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1.
Cyclin E2, the cycle continues   总被引:3,自引:0,他引:3  
The eukaryotic cell cycle is regulated by a family of serine/threonine protein kinases known as cyclin-dependent kinases (CDKs). The activation of a CDK is dependent on its association with a cyclin regulatory subunit. The formation of distinct cyclin-CDK complexes controls the progression through the first gap phase (G(1)) and initiation of DNA synthesis (S phase). These complexes are in turn regulated by protein phosphorylation and cyclin-dependent kinase inhibitors (CKIs). Cyclin E2 has emerged as the second member of the E-type cyclin family. Cyclin E2-associated kinase activity is regulated in a cell cycle dependent manner with peak activity at the G(1) to S transition. Ectopic expression of cyclin E2 in human cells accelerates G(1), suggesting that cyclin E2 is rate limiting for G(1) progression. Although the pattern and level of cyclin E2 expression in some primary tumor and normal tissue RNAs are distinct from cyclin E1, both E-type cyclins appear to have inherent functional redundancies. This functional redundancy has facilitated the rapid characterization of cyclin E2 and uncovered unique features associated with each E-type cyclin.  相似文献   

2.
Regulation of Exit from Quiescence by p27 and Cyclin D1-CDK4   总被引:13,自引:9,他引:4       下载免费PDF全文
The synthesis of cyclin D1 and its assembly with cyclin-dependent kinase 4 (CDK4) to form an active complex is a rate-limiting step in progression through the G1 phase of the cell cycle. Using an activated allele of mitogen-activated protein kinase kinase 1 (MEK1), we show that this kinase plays a significant role in positively regulating the expression of cyclin D1. This was found both in quiescent serum-starved cells and in cells expressing dominant-negative Ras. Despite the observation that cyclin D1 is a target of MEK1, in cycling cells, activated MEK1, but not cyclin D1, is capable of overcoming a G1 arrest induced by Ras inactivation. Either wild-type or catalytically inactive CDK4 cooperates with cyclin D1 in reversing the G1 arrest induced by inhibition of Ras activity. In quiescent NIH 3T3 cells expressing either ectopic cyclin D1 or activated MEK1, cyclin D1 is able to efficiently associate with CDK4; however, the complex is inactive. A significant percentage of the cyclin D1-CDK4 complexes are associated with p27 in serum-starved activated MEK1 or cyclin D1 cell lines. Reduction of p27 levels by expression of antisense p27 allows for S-phase entry from quiescence in NIH 3T3 cells expressing ectopic cyclin D1, but not in parental cells.  相似文献   

3.
The Raf family of protein kinases display differences in their abilities to promote the entry of quiescent NIH 3T3 cells into the S phase of the cell cycle. Although conditional activation of deltaA-Raf:ER promoted cell cycle progression, activation of deltaRaf-1:ER and deltaB-Raf:ER elicited a G1 arrest that was not overcome by exogenously added growth factors. Activation of all three deltaRaf:ER kinases led to elevated expression of cyclin D1 and cyclin E and reduced expression of p27Kip1. However, activation of deltaB-Raf:ER and deltaRaf-1:ER induced the expression of p21Cip1, whereas activation of deltaA-Raf:ER did not. A catalytically potentiated form of deltaA-Raf:ER, generated by point mutation, strongly induced p21Cip1 expression and elicited cell cycle arrest similarly to deltaB-Raf:ER and deltaRaf-1:ER. These data suggested that the strength and duration of signaling by Raf kinases might influence the biological outcome of activation of this pathway. By titration of deltaB-Raf:ER activity we demonstrated that low levels of Raf activity led to activation of cyclin D1-cdk4 and cyclin E-cdk2 complexes and to cell cycle progression whereas higher Raf activity elicited cell cycle arrest correlating with p21Cip1 induction and inhibition of cyclin-cdk activity. Using green fluorescent protein-tagged forms of deltaRaf-1:ER in primary mouse embryo fibroblasts (MEFs) we demonstrated that p21Cip1 was induced by Raf in a p53-independent manner, leading to cell cycle arrest. By contrast, activation of Raf in p21Cip1(-/-) MEFs led to a robust mitogenic response that was similar to that observed in response to platelet-derived growth factor. These data indicate that, depending on the level of kinase activity, Raf can elicit either cell cycle progression or cell cycle arrest in mouse fibroblasts. The ability of Raf to elicit cell cycle arrest is strongly associated with its ability to induce the expression of the cyclin-dependent kinase inhibitor p21Cip1 in a manner that bears analogy to alpha-factor arrest in Saccharomyces cerevisiae. These data are consistent with a role for Raf kinases in both proliferation and differentiation of mammalian cells.  相似文献   

4.
D-type cyclins regulate G1 cell cycle progression by enhancing the activities of cyclin-dependent kinases (CDKs), and their expression is frequently altered in malignant cells. We and others have previously shown that cyclin D1 is up-regulated in melanoma cells through adhesion-independent MEK-ERK1/2 signaling initiated by mutant B-RAF. Here, we describe the regulation and role of cyclin D3 in human melanoma cells. Cyclin D3 expression was enhanced in a cell panel of human melanoma cell lines compared with melanocytes and was regulated by fibronectin-mediated phosphatidylinositol 3-kinase/Akt signaling but not MEK activity. RNA interference experiments demonstrated that cyclin D3 contributed to G1-S cell cycle progression and proliferation in melanoma cells. Overexpression of cyclin D1 did not recover the effects of cyclin D3 knockdown. Finally, immunoprecipitation studies showed that CDK6 is a major binding partner for cyclin D3, whereas CDK4 preferentially associated with cyclin D1. Together, these findings demonstrate that cyclin D3 is an important regulator of melanoma G1-S cell cycle progression and that D-type cyclins are differentially regulated in melanoma cells.  相似文献   

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Mechanisms of Cyclin-Dependent Kinase Inactivation by Progestins   总被引:6,自引:2,他引:6       下载免费PDF全文
The steroid hormone progesterone regulates proliferation and differentiation in the mammary gland and uterus by cell cycle phase-specific actions. In breast cancer cells the predominant effect of synthetic progestins is long-term growth inhibition and arrest in G1 phase. Progestin-mediated growth arrest of T-47D breast cancer cells was preceded by inhibition of cyclin D1-Cdk4, cyclin D3-Cdk4, and cyclin E-Cdk2 kinase activities in vitro and reduced phosphorylation of pRB and p107. This was accompanied by decreases in the expression of cyclins D1, D3, and E, decreased abundance of cyclin D1- and cyclin D3-Cdk4 complexes, increased association of the cyclin-dependent kinase (CDK) inhibitor p27 with the remaining Cdk4 complexes, and changes in the molecular masses and compositions of cyclin E complexes. In control cells cyclin E eluted from Superdex 200 as two peaks of ~120 and ~200 kDa, with the 120-kDa peak displaying greater cyclin E-associated kinase activity. Following progestin treatment, almost all of the cyclin E was in the 200-kDa, low-activity form, which was associated with the CDK inhibitors p21 and p27; this change preceded the inhibition of cell cycle progression. These data suggest preferential formation of this higher-molecular-weight, CDK inhibitor-bound form and a reduced number of cyclin E-Cdk2 complexes as mechanisms for the decreased cyclin E-associated kinase activity following progestin treatment. Ectopic expression of cyclin D1 in progestin-inhibited cells led to the reappearance of the 120-kDa active form of cyclin E-Cdk2 preceding the resumption of cell cycle progression. Thus, decreased cyclin expression and consequent increased CDK inhibitor association are likely to mediate the decreases in CDK activity accompanying progestin-mediated growth inhibition.  相似文献   

7.
Cyclin dependent kinases 1 and 2 (CDK1 and CDK2) play crucial roles in regulating cell cycle progression from G1 to S, through S, and G2 to M phase. Both inhibition and aberrant activation of CDK1/2 can be detrimental to cancer cell growth. However, the tools routinely employed to discriminate between the activities of these 2 kinases do not have the selectivity commonly attributed to them. Activation of these kinases is often assayed as a decrease of the inhibitory tyrosine-15 phosphorylation, yet the antibodies used cannot discriminate between phosphorylated CDK1 and CDK2. Inhibitors of these kinases, while partially selective against purified kinases, may lack selectivity when applied to intact cells. High levels of cyclin E are often considered a marker of increased CDK2 activity, yet active CDK2 targets cyclin E for degradation, hence high levels usually reflect inactive CDK2. Finally, inhibition of CDK2 does not arrest cells in S phase suggesting CDK2 is not required for S phase progression. Furthermore, activation of CDK2 in S phase can rapidly induce DNA double-strand breaks in some cell lines. The misunderstandings associated with the use of these tools has led to misinterpretation of results. In this review, we highlight these challenges in the field.  相似文献   

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Cyclin G2, together with cyclin G1 and cyclin I, defines a novel cyclin family expressed in terminally differentiated tissues including brain and muscle. Cyclin G2 expression is up-regulated as cells undergo cell cycle arrest or apoptosis in response to inhibitory stimuli independent of p53 (Horne, M., Donaldson, K., Goolsby, G., Tran, D., Mulheisen, M., Hell, J. and Wahl, A. (1997) J. Biol. Chem. 272, 12650-12661). We tested the hypothesis that cyclin G2 may be a negative regulator of cell cycle progression and found that ectopic expression of cyclin G2 induces the formation of aberrant nuclei and cell cycle arrest in HEK293 and Chinese hamster ovary cells. Cyclin G2 is primarily partitioned to a detergent-resistant compartment, suggesting an association with cytoskeletal elements. We determined that cyclin G2 and its homolog cyclin G1 directly interact with the catalytic subunit of protein phosphatase 2A (PP2A). An okadaic acid-sensitive (<2 nm) phosphatase activity coprecipitates with endogenous and ectopic cyclin G2. We found that cyclin G2 also associates with various PP2A B' regulatory subunits, as previously shown for cyclin G1. The PP2A/A subunit is not detectable in cyclin G2-PP2A-B'-C complexes. Notably, cyclin G2 colocalizes with both PP2A/C and B' subunits in detergent-resistant cellular compartments, suggesting that these complexes form in living cells. The ability of cyclin G2 to inhibit cell cycle progression correlates with its ability to bind PP2A/B' and C subunits. Together, our findings suggest that cyclin G2-PP2A complexes inhibit cell cycle progression.  相似文献   

10.
Cyclic AMP is a second messenger for various hormones that inhibits cell multiplication and DNA synthesis in cultured astrocytes. We examined the effects of increasing intracellular cyclic AMP on the catalytic (cdks) and regulatory (cyclins and ckis) components of cyclin-dependent protein kinases, which regulate progression of the cell cycle before completion of DNA synthesis, in primary cultured astrocytes and in an astrocytic cell line C.LT.T.1.1. The amount of cdk4 changed little during the cell cycle and was not affected by cyclic AMP. There was little cdk1 and cdk2 in quiescent cells, and their expression increased during the G1-S phases. Cyclic AMP strongly inhibited cdk1 and cdk2 expression. Transforming growth factor beta also inhibited cdk1 expression in primary astrocytes. Cyclic AMP did not affect the two ckis p27KIP1 and p21CIP1. There was little cyclin D1 in quiescent cells, but it increased during the G1 phase and was reduced by cyclic AMP. Cyclin E increased during the G1-S phases and was not affected by cyclic AMP in primary astrocytes. The amount of cyclin A was low in quiescent cells and increased during the G1-S phases. Expression of its mRNA and protein was inhibited by cyclic AMP. The protein kinase activities associated with complexes of cyclins and cdks were increased by growth factors and prevented by cyclic AMP. We conclude that cyclic AMP inhibits progression of the cell cycle in astrocytes at least by preventing the expression of the regulatory subunits, cyclins D1 and A, and catalytic subunits, cdk1 and cdk2, of cyclin-regulated protein kinases. Key Words: Cyclin-dependent protein kinases-Glial cells-Cyclic AMP.  相似文献   

11.
BACKGROUND: D cyclins are essential for the progression of cells through the G1 phase of the cell cycle. There are three distinct D cyclins. Cyclin D1 has been shown to be expressed by many different types of cells but not by lymphocytes. Cyclins D2 and D3 have been found in lymphocytes. METHODS: We used high-resolution enzymatic amplification staining technology in conjunction with flow cytometry and confocal microscopy and with immunoblotting to reassess the expression of the D cyclins in human lymphocytes. RESULTS: Using high-resolution technology for flow cytometry, we found all three D cyclins in quiescent human peripheral blood lymphocytes. Cyclin D1 was expressed in quiescent and activated cells at levels commensurate with those of actively proliferating tumor cell lines. Cyclin D1 was functional inasmuch as it was complexed with CDK4. In the quiescent cells, cyclin D1 was expressed in the cytoplasm but, after activation, was found in the nucleus. CONCLUSIONS: These findings demonstrate that lymphocytes express cyclin D1 and necessitate a reappraisal of the hypothesis that the D cyclins subsume redundant activities with tissue-specific expression.  相似文献   

12.
Ordered cell cycle progression requires the expression and activation of several cyclins and cyclin-dependent kinases (Cdks). Hyperosmotic stress causes growth arrest possibly via proteasome-mediated degradation of cyclin D1. We studied the effect of hyposmotic conditions on three colonic (Caco2, HRT18, HT29) and two pancreatic (AsPC-1 and PaCa-2) cell lines. Hyposmosis caused reversible cell growth arrest of the five cell lines in a cell cycle-independent fashion, although some cell lines accumulated at the G(1)/S interface. Growth arrest was followed by apoptosis or by formation of multinucleated giant cells, which is consistent with cell cycle catastrophe. Hyposmosis dramatically decreased Cdc2, Cdk2, Cdk4, cyclin B1, and cyclin D3 expression in a time-dependent fashion, in association with an overall decrease in cellular protein synthesis. However, some protein levels remained unaltered, including cyclin E and keratin 8. Selective proteasome inhibition prevented Cdk and cyclin degradation and reversed hyposmotic stress-induced growth arrest, whereas calpain and lysosome enzyme inhibitors had no measurable effect on cell cycle protein degradation. Therefore, hyposmotic stress inhibits cell growth and, depending on the cell type, causes cell cycle catastrophe with or without apoptosis. The growth arrest is due to decreased protein synthesis and proteasome activation, with subsequent degradation of several cyclins and Cdks.  相似文献   

13.
The present study examines the molecular mechanisms by which a member of a novel series of pyrrolo-1,5-benzoxazepines, PBOX-21, induces G1 arrest in 1321N1 cells. PBOX-21-induced G1 arrest is preceded by both a decrease in CDK2 kinase activity, which is critical for the G1/S transition, and a downregulation in cyclin D(3) protein expression levels, suggesting that these two events may be crucially involved in the mediation of the cell cycle arrest. The decrease in CDK2 activity may be due to an observed decrease in CDK2 protein levels following PBOX-21 treatment. Coinciding with the arrest is a reduction in the activity of CDK4, due to either the observed PBOX-21 induced downregulation in CDK4 expression, or a reduction in complex formation between cyclin D(3)-CDK4 leading to a decrease in the levels of active cyclin D(3)-CDK4 complexes with kinase activity. The level of CDK6 activity was also seen to be reduced following PBOX-21 treatment, also possibly due to a reduction in complex formation with cyclin D(3). However, this reduction in CDK6 kinase activity was not seen until after PBOX-21-induced G1 arrest has reached its maximum, and therefore may be viewed as a consequence of, and a method of maintaining the PBOX-21-induced arrest, rather than a cause. Also in parallel with the G1 arrest elicited by PBOX-21 is an upregulation in the universal CDK inhibitor, p21. Furthermore, the retinoblastoma protein (Rb), a substrate of CDK2 and CDK6, whose phosphorylation is necessary for cell cycle progression, becomes hypophosphorylated. These results indicate that PBOX-21 exerts its growth inhibitory effects through the modulation of the expression and activity of several key G1 regulatory proteins.  相似文献   

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15.
Cell cycle control is regulated through the temporal action of both cyclin-dependent kinases and cyclin binding partners. Previously, we have demonstrated that low doses of oligomycin result in a cell cycle arrest of HL-60 cells in G(1) [S. Sweet, G. Singh, Accumulation of human promyelocytic leukemic (HL-60) cells at two energetic cell cycle checkpoints, Cancer Res. 55 (1995) 5164-5167]. In this study, we provide the molecular mechanisms for the observed G(1) arrest following mitochondrial ATPase inhibition. Protein expression of cyclin E and CDK2, the kinase activity of complexed cyclin E/CDK2, and protein expression of p16, p21, and p27 were all unaffected by oligomycin administration. While CDK4 levels were unchanged following oligomycin treatment, a dramatic reduction in cyclin D(1) was observed. Moreover, increased amounts of hypo-phosphorylated retinoblastoma protein (Rbp) and Rbp bound E2F were observed following mitochondrial ATP synthase inhibition. These data provide further evidence that surveillance of available energy occurs during G(1) and ATP deprivation results in cell cycle arrest via a reduction in cyclin D.  相似文献   

16.
Cyclin G2 is an atypical cyclin that associates with active protein phosphatase 2A. Cyclin G2 gene expression correlates with cell cycle inhibition; it is significantly upregulated in response to DNA damage and diverse growth inhibitory stimuli, but repressed by mitogenic signals. Ectopic expression of cyclin G2 promotes cell cycle arrest, cyclin dependent kinase 2 inhibition and the formation of aberrant nuclei [Bennin, D. A., Don, A. S., Brake, T., McKenzie, J. L., Rosenbaum, H., Ortiz, L., DePaoli-Roach, A. A., and Horne, M. C. (2002). Cyclin G2 associates with protein phosphatase 2A catalytic and regulatory B' subunits in active complexes and induces nuclear aberrations and a G(1)/S-phase cell cycle arrest. J Biol Chem 277, 27449-67]. Here we report that endogenous cyclin G2 copurifies with centrosomes and microtubules (MT) and that ectopic G2 expression alters microtubule stability. We find exogenous and endogenous cyclin G2 present at microtubule organizing centers (MTOCs) where it colocalizes with centrosomal markers in a variety of cell lines. We previously reported that cyclin G2 forms complexes with active protein phosphatase 2A (PP2A) and colocalizes with PP2A in a detergent-resistant compartment. We now show that cyclin G2 and PP2A colocalize at MTOCs in transfected cells and that the endogenous proteins copurify with isolated centrosomes. Displacement of the endogenous centrosomal scaffolding protein AKAP450 that anchors PP2A at the centrosome resulted in the depletion of centrosomal cyclin G2. We find that ectopic expression of cyclin G2 induces microtubule bundling and resistance to depolymerization, inhibition of polymer regrowth from MTOCs and a p53-dependent cell cycle arrest. Furthermore, we determined that a 100 amino acid carboxy-terminal region of cyclin G2 is sufficient to both direct GFP localization to centrosomes and induce cell cycle inhibition. Colocalization of endogenous cyclin G2 with only one of two GFP-centrin-tagged centrioles, the mature centriole present at microtubule foci, indicates that cyclin G2 resides primarily on the mother centriole. Copurification of cyclin G2 and PP2A subunits with microtubules and centrosomes, together with the effects of ectopic cyclin G2 on cell cycle progression, nuclear morphology and microtubule growth and stability, suggests that cyclin G2 may modulate the cell cycle and cellular division processes through modulation of PP2A and centrosomal associated activities.  相似文献   

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PTEN is a tumor suppressor frequently inactivated in brain, prostate, and uterine cancers that acts as a phosphatase on phosphatidylinositol-3,4,5-trisphosphate, antagonizing the activity of the phosphatidylinositol 3'-OH kinase. PTEN manifests its tumor suppressor function in most tumor cells by inducing G(1)-phase cell cycle arrest. To study the mechanism of cell cycle arrest, we established a tetracycline-inducible expression system for PTEN in cell lines lacking this gene. Expression of wild-type PTEN but not of mutant forms unable to dephosphorylate phosphoinositides reduced the expression of cyclin D1. Cyclin D1 reduction was accompanied by a marked decrease in endogenous retinoblastoma (Rb) protein phosphorylation on cyclin D/CDK4-specific sites, showing an early negative effect of PTEN on Rb inactivation. PTEN expression also prevented cyclin D1 from localizing to the nucleus during the G(1)- to S-phase cell cycle transition. The PTEN-induced localization defect and the cell growth arrest could be rescued by the expression of a nucleus-persistent mutant form of cyclin D1, indicating that an important effect of PTEN is at the level of nuclear availability of cyclin D1. Constitutively active Akt/PKB kinase counteracted the effect of PTEN on cyclin D1 translocation. The data are consistent with an oncogenesis model in which a lack of PTEN fuels the cell cycle by increasing the nuclear availability of cyclin D1 through the Akt/PKB pathway.  相似文献   

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