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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The cyclin D-dependent kinases CDK4 and CDK6 trigger phosphorylation of the retinoblastoma protein (RB) late in G1 phase, helping to cancel its growth-suppressive function and thereby facilitating S-phase entry. Although specific inhibition of cyclin D-dependent kinase activity in vivo can prevent cells from entering S phase, it does not affect S-phase entry in cells lacking functional RB, implying that RB may be the only substrate of CDK4 and CDK6 whose phosphorylation is necessary for G1 exit. Using a yeast two-hybrid interactive screen, we have now isolated a novel cyclin D-interacting myb-like protein (designated DMP1), which binds specifically to the nonamer DNA consensus sequences CCCG(G/T)ATGT to activate transcription. A subset of these DMP1 recognition sequences containing a GGA trinucleotide core can also function as Ets-responsive elements. DMP1 mRNA and protein are ubiquitously expressed throughout the cell cycle in mouse tissues and in representative cell lines. DMP1 binds to D-type cyclins directly in vitro and when coexpressed in insect Sf9 cells. In both settings, it can be phosphorylated by cyclin D-dependent kinases, suggesting that its transcriptional activity may normally be regulated through such mechanisms. These results raise the possibility that cyclin D-dependent kinases regulate gene expression in an RB independent manner, thereby serving to link other genetic programs to the cell cycle clock.
Mol Cell Biol 1996 Nov
PMID:Interaction of D-type cyclins with a novel myb-like transcription factor, DMP1. 888 74

Terminal cell differentiation involves permanent withdrawal from the cell division cycle. The inhibitors of cyclin-dependent kinases (CDKs) are potential molecules functioning to couple cell cycle arrest and cell differentiation. In murine C2C12 myoblast cells, G1 CDK enzymes (CDK2, CDK4, and CDK6) associate with four CDK inhibitors: p18INK4c, p19INK4d, p21, and p27Kip1. During induced myogenesis, p21 and its associated CDK proteins underwent an initial increase followed by a decrease as cells became terminally differentiated. The level of p27 protein gradually increased, but the amount of total associated CDK proteins remained unchanged. p19 protein decreased gradually during differentiation, as did its associated CDK4 protein. In contrast, p18 protein increased 50-fold, from negligible levels in proliferating myoblasts to clearly detectable levels within 8-12 h of myogenic induction. This initial rise was followed by a precipitous increase between 12 and 24 h postinduction, with p18 protein finally accumulating to its highest level in terminally differentiated cells. Induction of p18 correlated with increased and sequential complex formation--first increasing association with CDK6 and then with CDK4 over the course of myogenic differentiation. All of the CDK6 and half of the CDK4 were complexed with p18 in terminally differentiated C2C12 cells as well as in adult mouse muscle tissue. Finally, kinase activity of CDK2 and CDK4 decreases as C2C12 cells differentiate, whereas the CDK6 kinase activity is low in both proliferating myoblasts and differentiated myotubes. Our results indicate that p18 may play a critical role in causing and/or maintaining permanent cell cycle arrest associated with mature muscle formation.
Mol Biol Cell 1996 Oct
PMID:Induction of p18INK4c and its predominant association with CDK4 and CDK6 during myogenic differentiation. 889 64

Glucocorticoids inhibit proliferation of many cell types, but the events leading from the activated glucocorticoid receptor (GR) to growth arrest are not understood. Ectopic expression and activation of GR in human osteosarcoma cell lines U2OS and SAOS2, which lack endogenous receptors, result in a G1 cell cycle arrest. GR activation in U2OS cells represses expression of the cyclin-dependent kinases (CDKs) CDK4 and CDK6 as well as their regulatory partner, cyclin D3, leading to hypophosphorylation of the retinoblastoma protein (Rb). We also demonstrate a ligand-dependent reduction in the expression of E2F-1 and c-Myc, transcription factors involved in the G1-to-S-phase transition. Mitogen-activated protein kinase, CDK2, cyclin E, and the CDK inhibitors (CDIs) p27 and p21 are unaffected by receptor activation in U2OS cells. The receptor's N-terminal transcriptional activation domain is not required for growth arrest in U2OS cells. In Rb-deficient SAOS2 cells, however, the expression of p27 and p21 is induced upon receptor activation. Remarkably, in SAOS2 cells that express a GR deletion derivative lacking the N-terminal transcriptional activation domain, induction of CDI expression is abolished and the cells fail to undergo ligand-dependent cell cycle arrest. Similarly, murine S49 lymphoma cells, which, like SAOS2 cells, lack Rb, require the N-terminal activation domain for growth arrest and induce CDI expression upon GR activation. These cell-type-specific differences in receptor domains and cellular targets linking GR activation to cell cycle machinery suggest two distinct regulatory mechanisms of GR-mediated cell cycle arrest: one involving transcriptional repression of G1 cyclins and CDKs and the other involving enhanced transcription of CDIs by the activated receptor.
Mol Cell Biol 1997 Jun
PMID:Glucocorticoid receptor-mediated cell cycle arrest is achieved through distinct cell-specific transcriptional regulatory mechanisms. 915 17

Activins, members of the transforming growth factor-beta family, have been implicated in the regulation of growth and differentiation of various types of cells. We have recently found that activin A induces apoptotic cell death of plasmacytic cells including B cell hybridoma cells and myeloma cells. In the present study, we demonstrated that activin A caused cell-cycle arrest in the G1 phase before appearance of apoptotic cells in mouse B cell hybridoma cells. Phosphorylation of retinoblastoma protein (Rb) and in vitro Rb kinase activity of cyclin-dependent kinase (CDK)4 was inhibited in activin A-treated cells. Analysis of expression of genes regulating Rb phosphorylation revealed that activin A suppressed cyclin D2, the sole D-type cyclin gene expressed in the hybridoma cells, and activated p21CIP1/WAF1 but had no effect on expression of cyclin-dependent kinases (CDK2, CDK4, CDK6) and other CDK inhibitors (p27KIP1, p16INK4a, p15INK4b). Modulation of cyclin D2 and p21CIP1/WAF1 expression resulted in a decrease in level of cyclin D2-CDK4 complex and an increase in level of CDK4 complexed with p21CIP1/WAF1. Moreover, overexpression of cyclin D2 partially abrogated inhibition of Rb phosphorylation and G1 arrest in the hybridoma cells.
Mol Endocrinol 1997 Jul
PMID:Activin A induction of cell-cycle arrest involves modulation of cyclin D2 and p21CIP1/WAF1 in plasmacytic cells. 921 52

The molecular mechanisms that arrest cardiomyocytes in the cell cycle during the postnatal period remain largely unknown. We have examined changes of the expression of cyclins and CDKs, the activity of each CDK in cardiomyocytes during the postnatal period, and have compared those changes with rate of binucleation formation of cardiomyocytes in rats. The mRNA and protein levels of cyclin D1, A and B in cardiomyocytes were high at day 1, then the levels decreased at different rates during the postnatal period. While the protein levels of cyclin A and B rapidly decreased, the protein level of cyclin D1 was relatively constant. The protein levels of CDK4, CDK2, and cdc2 in cardiomyocytes were high at day 1, then their levels gradually decreased. However, the activity of CDK4, which is responsible for G1 phase of cell cycle, was detectable only at day 1. The activity of CDK2 activity, which is responsible for the S phase of cell cycle, was relatively high at day 1, decreased at day 2, abruptly decreased at day 4, maintained the same low level until day 10, and barely or not detectable thereafter in cardiomyocytes. The activity of cdc2 was high at day 1, increased by 20% at day 2, and then gradually decreased thereafter, although approximately 50% of maximum activity was present at day 6. Most cardiomyocytes were mononucleated during the first 2 days postnatal. The percentage of binucleated cardiomyocytes increased from 2.5% at day 2, 14% at day 4, 50% at day 8, 80% at day 14, and had reached adult levels at day 21 after birth. During active binucleation formation in neonatal (from days 1-14) cardiomyocytes, CDK4 or CDK2 was functionally negligible, while cdc2 was functionally active. These data suggest that there were differential and dramatic decrease of CDK4 and CDK2 activities in cardiomyocytes during neonatal period, and the functionally active cdc2 in neonatal cardiomyocytes may be involved in binucleation formation.
J Mol Cell Cardiol 1997 Jul
PMID:Differential and dramatic changes of cyclin-dependent kinase activities in cardiomyocytes during the neonatal period. 1160 19

The molecular mechanisms that regulate the cardiomyocyte cell cycle and its terminal differentiation remain largely unknown. To determine which cyclins or cyclin dependent kinases (CDKs) are important for cardiomyocyte proliferation, we examined the expression of cyclins and CDKs during normal cardiac development. All cyclins and CDKs were highly expressed during embryonic cardiac development, then they decreased at different rates after birth. The mRNAs and proteins of cyclins A and B (G2 and M phase cyclins) were found in embryonic and neonatal hearts, but were not detected in young or adult hearts. In contrast, while the mRNAs of cyclins D1, D2, D3, and E (G1 and S phase cyclins) were observed during all stages of development, the proteins of cyclins D1, D3, and E were observed in hearts at the young growth stage, although the levels decreased differently. Reverse transcriptase-polymerase chain reaction (RT-PCR) using specific cyclin B and D3 primers revealed that cyclins B and D3 originated from cardiomyocytes and noncardiomyocytes. The CDKs (cdc2, CDK2, and CDK4) were highly expressed during embryonic cardiac development and maintained almost constant levels during neonatal periods. However, they were expressed at very low levels at the young and adult stages. The pattern of proliferating cell nuclear antigen (PCNA) expression during cardiac development was similar to the expression of CDKs. These findings suggest that all cyclins and CDKs are involved in the cardiac cell cycle, and that marked and rapid reduction of mitotic cyclins may be associated with the withdrawal of the cardiac cell cycle after birth.
Mol Cells 1997 Jun 30
PMID:Cyclins and cyclin dependent kinases during cardiac development. 926 23

It is well documented that Ras functions as a molecular switch for reentry into the cell cycle at the border between G0 and G1 by transducing extracellular growth stimuli into early G1 mitogenic signals. In the present study, we investigated the role of Ras during the late stage of the G1 phase by using NIH 3T3 (M17) fibroblasts in which the expression of a dominant negative Ras mutant, p21(Ha-Ras[Asn17]), is induced in response to dexamethasone treatment. We found that delaying the expression of Ras(Asn17) until late in the G1 phase by introducing dexamethasone 3 h after the addition of epidermal growth factor (EGF) abolished the downregulation of the p27kip1 cyclin-dependent kinase (CDK) inhibitor which normally occurred during this period, with resultant suppression of cyclin Ds/CDK4 and cyclin E/CDK2 and G1 arrest. The immunodepletion of p27kip1 completely eliminated the CDK inhibitor activity from EGF-stimulated, dexamethasone-treated cell lysate. The failure of p27kip1 downregulation and G1 arrest was also observed in cells in which Ras(Asn17) was induced after growth stimulation with a phorbol ester or alpha-thrombin and was mimicked by the addition late in the G1 phase of inhibitors for phosphatidylinositol-3-kinase. Ras-mediated downregulation of p27kip1 involved both the suppression of synthesis and the stimulation of the degradation of the protein. Unlike the earlier expression of Ras(Asn17) at the border between G0 and G1, its delayed expression did not compromise the EGF-stimulated transient activation of extracellular signal-regulated kinases or inhibit the stimulated expression of a principal D-type cyclin, cyclin D1, until close to the border between G1 and S. We conclude that Ras plays temporally distinct, phase-specific roles throughout the G1 phase and that Ras function late in G1 is required for p27kip1 downregulation and passage through the restriction point, a prerequisite for entry into the S phase.
Mol Cell Biol 1997 Sep
PMID:Ras activity late in G1 phase required for p27kip1 downregulation, passage through the restriction point, and entry into S phase in growth factor-stimulated NIH 3T3 fibroblasts. 927 12

The molecular mechanisms responsible for the alterations in proliferative capacity of cardiac myocytes during development remain unknown; however, cell cycle dependent molecules may be involved. We have determined the expression of cyclins A, D1-3 and E, and cyclin-dependent kinases (CDKs) 2, 4, 5 and 6 and cdc2 in freshly isolated rat cardiac myocytes from fetal (18 days gestation), neonatal (2 days post-natal) and adult animals by immunoblotting. Our results show a dramatic decrease in expression of these proteins during normal cardiac development, such that levels are highest in fetal myocytes but are significantly down-regulated in adult cells (P<0.05, in each case). We also have determined the in vitro kinase activities of cdc2, CDK2, CDK4, CDK5 and CDK6 immunocomplexes in fetal, neonatal and adult myocytes. There was a consistent and significant loss of cdc2, CDK2, CDK4 and CDK6 kinase activities in adult cardiac cell lysates (5.3-, 10.6-, 1.5- and 1.9-fold decreases, respectively) when compared to neonatal samples (P<0.05); CDK5 activity showed a similar trend but failed to reach significance. In conclusion, our results show that the expression and activities of various positive regulators of the cell cycle are down-regulated significantly during development of the cardiac myocyte, concomitant with the loss of proliferative capacity in adult myocytes. Down-regulation of these proteins may be pivotal in the withdrawal of the cardiac myocyte from the cell cycle.
J Mol Cell Cardiol 1997 Aug
PMID:Expression and activities of cyclins and cyclin-dependent kinases in developing rat ventricular myocytes. 928 57

Germline mutations in CDKN2 on chromosome 9p21, which codes for the cyclin D kinase inhibitor p16, and more rarely, mutations in the gene coding for CDK4, the protein to which p16 binds, underlie susceptibility in some melanoma families. We have sequenced all exons of CDKN2 and analysed the CDK4 gene for mutations in 27 UK families showing evidence of predisposition to melanoma. Five different germline mutations in CDKN2 were found in six families. Three of the mutations (Met53Ile, Arg24Pro and 23ins24) have been reported previously. We have identified two novel CDKN2 mutations (88delG and Ala118Thr) which are likely to be associated with the development of melanoma, because of their co-segregation with the disease and their likely functional effect on the CDKN2 protein. In binding assays the protein expressed from the previously described mutation, Met53Ile, did not bind to CDK4/CDK6, confirming its role as a causal mutation in the development of melanoma. Ala118Thr appeared to be functional in this assay. Arg24Pro appeared to bind to CDK6, but not to CDK4. No mutations were detected in exon 2 of CDK4, suggesting that causal mutations in this gene are uncommon. The penetrance of these mutant CDKN2 genes is not yet established, nor is the risk of non-melanoma cancer to gene carriers.
Hum Mol Genet 1997 Nov
PMID:Germline mutations of the CDKN2 gene in UK melanoma families. 932 69

Cyclins contain two characteristic cyclin folds, each consisting of five alpha-helical bundles, which are connected to one another by a short linker peptide. The first repeat makes direct contact with cyclin-dependent kinase (CDK) subunits in assembled holoenzyme complexes, whereas the second does not contribute directly to the CDK interface. Although threonine 156 in mouse cyclin D1 is predicted to lie at the carboxyl terminus of the linker peptide that separates the two cyclin folds and is buried within the cyclin subunit, mutation of this residue to alanine has profound effects on the behavior of the derived cyclin D1-CDK4 complexes. CDK4 in complexes with mutant cyclin D1 (T156A or T156E but not T156S) is not phosphorylated by recombinant CDK-activating kinase (CAK) in vitro, fails to undergo activating T-loop phosphorylation in vivo, and remains catalytically inactive and unable to phosphorylate the retinoblastoma protein. Moreover, when it is ectopically overexpressed in mammalian cells, cyclin D1 (T156A) assembles with CDK4 in the cytoplasm but is not imported into the cell nucleus. CAK phosphorylation is not required for nuclear transport of cyclin D1-CDK4 complexes, because complexes containing wild-type cyclin D1 and a CDK4 (T172A) mutant lacking the CAK phosphorylation site are efficiently imported. In contrast, enforced overexpression of the CDK inhibitor p21Cip1 together with mutant cyclin D1 (T156A)-CDK4 complexes enhanced their nuclear localization. These results suggest that cyclin D1 (T156A or T156E) forms abortive complexes with CDK4 that prevent recognition by CAK and by other cellular factors that are required for their nuclear localization. These properties enable ectopically overexpressed cyclin D1 (T156A), or a more stable T156A/T286A double mutant that is resistant to ubiquitination, to compete with endogenous cyclin D1 in mammalian cells, thereby mobilizing CDK4 into cytoplasmic, catalytically inactive complexes and dominantly inhibiting the ability of transfected NIH 3T3 fibroblasts to enter S phase.
Mol Cell Biol 1997 Dec
PMID:A dominant-negative cyclin D1 mutant prevents nuclear import of cyclin-dependent kinase 4 (CDK4) and its phosphorylation by CDK-activating kinase. 937 67


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