 shortening caused by cyclin E overexpression. Microinjection of affinity-purified anti-cyclin E antibodies during G1 inhibited entry into S phase, whereas microinjection performed near the G1/S transition was ineffective. These results demonstrate that cyclin E is necessary for entry into S phase. Moreover, we found that cyclin E, in contrast to cyclin D1, was required for the G1/S transition even in cells lacking retinoblastoma protein function. Therefore, cyclins E and D1 control two different transitions within the human cell cycle.

Differential expression and regulation of Cyclin D1 protein in normal and tumor human cells: association with Cdk4 is required for Cyclin D1 function in G1 progression.

In this study we have surveyed by immunoblotting the protein levels of Cyclin D1, D2, D3 and their catalytic partners, Cdk4 and Cdk6 in normal and transformed human cells. We found that all these proteins were differentially expressed in diploid cells derived from different tissues, in contrast to Cyclin E, Cyclin A and Cdk2 which are ubiquitously expressed. D-type Cyclins were never dramatically overexpressed and often very poorly expressed in tumor cell lines when compared to the levels in their normal counterparts. In contrast, Cdk4 was expressed at high levels in several tumor cell lines and Cdk6 was ectopically expressed in two sarcoma lines, suggesting a possible involvement of these two Cdks in oncogenesis. Interestingly, low levels of Cyclin D1 and D3 proteins always correlated with functional inactivation of the retinoblastoma gene product (pRb). In cells displaying active pRb, Cyclin D1 was found associated with Cdk4 regardless of whether the p53 gene was wild-type or mutant. Microinjection during G1 of Cyclin D1 anti-sense cDNA or anti-Cyclin D1 antibody in these cells arrested the cell cycle in G1. In cells lacking pRb function, Cyclin D1 was dissociated from Cdk4. Microinjection during G1 of Cyclin D1 antisense cDNA or anti-Cyclin D1 antibody in these cells did not affect G1 progression. These results show that (i) in the absence of pRb, Cyclin D1 is expressed at low levels, is dissociated from Cdk4 and becomes dispensable in G1; (ii) Cyclin D1 needs to be associated with its catalytic subunit, Cdk4, to function as a positive regulator of G1 progression.

Cyclin D1-mediated inhibition of repair and replicative DNA synthesis in human fibroblasts.

Cyclin D1 is a key regulator of the G1 phase of the cell cycle. Inhibition of cyclin D1 function results in cell cycle arrest, whereas unregulated expression of the protein accelerates G1. Cyclin D1 is localized to the nucleus during G1. We found that during repair DNA synthesis, subsequent to UV-induced DNA damage, G1 cells readily lost their cyclin D1 while the proliferating cell nuclear antigen (PCNA) tightly associated with nuclear structures. Microinjection of cyclin D1 antisense accelerated DNA repair, whereas overexpression of cyclin D1 prevented DNA repair and the relocation of PCNA after DNA damage. Coexpression of cyclin D1 with its primary catalytic subunit, Cdk4, or with Cdk2, also prevented repair. In contrast, coexpression of PCNA, which is also a cyclin D1-associated protein, restored the ability of cells to repair their DNA. Acute overexpression of cyclin D1 in fibroblasts prevented them from entering S phase. Again, these effects were abolished by coexpression of cyclin D1 together with PCNA, but not with Cdk4 or Cdk2. Altogether, these results indicate that down-regulation of cyclin D1 is necessary for PCNA relocation and repair DNA synthesis as well as for the start of DNA replication. Cyclin D1 appears to be an essential component of a G1-checkpoint.