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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)

The Xenopus cdk2 gene encodes a 32-kDa protein kinase with sequence similarity to the 34-kDa product of the cdc2 gene. Previous studies have shown that the kinase activity of the protein product of the cdk2 gene oscillates in the Xenopus embryonic cell cycle with a high in M-phase and a low in interphase. In the present study cdk2 was found not to be associated with any newly synthesized proteins during the cell cycle, but the enzyme did undergo periodic changes in phosphorylation. Upon exit from metaphase, cdk2 became increasingly phosphorylated on both tyrosine and serine residues, and labeling on these residues increased progressively until entry into mitosis, when tyrosine residues were markedly dephosphorylated. Phosphopeptide mapping of cdk2 demonstrated the major sites of phosphorylation were in a phosphopeptide with a pI of 3.7 that contained both phosphoserine and phosphotyrosine. This phosphopeptide accumulated in egg extracts blocked in S-phase with aphidicolin and was not evident in cdc2 immunoprecipitated under the same conditions. Under the same conditions cdc2 was phosphorylated primarily on a phosphopeptide containing both phosphothreonine and phosphotyrosine residues, most likely threonine 14 and tyrosine 15. Affinity-purified human GST-cdc25 was able to dephosphorylate and activate cdk2 isolated from interphase cells. Phosphopeptide mapping demonstrated that the phosphate was specifically removed from the same phosphopeptide identified as the major in vivo site of phosphorylation. These results demonstrate that cdk2 is regulated in the cell cycle by phosphorylation and dephosphorylation on both serine and tyrosine residues. Moreover, the increased phosphorylation of cdk2 in aphidicolin-blocked extracts and the ability of cdc25 to mediate cdk2 dephosphorylation in vitro suggest the possibility that cdk2 is part of the mechanism ensuring mitosis is not initiated until completion of DNA replication. It also implies cdc25 may have other functions in addition to the regulation of cdc2 kinase activity.
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PMID:Cdc25 regulates the phosphorylation and activity of the Xenopus cdk2 protein kinase complex. 151 36

The cyclin-dependent kinase (Cdk) inhibitor p21SDI1/WAF1/CIP1 has been found to be involved in cell senescence, cell cycle arrest, and differentiation. p21SDI1 inhibits the activity of several Cdks, in contrast to other inhibitors such as p15INK4B and p16INK4A, which act on specific cyclin-Cdk complexes. Of interest were reports that p21SDI1 also bound proliferating cell nuclear antigen (PCNA), an auxiliary protein for DNA polymerase delta, and inhibited DNA replication but not DNA repair in vitro. To better understand the function of this interaction in vivo, we first determined the region of p21SDI1 that was needed for PCNA binding. Analysis of deletion mutants of p21SDI1, which covered the majority of the protein, revealed that deletion of either amino acids 142-147 or 149-154 resulted in loss of ability to bind a glutathione S-transferase-PCNA fusion protein. Site-directed mutagenesis in this region led to the identification of the PCNA binding motif RQXXMTXFYXXXR and demonstrated that mutation of either amino acid Met-147 or Phe-150 resulted in almost complete ablation of PCNA binding. Interestingly, when we determined DNA synthesis inhibitory activity of deletion mutants or point mutants that were unable to bind Cdk2 and/or PCNA, we found that loss of binding to PCNA did not affect inhibitory activity, whereas lack of Cdk2 binding greatly reduced the same. This result suggests that the primary mechanism for inhibition of DNA synthesis by p21SDI1 occurs via inhibition of Cdk activity.
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PMID:The C-terminal region of p21SDI1/WAF1/CIP1 is involved in proliferating cell nuclear antigen binding but does not appear to be required for growth inhibition. 761 95

DNA damage increases p53 protein levels and activates transcription of the p21 gene. The p21 protein binds to and inhibits cdk2 kinase, causing G1 arrest. Here, we have investigated if a p53 fusion protein is a substrate for cdk2 kinase in vitro. Cdk2 kinase was immunoprecipitated from NIH3T3 cells and allowed to phosphorylate a human p53-GST (glutathione-s-transferase) fusion protein. Cdk2 and cyclin E-cdk2 efficiently phosphorylated both wild-type (wt) and mutant p53-GST. Cdk2 immunoprecipitated from cells in Go and early G1 exhibited minimal p53 kinase activity, whereas cells in S-phase displayed high levels of p53 kinase activity. If NIH3T3 cells were X-ray irradiated to induce DNA damage, cdk2 p53 kinase activity was rapidly inhibited within 1 h, but had recovered by 4 h post irradiation. Mutation of serine 315 of p53 to alanine (p53-S315A) abolished phosphorylation by cdk2 kinase. However, wtp53 and p53-S315A were equally effective at activating transcription when cotransfected with a p53 reporter construct. The results demonstrate that ser 315 of p53 is phosphorylated by cdk2 in vitro. However, ser 315 of wtp53 is not required for transcriptional activity in vivo, suggesting that cdk2 phosphorylation of p53 may be involved in regulating other cellular functions of wtp53.
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PMID:Cdk2 kinase phosphorylates serine 315 of human p53 in vitro. 762 34

We have shown that staurosporine (STSP) arrests normal human diploid fibroblasts in the G1 phase of the cell cycle at a time approximately 3 h after release from low-serum-induced G0 arrest. This initial temporal mapping of the STSP-induced restriction point was based on flow cytometric analyses that measured the onset of DNA synthesis after release from STSP and low-serum treatment. Here we show that the STSP-mediated arrest point distinctly differs from low-serum G0 arrest. We have found that cyclin D1 is expressed in STSP-arrested G1 fibroblasts but not in low-serum-arrested G0 fibroblasts, whereas cyclin-dependent kinase 4 (cdk4) and proliferating cell nuclear antigen (PCNA) are equivalently expressed under conditions of both STSP treatment and serum deprivation. Cdk4/cyclin D1/PCNA complexes are also formed in STSP-arrested G1 fibroblasts, but they are absent in serum-deprived G0 cells. The formation of cdk4/cyclin D1/PCNA complexes was found to coincide with the transcription and synthesis of cyclin D1, which indicates that the lack of available cyclin D1 is the limiting factor in cdk4/cyclin D1/PCNA complex formation in serum-deprived fibroblasts. This conclusion was further supported by the observation that cyclin D1-GST fusion protein binds cdk4 and PCNA when added to G0 cell extracts. Circumstantial evidence obtained in our studies and by other investigators suggests that STSP-induced arrest may be due to the inhibition of cdk-activating kinase.
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PMID:CDK4/cyclin D1/PCNA complexes during staurosporine-induced G1 arrest and G0 arrest of human fibroblasts. 766 38

Characterization of cdk (cyclin dependent kinases) substrates and studies of their regulation require purified enzymatic complexes of cdc2-related catalytic and cyclin regulatory subunits. We produced human Cdc2 kinase in the fission yeast Schizosaccharomyces pombe as a fusion protein with glutathione S-transferase (GST). The GST-human Cdc2p fusion protein was active in vivo since it rescued a temperature-sensitive allele of cdc2. The fusion protein was purified using a one-step chromatography procedure with glutathione-Sepharose and exhibited a catalytic activity in vitro. Yeast cyclin B and suc1 were found in association with GST-Cdc2. A 17-fold stimulation of GST-Cdc2 kinase activity was obtained by incubation of recombinant human cyclin A with the S. pombe cellular extract prior to affinity purification. This indicates that cyclin concentration is limiting in this overexpression system. These findings describe a fast and easy production of active recombinant human Cdc2 kinase in yeast that can be used for biochemical studies.
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PMID:Characterization of an active GST-human Cdc2 fusion protein kinase expressed in the fission yeast Schizosaccharomyces pombe: a new approach to the study of cell cycle control proteins. 772 98

Members of the recently discovered family of cyclin-dependent kinases inhibitors (CKIs) appear to play an essential regulatory role in the control of cell proliferation. To investigate the molecular basis of the interaction between these proteins and the cyclin-dependent kinases (CDKs), we performed a systematic mutagenesis of the CKI family member p21Cip1 using the alanine-scanning strategy. We have examined the interaction between in vitro translated human cdk2, cyclins A and D1, purified proliferating cell nuclear antigen (PCNA) and a set of human p21Cip1 mutants fused to glutathione S-transferase. Independent domains that are required for the interaction with cdk2 and with PCNA have been identified. The cdk2 binding domain is located in the N-terminal part of the protein, between residues 45 and 60, a region that is fully conserved in the p27Kip1 inhibitor. A PCNA binding region was localised to the C-terminus of the protein, between residues 142 and 163. These findings define protein motifs that are highly conserved between members of the CKI family and that are likely to play an essential function in the regulation of the G1/S transition.
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PMID:Identification of binding domains on the p21Cip1 cyclin-dependent kinase inhibitor. 778 76

L6 cells are committed skeletal muscle precursors which can be induced to differentiate into multinucleated, terminally differentiated myotubes. Upon differentiation, these immature skeletal myotubes enter a quiescent state and are unable to reenter the cell cycle. We have examined expression of a series of genes involved in regulation of progression through the G1/S boundary in undifferentiated L6 cells and during terminal differentiation of L6 myoblasts. While no change in the level of cyclin D1 transcript and a transient increase in cyclin D2 transcript were observed, a large increase in cyclin D3 expression was found. Immunohistochemistry demonstrated strong staining for cyclin D3 protein in the nuclei of the multinucleated myotubes from 4 independent myoblast cell lines; L6, L8, G8 and C2C12. Immunoprecipitation confirmed a greater than 20-fold increase in the levels of cyclin D3 protein in the differentiated L6 myotubes as well as its association with a number of proteins. Western assays demonstrated, further, that cyclin D3 was complexed with the cyclin dependent-kinases, cdk2 and cdk4, in differentiated L6 cells. However, while kinase activity specific for a GST-pRB fusion protein was seen for cyclin D3-containing complexes isolated from undifferentiated cells, the high levels of cyclin D3 in the differentiated myotubes had no associated kinase activity. These data demonstrate that cyclin D3 may also have a function in terminally differentiated, quiescent cells. The lack of cyclin D3-associated kinase activity and its association with a number of different proteins suggest that cyclin D3 may regulate the function of other proteins by direct interaction with these factors.
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PMID:Expression of the positive regulator of cell cycle progression, cyclin D3, is induced during differentiation of myoblasts into quiescent myotubes. 782 68

Transcription factor E2F-1 has a putative consensus sequence for phosphorylation by cyclin dependent kinase (Ser-Pro-X-Lys/Arg). Therefore, we studied the phosphorylation of E2F-1 in vivo and in vitro and its biological functions. E2F-1 was prepared by immunoprecipitation with anti-E2F-1 antibody from IMR32 lysates and was effectively phosphorylated by human cyclin A-cdk2 which was expressed in insect cells using baculovirus system. GST-E2F-1 was phosphorylated by cyclin A-cdk2 more efficiently than by cyclin E-cdk2. Cyclin D1-cdk4 phosphorylated pRB but scarcely phosphorylated GST-E2F-1 or H1 histone. The 60 kd protein precipitated with anti-E2F-1 antibody was phosphorylated in vivo. Phospho-peptide mapping indicated that its cleavage profile was identical with that of E2F-1 phosphorylated by cyclin A-cdk2 in vitro. This 60 kd protein, which is likely to be E2F-1, was not phosphorylated during the G0 and early G1 phase. Phosphorylation of E2F-1 began from the S phase while phosphorylation of pRB started nearly at G1/S. The in vivo phosphorylation of E2F-1 was inhibited by butyrolactone I, a cyclin-dependent kinase inhibitor (Kitagawa et al., 1993, Oncogene, 8, 2425-2432). The binding of E2F-1 to E2 promoter was found to be reduced by phosphorylation of E2F-1 by cyclin A-cdk2, suggesting that phosphorylation of E2F-1 may induce shut off of gene expression at the transcriptional level. These results suggest that E2F-1 is phosphorylated by cyclin A-cdk2 in the S phase in vivo as well as in vitro and that its phosphorylation by cyclin A-cdk2 may modulate its activity.
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PMID:Phosphorylation of E2F-1 by cyclin A-cdk2. 783 23

In Saccharomyces cerevisiae, transient accumulation of G1 cyclin/p34CDC28 (Cdc28p) complexes induces cells to traverse the cell cycle Start checkpoint and commit to a round of cell division. To investigate posttranslational controls that modulate Cdc28p activity during the G1 phase, we have reconstituted cyclin-dependent activation of Cdc28p in a cyclin-depleted G1 extract. A glutathione S-transferase-G1 cyclin chimera (GST-Cln2p) efficiently binds to and activates Cdc28p as a histone H1 kinase. Activation of Cdc28p by GST-Cln2p requires ATP, crude yeast cytosol, and the conserved Thr-169 residue that serves in other organisms as a substrate for phosphorylation by cyclin-dependent protein kinase-activating kinase. This assay may be useful for distinguishing genes that promote directly the posttranslational assembly of active Cln2p/Cdc28p kinase complexes from those that stimulate the accumulation of active complexes via a positive-feedback loop that governs synthesis of G1 cyclins.
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PMID:G1 cyclin-dependent activation of p34CDC28 (Cdc28p) in vitro. 786 57

The exchange rate of cyclin A and cyclin B between endogenous p34cdc2 and bacterially expressed GST-cdc2 was measured in concentrated Xenopus egg extracts. The half-lives of the cyclin A.p34cdc2 and the cyclin B.p34cdc2 complexes are estimated as 4 and 15 h, respectively. There is no significant difference in these affinities when they are measured in mitosis, interphase, or during transition between these two states. The tight association between cyclin and p34cdc2 in the cell cycle may be significant for mechanisms of cyclin destruction.
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PMID:Cyclin A and cyclin B dissociate from p34cdc2 with half-times of 4 and 15 h, respectively, regardless of the phase of the cell cycle. 796 81


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