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Query: EC:2.7.11.22 (cdc2)
 8,319 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Cyclin-dependent kinases (Cdks) are required for cell cycle progression. Two potentially significant Cdk substrates in human cells are the human single-stranded binding protein (HSSB or RPA), which plays an essential role in DNA replication, repair, and recombination, and the tumor suppressor p107 which acts to negatively regulate cell growth. In this report we describe the in vitro phosphorylation of these two proteins by Cdks in an attempt to understand how cyclin-substrate interactions direct phosphorylation efficiencies. We show that cyclin A-Cdk2 efficiently phosphorylates the p34 subunit of HSSB (HSSB-p34) alone or as a part of the heterotrimeric complex. In contrast, cyclin E-Cdk2 that is active in phosphorylating histone H1, does not support the phosphorylation of the p34 subunit of HSSB. We provide evidence that this differential phosphorylation results from a specific interaction between HSSB-p34 and cyclin A, but not cyclin E. Thus the observed cell cycle-dependent phosphorylation of HSSB-p34 at the G1 to S transition is most likely catalyzed by cyclin A-Cdk2 initiated by the direct interaction between cyclin A and the HSSB-p34 subunit. These studies are consistent with our previous observation that p107, which directly binds cyclin A, is efficiently phosphorylated by cyclin A-Cdk2 but not cyclin B-associated kinases. Here we further demonstrate that cyclin A only complexes with p107 in its unphosphorylated form. These data suggest a catalytic mechanism by which Cdk acts: substrate targeting by a cyclin-substrate interaction followed by dissociation of the Cdk upon phosphate incorporation allowing the Cdk to become available for the next cycle of phosphorylation.
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PMID:Studies on the in vitro phosphorylation of HSSB-p34 and -p107 by cyclin-dependent kinases. Cyclin-substrate interactions dictate the efficiency of phosphorylation. 879 63

Cyclin-dependent kinase 5(cdk5) is highly homologous to other members of the cdk family that are known to function in proliferating cells. Despite the structural similarity, cdk5-associated histone H1 kinase activity is only detectable in postmitotic neurons of the central nervous system (CNS). p35 is a neuronal-specific cdk5 regulator that activates cdk5 kinase activity upon association. The cdk5/p35 kinase activity increases during the progression of CNS neurogenesis, suggesting a function of cdk5 in neuronal differentiation. Here we show that both cdk5 and p35 proteins are present in the growth cones of developing neurons. The staining pattern of cdk5 in the growth cones is similar to that of actin filaments but not microtubules. To address the functional significance of the cdk5/p35 kinase in neurogenesis, we ectopically expressed wild-type or mutant kinases in cortical cultures. Expression of dominant-negative mutants of cdk5 (cdk5N144 and cdk5T33) inhibited neurite outgrowth, which was rescued by coexpression of the wild-type proteins. A similar extent of neurite outgrowth inhibition was obtained by transfection of an antisense p35 construct, which in turn was only rescued by p35 but not cdk5 coexpression. In contrast, longer neurites were elaborated in neurons that coexpressed exogenous cdk5 and p35. These observations suggest that the cdk5/p35 kinase plays a critical role in neurite outgrowth during neuronal differentiation.
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PMID:The cdk5/p35 kinase is essential for neurite outgrowth during neuronal differentiation. 884 18

The sequential transcriptional activation of cyclins, the regulatory subunits of cell cycle specific kinases, regulates progress through the cell cycle. In mitogen-stimulated cells cyclin D1 induction in early G1 is followed by induction of cyclin E, activation of the cyclin-dependent kinase Cdk2, and hyperphosphorylation of the retinoblastoma gene product (pRB) in mid-to-late G1 phase. T-47D breast cancer cells expressing cyclin D1 under the control of a metal-responsive metallothionein promoter were used to determine whether Cdk2 activation and pRB hyperphosphorylation are consequences of cyclin D1 induction. A 4-5-fold increase in cyclin D1 protein abundance was followed by approximately 2-fold increases in cyclin E protein abundance and Cdk2 activity and by hyperphosphorylation of pRB. These responses were apparent approximately 3 h after the increase in cyclin D1 protein, and approximately 3 h prior to the entry of cyclin D1-stimulated cells into S phase 12 h after zinc treatment. Cyclin D1 immunoprecipitates contained Cdk4 but no detectable Cdk2 and displayed pRb but not histone H1 kinase activity. Cdk2 activation was therefore likely to be due to increased abundance of cyclin E/Cdk2 complexes rather than formation of active cyclin D1/Cdk2 complexes. The sequence of events following zinc induction of cyclin D1 thus mimicked that following mitogen induction of cyclin D1. These data show that cyclin D1 induction is sufficient for Cdk2 activation and pRB hyperphosphorylation in T-47D human breast cancer cells, providing evidence that cyclin D1 induction is a critical event in G1 phase progression.
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PMID:Inducible expression of cyclin D1 in T-47D human breast cancer cells is sufficient for Cdk2 activation and pRB hyperphosphorylation. 886 12

The experiments described in this report were undertaken to define the parameters that regulate cyclin E/cyclin-dependent kinase 2 (Cdk2) kinase activity in mitotically quiescent, serum-starved fibroblastic cells and in cells that had been stimulated to enter the cell cycle and progress through G1 into S phase. We have analyzed the expression of cyclin E and Cdk2, the extent to which these two proteins form complexes, and the enzymatic activity of cyclin E/cdk2 kinase. Particular attention was focused upon subcellular localization and the effect of compartmentalization on the association between cyclin E and Cdk2. In addition, we have examined the interaction of cyclin E/Cdk2 complexes with two well-characterized inhibitors of Cdk2 kinase activity, Cip1 and Kip1. This represents the first report in which all of these parameters have been measured simultaneously in a single, normal diploid cell line. In G0 cells, there is abundant cyclin E and Cdk2, yet there is little or no detectable Cdk2-dependent histone H1 kinase activity. After serum stimulation, there is a rapid increase in the amount of cyclin E that is bound to Cdk2, although there is no significant change in the abundance of either the cyclin or the Cdk. Immunocytochemical data indicate that cyclin E, Cip1, and Kip1 are located within the nuclei of cell in G0, but very little Cdk2 is observed within the nuclei of serum-starved cells. Cdk2 rapidly enters the nucleus upon serum stimulation. The abundance of the cyclin E/Cdk2 complex increases to the extent that the binding capacity of Cip1 is exceeded about 8-12 h after serum stimulation. The abundance of Kip1 decreases at the same time that the Cip1 threshold is exceeded, so that cyclin E/Kip1-containing complexes decrease by 90% within 8-12 h. Cyclin E/Cdk2 kinase activity begins to increase rapidly thereafter, reaching a maximum level about 16 h after serum stimulation. We have been unable to detect histone H1 kinase activity in complexes that contain cyclin E bound to Kip1 or Cip1. We conclude that compartmentalization is the predominant barrier to activation of cyclin E-dependent kinases in quiescent cells. Cip1 and Kip1 serve to prevent premature activation of cyclin E/Cdk2 complexes that form during G0 or early G1.
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PMID:Cyclin E/Cdk2 activity is controlled by different mechanisms in the G0 and G1 phases of the cell cycle. 889 32

Despite intensive efforts, the exact cellular mechanisms leading to gut differentiation and development remain largely undefined. The cyclins, the cyclin-dependent kinases (Cdks), and the Cdk inhibitors (e.g., p21 and p27) are proteins that are important for cell cycle progression, subsequent growth inhibition, and differentiation of various cell types. The purpose of our study was to better define the role of these cell cycle proteins in gut differentiation using the Caco-2 human cell line, which spontaneously differentiates to a small bowel phenotype, as demonstrated by induction of sucrase-isomaltase (SI) gene expression. We found that protein levels of the cyclins (both D- and E-type) and the Cdks (both Cdk2 and Cdk4) progressively decreased in postconfluent Caco-2 cells. Moreover, cyclin E-associated histone H1 kinase activity decreased in an analogous fashion as the cyclins and Cdks. In contrast, induction of the Cdk inhibitor p21 occurred by 3 days postconfluency, which was before the increase in SI mRNA levels. These changes in the cell cycle proteins, which include a progressive decrease of the cyclins and Cdks and a concomitant induction of p21, suggest an important role for these proteins in Caco-2 cell differentiation. Identifying the cell cycle mechanisms responsible for intestinal cell differentiation will be important to our understanding of both normal gut development as well as gut neoplasia, which involves aberrant regulation of cell cycle arrest.
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PMID:Cell cycle protein suppression and p21 induction in differentiating Caco-2 cells. 889 94

Apoptosis has recently been hypothesized to be the result of aberrant cell cycle control. In this study, we have investigated the role of cell cycle-regulatory elements in Fas-induced apoptosis of hematopoietic cells. When HL-60 cells were treated with anti-Fas antibody, rapid activation of growth-associated histone H1 kinase was observed without any change in cell cycle distribution. This was accompanied by the increase in cdc2 mRNA expression and Cdc2 kinase activity. Up-regulation of cdc2 mRNA was similarly induced in BCL-2-overexpressing HL-60 subline by anti-Fas treatment independently of the appearance of apoptotic phenotypes. Fas-induced apoptosis was completely inhibited by butyrolactone I, a specific inhibitor of Cdc2 kinase. Moreover, the same phenomenon was observed during Fas-induced but not spontaneous apoptosis of postmitotic granulocytes. Finally, we have found that "Fas-responsive element" was located between nucleotides -730 and -552 of the cdc2 promoter and was responsive for transcriptional activation of the cdc2 gene during Fas-induced apoptosis. These results indicate that aberrant activation of Cdc2 is associated with Fas-induced apoptosis of hematopoietic cells, and that the mechanism of cdc2 transcription during Fas-induced apoptosis is different from that in normal cell cycle control.
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PMID:Transcriptional activation of the cdc2 gene is associated with Fas-induced apoptosis of human hematopoietic cells. 891 Apr 74

Saccharomyces cerevisiae, like most eucaryotic cells, can prevent the onset of anaphase until chromosomes are properly aligned on the mitotic spindle. We determined that Cdc55p (regulatory B subunit of protein phosphatase 2A [PP2A]) is required for the kinetochore/spindle checkpoint regulatory pathway in yeast. ctf13 cdc55 double mutants could not maintain a ctf13-induced mitotic delay, as determined by antitubulin staining and levels of histone H1 kinase activity. In addition, cdc55::LEU2 mutants and tpd3::LEU2 mutants (regulatory A subunit of PP2A) were nocodazole sensitive and exhibited the phenotypes of previously identified kinetochore/spindle checkpoint mutants. Inactivating CDC55 did not simply bypass the arrest that results from inhibiting ubiquitin-dependent proteolysis because cdc16-1 cdc55::LEU2 and cdc23-1 cdc55::LEU2 double mutants arrested normally at elevated temperatures. CDC55 is specific for the kinetochore/spindle checkpoint because cdc55 mutants showed normal sensitivity to gamma radiation and hydroxyurea. The conditional lethality and the abnormal cellular morphogenesis of cdc55::LEU2 were suppressed by cdc28F19, suggesting that the cdc55 phenotypes are dependent on the phosphorylation state of Cdc28p. In contrast, the nocodazole sensitivity of cdc55::LEU2 was not suppressed by cdc28F19. Therefore, the mitotic checkpoint activity of CDC55 (and TPD3) is independent of regulated phosphorylation of Cdc28p. Finally, cdc55::LEU2 suppresses the temperature sensitivity of cdc20-1, suggesting additional roles for CDC55 in mitosis.
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PMID:Cdc55p, the B-type regulatory subunit of protein phosphatase 2A, has multiple functions in mitosis and is required for the kinetochore/spindle checkpoint in Saccharomyces cerevisiae. 900 Dec 15

Maturation-promoting factor (MPF) is known to be a key regulator of both mitotic and meiotic cell cycles. MPF is a complex of a B cyclin and the cyclin-dependent kinase cdkl (p34cdc2). Oocyte maturation and its arrest at metaphase of meiosis II (MII) are regulated by changes in MPF activity. In this study, experiments were conducted to examine the dynamics of MPF activity and its constituent proteins during in vitro maturation of bovine oocytes. Bovine oocytes displayed relatively low levels of MPF (histone H1 kinase) activity at the germinal vesicle stage during the first 8 h of maturation. MPF activity increased gradually thereafter, and its first peak of activity occurred at 12-14 h of maturation (presumptive metaphase I), which was followed by an abrupt reduction in activity at 16-18 h, during presumptive anaphase and telophase. MPF activity then increased, reaching a plateau at 20-24 h of maturation (MII stage). This high level of MPF activity was maintained for several hours but decreased gradually after 30 h of maturation and became barely detectable by 48 h of in vitro maturation (IVM) culture. At each time point, there was a significant variation among individual oocytes in histone H1 kinase activity, which was probably due to asynchronous maturation. Abundance of cdk1 increased gradually during the first 8 h and then remained relatively constant except for an apparent reduction at 18-22 h of IVM. The level of cyclin B2 increased quickly during the initial 2 h of culture, and this high level was maintained until 16 h, after which a significant reduction was observed between 18 and 22 h of IVM. The de novo synthesis of cyclin B2, however, exhibited a biphasic oscillation during maturation, with peaks before the onset of MI and of MII. These results have defined the profiles of MPF activity and its individual components during bovine oocyte maturation in vitro. We conclude that active MPF regulates bovine oocyte maturation and that de novo synthesis of cyclin B2 occurs during the process of maturation.
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PMID:Dynamics of maturation-promoting factor and its constituent proteins during in vitro maturation of bovine oocytes. 900 57

M-phase promoting factor or maturation promoting factor, a key regulator of the G2-->M transition of the cell cycle, is a complex of cdc2 and a B-type cyclin. We have previously shown that Xenopus cyclin B1 has five sites of Ser phosphorylation, four of which map to a recently identified cytoplasmic retention signal (CRS). The CRS appears to be responsible for the cytoplasmic localization of B-type cyclins, although the underlying mechanism is still unclear. Phosphorylation of cyclin B1 is not required for cdc2 binding or cdc2 kinase activity. However, when all of the Ser phosphorylation sites in the CRS are mutated to Ala to abolish phosphorylation, the mutant cyclin B1Ala is inactivated; activity can be enhanced by mutation of these residues to Glu to mimic phosphoserine, suggesting that phosphorylation of cyclin B1 is required for its biological activity. Here we show that biological activity can be restored to cyclin B1Ala by appending either a nuclear localization signal (NLS), or a second CRS domain with the Ser phosphorylation sites mutated to Glu, while fusion of a second CRS domain with the Ser phosphorylation sites mutated to Ala inactivates wild-type cyclin B1. Nuclear histone H1 kinase activity was detected in association with cyclin B1Ala targeted to the nucleus by a wild-type NLS, but not by a mutant NLS. These results demonstrate that nuclear translocation mediates the biological activity of cyclin B1 and suggest that phosphorylation within the CRS domain of cyclin B1 plays a regulatory role in this process. Furthermore, given the similar in vitro substrate specificity of cyclin-dependent kinases, this investigation provides direct evidence for the hypothesis that the control of subcellular localization of cyclins plays a key role in regulating the biological activity of cyclin-dependent kinase-cyclin complexes.
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PMID:Nuclear localization of cyclin B1 mediates its biological activity and is regulated by phosphorylation. 901 13

Recent studies have implicated the cell cycle kinase cdc2 and cyclin A in the inhibition of the fusion of endocytic vesicles in vitro during mitosis. However, the presence of cyclins or their associated cyclin dependent kinases (cdks) in the endocytic fractions have not been reported. Using Western-blotting and immunocytochemistry approaches with different anticyclin A antibodies we have detected cyclin A in the endocytic compartment of the rat liver. During the pre-replicative phase of liver regeneration the amount of cyclin A in endosomes increases significantly with a peak around 12 hours after partial hepatectomy. Cyclin A-dependent kinases, cdc2 and cdk2, were also found in isolated endosome fractions, showing a distinct kinetics of accumulation during the regenerative period. Finally, histone H1 kinase activity was detected associated with cyclin A in endocytic vesicles and increased in regenerating liver. These results suggest that changes in the organization and in the function of the endocytic compartment during the hepatocellular proliferation may be modulated by proteins involved in the regulation of the cell cycle.
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PMID:Cyclin A is present in the endocytic compartment of rat liver cells and increases during liver regeneration. 902 58


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