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

Cdc2-Cyclin B, the protein kinase that catalyzes the onset of mitosis, is subject to multiple forms of regulation. In the fission yeast Schizosaccharomyces pombe and most other species, a key mode of Cdc2-Cyclin B regulation is the inhibitory phosphorylation of Cdc2 on tyrosine-15. This phosphorylation is catalyzed by the protein kinases Wee1 and Mik1 and removed by the phosphatase Cdc25. These proteins are also regulated, a notable example being the inhibition of Wee1 by the protein kinase Nim1/Cdr1. The temperature-sensitive mutation cdc25-22 is synthetic lethal with nim1/cdr1 mutations, suggesting that a synthetic lethal genetic screen could be used to identify novel mitotic regulators. Here we describe that such a screen has identified cdr2(+), a gene that has an important role in the mitotic control. Cdr2 is a 775 amino acid protein kinase that is closely related to Nim1 and mitotic control proteins in budding yeast. Deletion of cdr2 causes a G2-M delay that is more severe than that caused by nim1/cdr1 mutations. Genetic studies are consistent with a model in which Cdr2 negatively regulates Wee1. This model is supported by experiments showing that Cdr2 associates with the N-terminal regulatory domain of Wee1 in cell lysates and phosphorylates Wee1 in vitro. Thus, Cdr2 is a novel mitotic control protein that appears to regulate Wee1.
Mol Biol Cell 1998 Dec
PMID:The protein kinase Cdr2, related to Nim1/Cdr1 mitotic inducer, regulates the onset of mitosis in fission yeast. 984 72

A novel cyclin gene was discovered by searching an expressed sequence tag database with a cyclin box profile. The human cyclin E2 gene encodes a 404-amino-acid protein that is most closely related to cyclin E. Cyclin E2 associates with Cdk2 in a functional kinase complex that is inhibited by both p27(Kip1) and p21(Cip1). The catalytic activity associated with cyclin E2 complexes is cell cycle regulated and peaks at the G1/S transition. Overexpression of cyclin E2 in mammalian cells accelerates G1, demonstrating that cyclin E2 may be rate limiting for G1 progression. Unlike cyclin E1, which is expressed in most proliferating normal and tumor cells, cyclin E2 levels were low to undetectable in nontransformed cells and increased significantly in tumor-derived cells. The discovery of a novel second cyclin E family member suggests that multiple unique cyclin E-CDK complexes regulate cell cycle progression.
Mol Cell Biol 1999 Jan
PMID:Cyclin E2, a novel G1 cyclin that binds Cdk2 and is aberrantly expressed in human cancers. 985 85

Cyclin A-Cdk2 complexes bind to Skp1 and Skp2 during S phase, but the function of Skp1 and Skp2 is unclear. Skp1, together with F-box proteins like Skp2, are part of ubiquitin-ligase E3 complexes that target many cell cycle regulators for ubiquitination-mediated proteolysis. In this study, we investigated the potential regulation of cyclin A-Cdk2 activity by Skp1 and Skp2. We found that Skp2 can inhibit the kinase activity of cyclin A-Cdk2 in vitro, both by direct inhibition of cyclin A-Cdk2 and by inhibition of the activation of Cdk2 by cyclin-dependent kinase (CDK)-activating kinase phosphorylation. Only the kinase activity of Cdk2, not of that of Cdc2 or Cdk5, is reduced by Skp2. Skp2 is phosphorylated by cyclin A-Cdk2 on residue Ser76, but nonphosphorylatable mutants of Skp2 can still inhibit the kinase activity of cyclin A-Cdk2 toward histone H1. The F box of Skp2 is required for binding to Skp1, and both the N-terminal and C-terminal regions of Skp2 are involved in binding to cyclin A-Cdk2. Furthermore, Skp2 and the CDK inhibitor p21(Cip1/WAF1) bind to cyclin A-Cdk2 in a mutually exclusive manner. Overexpression of Skp2, but not Skp1, in mammalian cells causes a G1/S cell cycle arrest.
Mol Cell Biol 1999 Jan
PMID:Regulation of cyclin A-Cdk2 by SCF component Skp1 and F-box protein Skp2. 985 87

DNA polymerase alpha-primase is known to be phosphorylated in human and yeast cells in a cell cycle-dependent manner on the p180 and p68 subunits. Here we show that phosphorylation of purified human DNA polymerase alpha-primase by purified cyclin A/cdk2 in vitro reduced its ability to initiate simian virus 40 (SV40) DNA replication in vitro, while phosphorylation by cyclin E/cdk2 stimulated its initiation activity. Tryptic phosphopeptide mapping revealed a family of p68 peptides that was modified well by cyclin A/cdk2 and poorly by cyclin E/cdk2. The p180 phosphopeptides were identical with both kinases. By mass spectrometry, the p68 peptide family was identified as residues 141 to 160. Cyclin A/cdk2- and cyclin A/cdc2-modified p68 also displayed a phosphorylation-dependent shift to slower electrophoretic mobility. Mutation of the four putative phosphorylation sites within p68 peptide residues 141 to 160 prevented its phosphorylation by cyclin A/cdk2 and the inhibition of replication activity. Phosphopeptide maps of the p68 subunit of DNA polymerase alpha-primase from human cells, synchronized and labeled in G1/S and in G2, revealed a cyclin E/cdk2-like pattern in G1/S and a cyclin A/cdk2-like pattern in G2. The slower-electrophoretic-mobility form of p68 was absent in human cells in G1/S and appeared as the cells entered G2/M. Consistent with this, the ability of DNA polymerase alpha-primase isolated from synchronized human cells to initiate SV40 replication was maximal in G1/S, decreased as the cells completed S phase, and reached a minimum in G2/M. These results suggest that the replication activity of DNA polymerase alpha-primase in human cells is regulated by phosphorylation in a cell cycle-dependent manner.
Mol Cell Biol 1999 Jan
PMID:Cell cycle-dependent regulation of human DNA polymerase alpha-primase activity by phosphorylation. 985 88

Cyclin G1 is a recently cloned transcriptional target of p53, it is located in neurons and ventricular ependymal cells and is elevated in neurons after axotomy and cerebral ischemia. The biological function for cyclin G1 in differentiated neurons has thus far not been elucidated. Recently, cyclin G1 has been shown to interact with the B' subunits of serine/threonine protein phosphatase 2A (PP2A) in a rat fibroblast cell line [K. Okamoto, C., Kamibayashi, M. Serrano, C. Prives, M.C. Mumby, D. Beach, p53-dependent association between cyclin G and the B' subunit of protein phosphatase 2A, Mol. Cell. Biol. 16 (1996) 6593-6602]. To further explore whether a similar interaction between cyclin G1 and PP2A B' subunits exists in the central nervous system, the present study compared the regional and developmental expression pattern, subcellular distribution and complex formation between cyclin G1 and the PP2A B' regulatory subunits in the rat brain. In situ hybridization of cyclin G1 and the B'alpha and B'beta subunits of PP2A showed an overlapping distribution in neurons of the cerebral cortex, hippocampus and thalamus at embryonic and early postnatal ages, but their developmental regulation differed. Whereas mRNA and protein levels of PP2A B' subunits were high in the cortical plate, subiculum, hippocampal areas and thalamus at E20 and decreased with age, those of cyclin G1 increased with age and were maximal in the adult cortex and hippocampus. In rat 14-day-old embryonic cortical cultures, cyclin G1 and PP2A B'alpha protein co-localized in nuclear and perinuclear areas of neurons, and both proteins were highly expressed in nuclei of cortical and hippocampal pyramidal cells and the mitral cell layer of the neonatal olfactory bulb. Both cyclin G1 and the PP2A regulatory B'alpha subunits were specifically expressed in neurons and not in glial cells. Antibodies raised against the B'alpha subunits of PP2A immunoprecipitated cyclin G1 in adult cortical lysates, indicating the presence of a complex involving cyclin G1 and the B'alpha subunits of PP2A. This study shows that the regional and subcellular localization of PP2A B' regulatory subunits and cyclin G1 are very similar at early postnatal stages. We discuss the possible functions of a cyclin G1-PP2A B'alpha complex in neurons.
Brain Res Mol Brain Res 1999 Jan 22
PMID:Developmental expression and co-localization of cyclin G1 and the B' subunits of protein phosphatase 2a in neurons. 988 95

The mechanism by which cyclin-dependent kinase 4 (CDK4) regulates cell cycle progression is not entirely clear. Cyclin D/CDK4 appears to initiate phosphorylation of retinoblastoma protein (Rb) leading to inactivation of the S-phase-inhibitory action of Rb. However, cyclin D/CDK4 has been postulated to act in a noncatalytic manner to regulate the cyclin E/CDK2-inhibitory activity of p27(Kip1) by sequestration. In this study we investigated the roles of CDK4 in cell cycle regulation by targeted disruption of the mouse CDK4 gene. CDK4(-/-) mice survived embryogenesis and showed growth retardation and reproductive dysfunction associated with hypoplastic seminiferous tubules in the testis and perturbed corpus luteum formation in the ovary. These phenotypes appear to be opposite to those of p27-deficient mice such as gigantism and gonadal hyperplasia. A majority of CDK4(-/-) mice developed diabetes mellitus by 6 weeks, associated with degeneration of pancreatic islets. Fibroblasts from CDK4(-/-) mouse embryos proliferated similarly to wild-type embryonic fibroblasts under conditions that promote continuous growth. However, quiescent CDK4(-/-) fibroblasts exhibited a substantial ( approximately 6-h) delay in S-phase entry after serum stimulation. This cell cycle perturbation by CDK4 disruption was associated with increased binding of p27 to cyclin E/CDK2 and diminished activation of CDK2 accompanied by impaired Rb phosphorylation. Importantly, fibroblasts from CDK4(-/-) p27(-/-) embryos displayed partially restored kinetics of the G(0)-S transition, indicating the significance of the sequestration of p27 by CDK4. These results suggest that at least part of CDK4's participation in the rate-limiting mechanism for the G(0)-S transition consists of controlling p27 activity.
Mol Cell Biol 1999 Oct
PMID:Targeted disruption of CDK4 delays cell cycle entry with enhanced p27(Kip1) activity. 1049 Jun 38

Promoters of growth and cell cycle regulated genes frequently carry binding sites for transcription factors of the E2F and Sp1 families. We have demonstrated recently that direct interaction between Sp1 and a subgroup of the E2F factors is essential for the regulation of certain promoters. We show here that the amino acids necessary for this interaction in both cases are located within the DNA binding domain. This is in line with the assumption, that the interaction between E2F and Sp-factors contributes to promoter-specificity. Cyclin A, which binds to E2F-1 in close vicinity to Sp1 does not interfere with this interaction. Moreover we have investigated the ability of other members of the Sp1 family to interact with E2F-1 and to regulate the activity of the E2F and Sp1 dependent murine thymidine kinase promoter. All four factors of the Sp1 family are able to bind E2F-1 in co-immunoprecipitation and GST-pull down experiments. Mobility shift assays with oligonucleotides comprising the Sp1, or both the Sp1 and the E2F binding site suggest that Sp1 and Sp3 supply most if not all activity binding to the GC-box of the thymidine kinase promoter in murine fibroblasts. Reporter gene assays in Drosophila melanogaster SL2 cells and murine fibroblast 3T6 cells demonstrate that the thymidine kinase promoter is activated strongly by Sp1 and Sp3, weakly by Sp4, and not at all by Sp2. Co-expression of E2F-1 results in synergistic activation in 3T6 but not in SL2 cells.
J Mol Biol 1999 Nov 12
PMID:Transcription factors of the Sp1 family: interaction with E2F and regulation of the murine thymidine kinase promoter. 1054 81

In order to investigate the hypothesis that aberrant expression of cell-cycle regulatory proteins may represent early events in the process of carcinogenesis, levels of expression of the negative regulators p21(waf1/cip1) (p21), p27(kip1) (p27), and p16(ink4a) (p16) and/or the positive regulators cyclin D(1) and cyclin E were examined by western blot analysis in cells transformed in vitro by ionizing radiation. The levels of these proteins in 12 independently derived mouse 10T(1/2) cell clones transformed by 1.5 Gy of alpha radiation were compared with those in nine similarly derived nontransformed control clones. Constitutive levels of p21 were very low in all control clones, whereas p21 expression was significantly elevated in nine of 12 transformed clones. Two of the three transformed clones displaying low levels of p21 expressed increased levels of p53. p21 regulation was also altered in response to radiation in transformed clones as compared with controls, only minimal induction was observed 4 h following gamma irradiation. Western blot analysis indicated a constant expression of p27 protein but slightly decreased levels of p16 in these transformed clones. Cyclin D(1) was overexpressed in 11 of 12 transformed clones; in only two of these were the levels of cyclin E elevated. Overall, the results suggest that alterations in the expression of cell cycle regulatory proteins may represent important events in radiation-induced oncogenic transformation in vitro. Although the specific alterations vary among different transformed clones, overexpression and aberrant regulation of p21 appear to be the most frequent ones.
Mol Carcinog 2000 Feb
PMID:Overexpression of p21 protein in radiation-transformed mouse 10T(1/2) cell clones. 1065 6

v-Jun accelerates G(1) progression and shares the capacity of the Myc, E2F, and E1A oncoproteins to sustain S-phase entry in the absence of mitogens; however, how it does so is unknown. To gain insight into the mechanism, we investigated how v-Jun affects mitogen-dependent processes which control the G(1)/S transition. We show that v-Jun enables cells to express cyclin A and cyclin A-cdk2 kinase activity in the absence of growth factors and that deregulation of cdk2 is required for S-phase entry. Cyclin A expression is repressed in quiescent cells by E2F acting in conjunction with its pocket protein partners Rb, p107, and p130; however, v-Jun overrides this control, causing phosphorylated Rb and proliferation-specific E2F-p107 complexes to persist after mitogen withdrawal. Dephosphorylation of Rb and destruction of cyclin A nevertheless occur normally at mitosis, indicating that v-Jun enables cells to rephosphorylate Rb and reaccumulate cyclin A without exogenous mitogenic stimulation each time the mitotic "clock" is reset. D-cyclin-cdk activity is required for Rb phosphorylation in v-Jun-transformed cells, since ectopic expression of the cdk4- and cdk6-specific inhibitor p16(INK4A) inhibits both DNA synthesis and cell proliferation. Despite this, v-Jun does not stimulate D-cyclin-cdk activity but does induce a marked deregulation of cyclin E-cdk2. In particular, hormonal activation of a conditional v-Jun-estrogen receptor fusion protein in quiescent, growth factor-deprived cells stimulates cyclin E-cdk2 activity and triggers Rb phosphorylation and DNA synthesis. Thus, v-Jun overrides the mitogen dependence of S-phase entry by deregulating Rb phosphorylation, E2F-pocket protein interactions, and ultimately cyclin A-cdk2 activity. This is the first report, however, that cyclin E-cdk2, rather than D-cyclin-cdk, is likely to be the critical Rb kinase target of v-Jun.
Mol Cell Biol 2000 Apr
PMID:v-Jun overrides the mitogen dependence of S-phase entry by deregulating retinoblastoma protein phosphorylation and E2F-pocket protein interactions as a consequence of enhanced cyclin E-cdk2 catalytic activity. 1071 76

The Saccharomyces cerevisiae genes PHO80 and PHO85 encode, respectively, a cyclin and cyclin-dependent kinase, which negatively regulate PHO5 gene transcription by phosphorylating the transcription activator Pho4p. Cyclin-dependent kinases (CDKs) are highly conserved proteins, both within and between species. It was previously demonstrated, using reporter genes activated in yeast by Pho4p, that hybrid proteins in which over two-thirds of Pho85p were replaced with the homologous region from human Cdk2 retained the function of native Pho85p with respect to promoter repression. In the present study, various truncated forms of the hybrid human-yeast CDKs were tested for function. Surprisingly, truncations in which significant portions of the C-terminal region of the 291-residue hybrid CDK were deleted retained activity. Genes encoding human Cdk2 proteins which terminated after amino acids 151, 140, 130, 120 and 90 each complement a chromosomal pho85 gene disruption in which the HIS3 gene is inserted at codon 49. Truncated Cdk2 proteins containing less than 60 amino acids failed to complement the pho85::HIS3 gene disruption. Although the functional C-terminal truncations disrupt the ATP-binding and active sites of Cdk2, reporter gene repression mediated by these truncated proteins is apparently due to phosphorylation of Pho4p, since a gene in which the essential lysine codon at position 33 was converted to an arginine codon does not complement the chromosomal gene disruption. The human Cdk2 truncations were demonstrated to function through intergenic complementation. The intact Cdk2-Pho85 hybrid CDK complemented the pho85 mutation in yeast strains in which the entire PHO85 coding region was deleted from chromosome XVI. The C-terminal Cdk2 truncations, however, were non-functional in these strains and thus dependent for activity on the pho85 coding region which remained in the mutant pho85::HIS3 chromosomal locus. These genetic results are consistent with a model involving protein fragment complementation in which the active site of the CDK is bisected.
Mol Gen Genet 2000 Mar
PMID:Intergenic complementation truncation mutants of cyclin-dependent kinase. 1077 40


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