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

The START cell cycle transition in the budding yeast Saccharomyces cerevisiae is catalyzed by the Cdc28 cyclin-dependent kinase associated with Cln-type cyclins. Since ectopic expression of the B-type cyclin CLB5 can efficiently rescue the inviability that results from CLN depletion, we tested the specificity of the CLN and CLB classes of cyclins for promoting START-associated events. Several aspects of the regulation of the mating factor response were compared for cells in which START activity was provided by either Cln-cyclins or Clb5. Unlike Cln1 and Cln2, high level expression of Clb5 was unable to repress the activity of the mating factor response pathway at START. Downregulation of Far1 protein at START is normal in cln- GAL1::CLB5 cells. Even though the Clb5-Cdc28 kinase activity in cln- GAL1::CLB5 cells is not downregulated in response to mating factor, cells arrest in the first cycle after addition of mating factor with a similar sensitivity as wild-type cells. However, whereas wild-type cells treated with mating factor arrest specifically in G1 phase as unbudded cells with unreplicated DNA (pre-START), most cln- GAL1::CLB5 cells arrest as budded post-START cells with replicated DNA. Our findings demonstrate the ability of post-START cells to arrest in response to mating factor and provide novel evidence for mechanisms that contribute to restrict mating factor-induced arrest in wild-type cells to the G1 phase of the cell cycle.
Mol Gen Genet 1998 May
PMID:Cyclin-specific START events and the G1-phase specificity of arrest by mating factor in budding yeast. 964 24

To explore the regulation and function of D-type cyclins in breast cancer cells, the mouse mammary hyperplastic epithelial cell line TM2H was treated with 5 mM hexamethylenebisacetamide (HMBA), a polar differentiation factor. The resulting growth-inhibitory effect of HMBA was completely reversible and was analyzed in terms of percent cells in G1; association of D-type cyclins with cyclin-dependent kinase (cdk) 4 and cdk6; G1 kinase activity; association of retinoblastoma protein (pRb) and phosphorylated pRb with D-type cyclins; and association of p16INK4a, p15INK4b, and p27Kip1 with cdk4 and cdk6. Synchronized TM2H cells were examined at 0, 3, 5, 9, 12, and 24 h after exposure to 5 mM HMBA. Inhibition of DNA synthesis, as measured by thymidine uptake, was first observed at 5 h (40%) and peaked at 24 h (80%). Flow cytometry at 9 h showed treated cells to be in G1 arrest. Western blot analysis showed weakly detectable cyclin D1 but readily detectable cyclin D2 and D3 proteins at 0 h; thereafter, cyclin D2 and D3 protein levels remained higher while cyclin D1 levels declined significantly in treated versus untreated cells. By 5 h (early G1), HMBA had markedly inhibited cdk4 and cdk6 kinase activity (67% and 75%, respectively) in treated versus untreated cells. By 9 and 12 h, pRb levels had increased 3.4-fold in treated versus untreated cells. At 5 h, cyclin D-associated pRb was totally hypophosphorylated in treated cells and hyperphosphorylated in untreated cells. The levels of pRb associated with cyclin D2 and D3 increased 2.89-fold and 4.6-fold, respectively, in treated versus untreated cells. At 5 h, treated cells showed a fivefold increase in cdk4-associated p27Kip1 and, at 9 h, a fourfold increase in cdk6-associated p27Kip1 over control levels. In confirmation of these data, HMBA was found to inhibit the growth of Rb-positive Du/145Rb cells but not their Rb-negative parental Du/145 cells. The data suggest that HMBA-induced growth inhibition is due to multifactorial mechanisms involving decreases in total cyclin D1 and inhibition of cdk4 and cdk6 kinase activities through elevation of levels of cdk4- and cdk6-associated p27Kip1 and concomitant increases in hypophosphorylated pRb and stable cyclin D2/pRb and cyclin D3/pRb complexes that help maintain pRb in a functional state.
Mol Carcinog 1998 Jun
PMID:Interaction of retinoblastoma protein and D cyclins during cell-growth inhibition by hexamethylenebisacetamide in TM2H mouse epithelial cells. 965 57

The molecular mechanism of androgen-independent growth of prostate cancer after androgen ablation was explored in LNCaP cells. An androgen-dependent clonal subline of the LNCaP human prostate carcinoma cell line, LNCaP 104-S, progressed to a slow growing stage (104-R1) and then to a faster growing stage (104-R2) during more than 2 yr of continuous culture in the absence of androgen. Androgen-induced proliferation of 104-S cells is inhibited by the antiandrogen Casodex, while proliferation of 104-R1 and 104-R2 cells is unaffected by Casodex. This indicates that proliferation of 104-R1 and 104-R2 cells is not supported by low levels of androgen in the culture medium. Compared with LNCaP 104-S cells, both 104-R1 and 104-R2 cells express higher basal levels of androgen receptor (AR), and proliferation of these two cell lines is paradoxically repressed by androgen. After continuous passage in androgen-containing medium, 104-R1 cells reverted back to an androgen-dependent phenotype. The mechanism of androgenic repression of 104-R1 and 104-R2 sublines was further evaluated by examining the role of critical regulatory factors involved in the control of cell cycle progression. At concentrations that repressed growth, androgen transiently induced the expression of the cyclin-dependent kinase (cdk) inhibitor p21waf1/cip1 in 104-R1 cells, while expression of the cdk inhibitor p27Kip1 was persistently induced by androgen in both 104-R1 and 104-R2 cells. Induced expression of murine p27Kip1 in 104-R2 cells resulted in G1 arrest. Specific immunoprecipitates of Cdk2 but not Cdk4 from androgen-treated 104-R1 cells contained both p21waf1/cip1 and p27Kip1. This observation was confirmed by in vitro assay of histone H1 and Rb (retinoblastoma protein) phosphorylation by the proteins associated with the immune complex. Furthermore, inhibition of Cdk2 activity correlated with the accumulation of p27Kip1 and not p21waf1/cip1. From these results we conclude that androgenic repression of LNCaP 104-R1 and 104-R2 cell proliferation is due to the induction of p27Kip1, which in turn inhibits Cdk2, a factor critical for cell cycle progression and proliferation.
Mol Endocrinol 1998 Jul
PMID:Progression of LNCaP prostate tumor cells during androgen deprivation: hormone-independent growth, repression of proliferation by androgen, and role for p27Kip1 in androgen-induced cell cycle arrest. 965 99

The Cdc28p cyclin-dependent kinase is thought to both catalyze the onset of DNA replication and prevent rereplication by blocking the reassembly of initiation complexes at replication origins. Budding yeast with mutations in the CDC16 gene represent an exception to this model, because they rereplicate DNA despite being in a G2-like arrest with continually elevated Cdc28p kinase activity. We show, in contradiction to Pichler et al. (1997), that the extra DNA that accumulates in cdc16 mutants is largely chromosomal, as we originally reported. Two-dimensional DNA electrophoresis shows that cdc16 mutants reinitiate DNA synthesis from normal chromosome replication origins, and density transfer experiments show that multiple chromosomal locations are affected. Rereplication from origins requires both Cdc6p and Cdc46/Mcm5p, initiation proteins that had been thought to be inactivated by the Cdc28p kinase. These results establish that CDC16 is required to prevent inappropriate firing of replication origins.
Mol Cell 1998 Feb
PMID:CDC16 controls initiation at chromosome replication origins. 966 Sep 30

MyoD is a basic helix-loop-helix transcription factor involved in the activation of genes encoding skeletal muscle-specific proteins. Independent of its ability to transactivate muscle-specific genes, MyoD can also act as a cell cycle inhibitor. MyoD activity is regulated by transcriptional and posttranscriptional mechanisms. While MyoD can be found phosphorylated, the functional significance of this posttranslation modification has not been established. MyoD contains several consensus cyclin-dependent kinase (CDK) phosphorylation sites. In these studies, we examined whether a link could be established between MyoD activity and phosphorylation at putative CDK sites. Site-directed mutagenesis of potential CDK phosphorylation sites in MyoD revealed that S200 is required for MyoD hyperphosphorylation as well as the normally short half-life of the MyoD protein. Additionally, we determined that turnover of the MyoD protein requires the proteasome and Cdc34 ubiquitin-conjugating enzyme activity. Results of these studies demonstrate that hyperphosphorylated MyoD is targeted for rapid degradation by the ubiquitin pathway. The targeted degradation of MyoD following CDK phosphorylation identifies a mechanism through which MyoD activity can be regulated coordinately with the cell cycle machinery (CDK2 and CDK4) and/or coordinately with the cellular transcriptional machinery (CDK7, CDK8, and CDK9).
Mol Cell Biol 1998 Sep
PMID:Phosphorylation of nuclear MyoD is required for its rapid degradation. 971 May 83

Vertebrate poly(A) polymerase (PAP) contains a catalytic domain and a C-terminal Ser-Thr-rich regulatory region. Consensus and nonconsensus cyclin-dependent kinase (cdk) sites are conserved in the Ser-Thr-rich region in vertebrate PAPs. PAP is phosphorylated by cdc2-cyclin B on these sites in vitro and in vivo and is inactivated by hyperphosphorylation in M-phase cells, when cdc2-cyclin B is active. In the experiments described here, we undertook a genetic approach in chicken DT40 cells to study the function of PAP phosphorylation. We found that PAP is highly conserved in chicken and is essential in DT40 cells. While cells could tolerate reduced levels of PAP, even modest overexpression of either wild-type PAP or a mutant PAP with two consensus cdk sites mutated (cdk- PAP) was highly deleterious and at a minimum resulted in reduced growth rates. Importantly, cells that expressed cdk- PAP had a significantly lower growth rate than did cells that expressed similar levels of wild-type PAP, which was reflected in increased accumulation of cells in the G0-G1 phase of the cell cycle. We propose that the lower growth rate is due to the failure of hyperphosphorylation and thus M-phase inactivation of cdk- PAP.
Mol Cell Biol 1998 Sep
PMID:Deregulation of poly(A) polymerase interferes with cell growth. 971 May 85

In the yeast Saccharomyces cerevisiae, Sic1, an inhibitor of Clb-Cdc28 kinases, must be phosphorylated and degraded in G1 for cells to initiate DNA replication, and Cln-Cdc28 kinase appears to be primarily responsible for phosphorylation of Sic1. The Pho85 kinase is a yeast cyclin-dependent kinase (Cdk), which is not essential for cell growth unless both CLN1 and CLN2 are absent. We demonstrate that Pho85, when complexed with Pcl1, a G1 cyclin homologue, can phosphorylate Sic1 in vitro, and that Sic1 appears to be more stable in pho85Delta cells. Three consensus Cdk phosphorylation sites present in Sic1 are phosphorylated in vivo, and two of them are required for prompt degradation of the inhibitor. Pho85 and other G1 Cdks appear to phosphorylate Sic1 at different sites in vivo. Thus at least two distinct Cdks can participate in phosphorylation of Sic1 and may therefore regulate progression through G1.
Mol Biol Cell 1998 Sep
PMID:Phosphorylation of sic1, a cyclin-dependent kinase (Cdk) inhibitor, by Cdk including Pho85 kinase is required for its prompt degradation. 972 2

A morphogenesis checkpoint in budding yeast delays cell cycle progression in response to perturbations of cell polarity that prevent bud formation (Lew, D.J., and S.I. Reed. 1995. J. Cell Biol. 129:739- 749). The cell cycle delay depends upon the tyrosine kinase Swe1p, which phosphorylates and inhibits the cyclin-dependent kinase Cdc28p (Sia, R.A.L., H.A. Herald, and D.J. Lew. 1996. Mol. Biol. Cell. 7:1657- 1666). In this report, we have investigated the nature of the defect(s) that trigger this checkpoint. A Swe1p- dependent cell cycle delay was triggered by direct perturbations of the actin cytoskeleton, even when polarity establishment functions remained intact. Furthermore, actin perturbation could trigger the checkpoint even in cells that had already formed a bud, suggesting that the checkpoint directly monitors actin organization, rather than (or in addition to) polarity establishment or bud formation. In addition, we show that the checkpoint could detect actin perturbations through most of the cell cycle. However, the ability to respond to such perturbations by delaying cell cycle progression was restricted to a narrow window of the cell cycle, delimited by the periodic accumulation of the checkpoint effector, Swe1p.
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PMID:A morphogenesis checkpoint monitors the actin cytoskeleton in yeast. 974 79

Exit from mitosis requires the inactivation of mitotic cyclin-dependent kinase-cyclin complexes, primarily by ubiquitin-dependent cyclin proteolysis. Cyclin destruction is regulated by a ubiquitin ligase known as the anaphase-promoting complex (APC). In the budding yeast Saccharomyces cerevisiae, members of a large class of late mitotic mutants, including cdc15, cdc5, cdc14, dbf2, and tem1, arrest in anaphase with a phenotype similar to that of cells expressing nondegradable forms of mitotic cyclins. We addressed the possibility that the products of these genes are components of a regulatory network that governs cyclin proteolysis. We identified a complex array of genetic interactions among these mutants and found that the growth defect in most of the mutants is suppressed by overexpression of SPO12, YAK1, and SIC1 and is exacerbated by overproduction of the mitotic cyclin Clb2. When arrested in late mitosis, the mutants exhibit a defect in cyclin-specific APC activity that is accompanied by high Clb2 levels and low levels of the anaphase inhibitor Pds1. Mutant cells arrested in G1 contain normal APC activity. We conclude that Cdc15, Cdc5, Cdc14, Dbf2, and Tem1 cooperate in the activation of the APC in late mitosis but are not required for maintenance of that activity in G1.
Mol Biol Cell 1998 Oct
PMID:A late mitotic regulatory network controlling cyclin destruction in Saccharomyces cerevisiae. 976 45

We earlier reported that TIS21 mRNA expression was markedly decreased in A549 and NCIH69 human lung cancer cells and in thymic carcinoma tissues obtained from transgenic mice containing simian virus 40 large T antigen (J Cancer Res Clin Oncol 121:279-284, 1995). To determine how TIS21 inhibits growth, we made 293 cells that constitutively expressed TIS21 protein. The constitutive TIS21 expresser lines C9 and C11 grew to a lower saturation density than did those in the vector-transfected clones (V7 and V10) and antisense-transfected clones (AS1 and AS4), and the size of the C9 and C11 cells increased significantly after transfection with TIS21 cDNA. The serum-stimulated cell cycle was analyzed by fluorescence-activated cell sorting after double thymidine treatment; V10 progressed normally through the cell division cycle, but C9 and C11 cells accumulated continuously in G1 phase until 36 h after treatment. On the other hand, the progression of cells that had already entered to S or G2/M phase was not inhibited. When cell-cycle regulatory proteins were measured, C9 and C11 cells showed significantly reduced synthesis of cyclin E and cyclin-dependent kinase (cdk) 4 as well as a decrease in cyclin E-associated cdk activity. These observations led us to conclude that TIS21 overexpression in G1 phase decreased the amounts of cyclin E and cdk4, thereby decreasing the activity of cdks at the G1-S transition.
Mol Carcinog 1998 Sep
PMID:Induction of growth inhibition of 293 cells by downregulation of the cyclin E and cyclin-dependent kinase 4 proteins due to overexpression of TIS21. 976 35


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