Cell cycle exit during terminal erythroid differentiation is associated with accumulation of p27(Kip1) and inactivation of cdk2 kinase.
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Progression through the mammalian cell cycle is regulated by cyclins, cyclin- dependent kinases (CDKs), and cyclin-dependent kinase inhibitors (CKIs). The function of these proteins in the irreversible growth arrest associated with terminally differentiated cells is largely unknown. The function of Cip/Kip proteins p21(Cip1) and p27(Kip1) during erythropoietin-induced terminal differentiation of primary erythroblasts isolated from the spleens of mice infected with the anemia-inducing strain of Friend virus was investigated. Both p21(Cip1) and p27(Kip1) proteins were induced during erythroid differentiation, but only p27(Kip1) associated with the principal G(1) CDKs-cdk4, cdk6, and cdk2. The kinetics of binding of p27(Kip1) to CDK complexes was distinct in that p27(Kip1) associated primarily with cdk4 (and, to a lesser extent, cdk6) early in differentiation, followed by subsequent association with cdk2. Binding of p27(Kip1) to cdk4 had no apparent inhibitory effect on cdk4 kinase activity, whereas inhibition of cdk2 kinase activity was associated with p27(Kip1) binding, accumulation of hypo-phosphorylated retinoblastoma protein, and G(1) growth arrest. Inhibition of cdk4 kinase activity late in differentiation resulted from events other than p27(Kip1) binding or loss of cyclin D from the complex. The data demonstrate that p27(Kip1) differentially regulates the activity of cdk4 and cdk2 during terminal erythroid differentiation and suggests a switching mechanism whereby cdk4 functions to sequester p27(Kip1) until a specified time in differentiation when cdk2 kinase activity is targeted by p27(Kip1) to elicit G(1) growth arrest. Further, the data imply that p21(Cip1) may have a function independent of growth arrest during erythroid differentiation. (Blood. 2000;96:2746-2754) (+info)
Differential expression of cell-cell adhesion proteins and cyclin D in MEK1-transdifferentiated MDCK cells.
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Overexpression of a constitutively active mutant of the mitogen-activated protein kinase kinase MEK1 (caMEK1) in epithelial Madin-Darby canine kidney (MDCK)-C7 cells disrupts morphogenesis, induces an invasive phenotype, and is associated with a reduced rate of cell proliferation. The role of cell-cell adhesion molecules and cell cycle proteins in these processes, however, has not been investigated. We now report loss of E-cadherin expression as well as a marked reduction of beta- and alpha-catenin expression in transdifferentiated MDCK-C7 cells stably expressing caMEK1 (C7caMEK1) compared with epithelial mock-transfected MDCK-C7 (C7Mock1) cells. At least part of the remaining alpha-catenin was coimmunoprecipitated with beta-catenin, whereas no E-cadherin was detected in beta-catenin immunoprecipitates. In both cell types, the proteasome-specific protease inhibitors N-acetyl-Leu-Leu-norleucinal (ALLN) and lactacystin led to a time-dependent accumulation of beta-catenin, including the appearance of high-molecular-weight beta-catenin species. Quiescent as well as serum-stimulated C7caMEK1 cells showed a higher cyclin D expression than epithelial C7Mock1 cells. The MEK inhibitor U-0126 inhibited extracellular signal-regulated kinase phosphorylation and cyclin D expression in C7caMEK1 cells and almost abolished their already reduced cell proliferation rate. We conclude that the transdifferentiated and invasive phenotype of C7caMEK1 cells is associated with a diminished expression of proteins involved in cell-cell adhesion. Although beta-catenin expression is reduced, C7caMEK1 cells show a higher expression of U-0126-sensitive cyclin D protein. (+info)
Protein kinase C signaling mediates a program of cell cycle withdrawal in the intestinal epithelium.
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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. (+info)
Hypoxia inhibits G1/S transition through regulation of p27 expression.
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Mammalian cellular responses to hypoxia include adaptive metabolic changes and a G1 cell cycle arrest. Although transcriptional regulation of metabolic genes by the hypoxia-induced transcription factor (HIF-1) has been established, the mechanism for the hypoxia-induced G1 arrest is not known. By using genetically defined primary wild-type murine embryo fibroblasts and those nullizygous for regulators of the G1/S checkpoint, we observed that the retinoblastoma protein is essential for the G1/S hypoxia-induced checkpoint, whereas p53 and p21 are not required. In addition, we found that the cyclin-dependent kinase inhibitor p27 is induced by hypoxia, thereby inhibiting CDK2 activity and forestalling S phase entry through retinoblastoma protein hypophosphorylation. Reduction or absence of p27 abrogated the hypoxia-induced G1 checkpoint, suggesting that it is a key regulator of G1/S transition in hypoxic cells. Intriguingly, hypoxic induction of p27 appears to be transcriptional and through an HIF-1-independent region of its proximal promoter. This demonstration of the molecular mechanism of hypoxia-induced G1/S regulation provides insight into a fundamental response of mammalian cells to low oxygen tension. (+info)
Phosphorylation-dependent and -independent functions of p130 cooperate to evoke a sustained G1 block.
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The retinoblastoma (pRb)-related p130 pocket protein is a regulator of cell growth and differentiation, and a candidate tumour suppressor. Both pRb and p130 operate through interactions with cellular proteins, including the E2F transcription factors. While such interactions are controlled by phosphorylation of multiple sites of pRb, regulation of p130 remains poorly understood. We now identify 22 in vivo phosphorylation sites of p130, targeted by diverse kinases, and present evidence for three cyclin-dependent kinase 4(6) [Cdk4(6)] specific phosphorylations, which appear critical for controlling the growth-restraining activity of p130. When expressed in U2OS cells, the phosphorylation-deficient mutant p130(Delta)(CDK4), in which the Cdk4 specific sites were mutated to alanine residues, imposed a more sustained G1 arrest than a constitutively active pRb(Delta)(CDK), known to repress all cellular E2F activity. Experiments using p130(Delta)(Cdk4) and another phosphorylation-deficient mutant, p130(PM19A), with 19 phosphorylation sites mutated, revealed that the p130-imposed G1 block reflects cooperative growth-suppressive effects of phosphorylation-regulated E2F binding and phosphorylation-independent sequestration of cyclin E(A)-Cdk2 through the N-terminal cyclin binding motif of p130. (+info)
The midblastula transition in Xenopus embryos activates multiple pathways to prevent apoptosis in response to DNA damage.
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Apoptosis is controlled by a complex interplay between regulatory proteins. Previous work has shown that Xenopus embryos remove damaged cells by apoptosis when irradiated before, but not after, the midblastula transition (MBT). Here we demonstrate that Akt/protein kinase B is activated and mediates an antiapoptotic signal only in embryos irradiated after the MBT. In addition, an increase in xBcl-2/xBax oligomerization and a decrease in xBax homodimerization promote a protective effect against apoptosis only after the MBT. The post-MBT survival mechanism arrests cells in G(1) phase by increasing expression of the cyclin-dependent kinase inhibitor p27(Xic1). p27(Xic1) associates with cyclin D/Cdk4 and cyclin A/Cdk2 complexes to cause G(1)/S arrest, perhaps allowing more time for DNA repair. Taken together, the results define the DNA damage response as an element of the MBT and indicate that multiple mechanisms prevent apoptosis after the MBT. (+info)
Inhibition of cyclin E-cyclin-dependent kinase 2 complex formation and activity is associated with cell cycle arrest and withdrawal in oligodendrocyte progenitor cells.
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Stimulatory and inhibitory signals regulate cell proliferation through the activity of specific enzymes that operate in distinct phases of the cell cycle. We have studied cell cycle progression, arrest, and withdrawal in the oligodendrocyte progenitor (OP) cell model system, focusing on the G(1) phase and G(1)-S transition. Not only were proliferating OPs found to display higher protein levels of cyclin E and D and cyclin-dependent kinases (cdk) 2, 4, and 6 than cells that had permanently withdrawn from the cycle, but the kinase activities of both cyclin D-cdk4/6 and cyclin E-cdk2 were also higher in dividing OPs. This was associated with a decrease in the formation of the cyclin E-cdk2 and cyclin D-cdk4/cyclin D-cdk6 complexes in differentiated oligodendrocytes that had permanently withdrawn from the cell cycle. Reversible cell cycle arrest in G(1) induced by glutamatergic and beta-adrenergic receptor activation or cell depolarization, however, did not modify cyclin E and cdk2 protein expression compared with proliferating OPs. Instead, these agents caused a selective decrease in cdk2 activity and an impairment of cyclin E-cdk2 complex formation. Although cyclin D protein levels were higher than in proliferating cells, cyclin D-associated kinase activity was not modified in G(1)-arrested OPs. Analysis in corpus callosum in vivo showed that cyclin E-cdk2 activity increased between postnatal days 3 and 15 and decreased between postnatal days 15 and 30. Our results indicate that the cyclin E-cdk2 complex is a major regulator of OP cell cycle progression and that the cdks involved in reversible cell cycle arrest are distinct from those implicated in permanent cell cycle withdrawal. (+info)
Independence of herpesvirus-induced T cell lymphoma from viral cyclin D homologue.
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Cyclin D family members are cellular protooncogenes, and their viral homologues in the Kaposi's sarcoma-associated herpesvirus (KSHV, human herpesvirus type 8 [HHV-8]) and the closely related Herpesvirus saimiri have been implicated as putative cofactors of viral transformation and pathogenesis. KSHV is regularly found in Kaposi's sarcoma and in the primary effusion B cell lymphoma and Castleman's disease associated with immunosuppression and AIDS. H. saimiri strain C488 transforms human and marmoset T cells in vitro and causes polyclonal T cell lymphoma in New World monkeys. The viral cyclins stimulate cell cycle progression of quiescent fibroblasts, and they form active cyclin-dependent kinase (CDK)6 complexes of broad substrate specificity that can resist and downregulate cellular CDK inhibitors. This study shows that the viral cyclin of H. saimiri strain C488 is not required for viral replication, T cell transformation, and pathogenicity in New World primates. (+info)