Gene/Protein Disease Symptom Drug Enzyme Compound
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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 p34cdc2 protein kinase is a component of maturation-promoting factor, the master regulator of the cell cycle in all eukaryotes. The activity of p34cdc2 is itself tightly regulated by phosphorylation and dephosphorylation. Predicted regulatory phosphorylation sites of Xenopus p34cdc2 were mutated in vitro, and in vitro-transcribed RNAs were injected into Xenopus oocytes. The cdc2 single mutants Thr-14----Ala and Tyr-15----Phe did not induce germinal vesicle breakdown (BVBD) upon microinjection into oocytes. In contrast, the cdc2 double mutant Ala-14/Phe-15 did induce GVBD. Both the Ala-14 and Ala-14/Phe-15p34cdc2 mutants were shown to coimmunoprecipitate cyclin B1 and to phosphorylate histone H1 in immune complex kinase assays. Microinjection of antisense oligonucleotides to c-mosXe was used to demonstrate the role of mos protein synthesis in the induction of GVBD by the Ala-14/Phe-15 cdc2 mutant. Thr-161 was also mutated. p34cdc2 single mutants Ala-161 and Glu-161 and triple mutants Ala-14/Phe-15/Ala-161 and Ala-14/Phe-15/Glu-161 failed to induce GVBD in oocytes and showed a decreased binding to cyclin B1 in coimmunoprecipitations. Each of the cdc2 mutants was also assayed by coinjection with cyclin B1 or c-mosXe RNA into oocytes. Several of the cdc2 mutants were found to affect the kinetics of cyclin B1 and/or mos-induced GVBD upon coinjection, although none affected the rate of progesterone-induced maturation. We demonstrate here the significance of Thr-14, Tyr-15, and Thr-161 of p34cdc2 in Xenopus oocyte maturation. In addition, these results suggest a regulatory role for mosXe in induction of oocyte maturation by the cdc2 mutant Ala-14/Phe-15.
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PMID:Requirement of mosXe protein kinase for meiotic maturation of Xenopus oocytes induced by a cdc2 mutant lacking regulatory phosphorylation sites. 137 75

Cys-cdc2(8-20), a synthetic peptide derived from p34cdc2, was previously reported to be a specific and efficient substrate of a pp60c-src-related tyrosine kinase isolated from bovine spleen (the spleen tyrosine kinase) (Litwin, C.M.E., Cheng, H.-C., and Wang, J.H. (1991) J. Biol. Chem. 266, 2557-2566). The longer peptide, cdc2(1-24), was found to be phosphorylated by the kinase with similar efficiency, and Tyr15 was the only amino acid residue phosphorylated. This indicated that the amino acid sequence of cdc2(8-20) peptide, EKI-GEGTYGVVYK, contained the structural features important for protein tyrosine kinase substrate activity. A stepwise procedure using synthetic peptides was employed to investigate such structural features. First, a computer search of protein sequences homologous to cdc2(8-20) uncovered five protein kinases containing homologous sequence with tyrosine at a position corresponding to Tyr15 of p34cdc2. Second, a peptide derived from ribosomal S6 protein kinase (rsk(436-456] was synthesized. The rsk(436-456) peptide contained a segment, ETIGVGSYSVCKR, which is highly homologous to that of cdc2(8-20). It was found to be a very poor substrate of the spleen tyrosine kinase. Third, peptide analogs of cdc2(6-20) with single substitutions of amino acid residues Lys9, Glu12, Thr14, Gly16, Val18, and Tyr19 by amino acid residues at corresponding positions of rsk were synthesized and tested as spleen tyrosine kinase substrates. Only Glu12 and Thr14 substituted peptide analogs showed decreased substrate activities. (The substrate activity of a peptide is the ability of the peptide to serve as the substrate of the spleen tyrosine kinase. It was determined of the spleen tyrosine kinase. It was determined either by the kinetic parameters (Km and Vmax) of phosphorylation of the peptide or by the initial phosphorylation rate of the peptide by the spleen tyrosine kinase.) An analog with double substitution at Glu12 An analog with double substitution at Glu12 and Thr14 was found to be almost as poor a substrate as the rsk peptide. In addition, peptide analogs with Tyr15 substituted by Phe or D-Tyr had poor substrate activities as well as weak inhibitory activities. Thus, Glu12, Thr14, and Tyr15 residues of p34cdc2 contained structural components essential for the efficient phosphorylation of the peptides derived from p34cdc2 by the pp60c-src-related spleen tyrosine kinase.
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PMID:Structural basis of specific and efficient phosphorylation of peptides derived from p34cdc2 by a pp60src-related protein tyrosine kinase. 171 44

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

G0S24 is a member of a set of genes (putative G0/G1 switch regulatory genes) that are expressed transiently within 1-2 hr of the addition of lectin or cycloheximide to human blood mononuclear cells. Comparison of a full-length cDNA sequence with the corresponding genomic sequence reveals an open reading frame of 326 amino acids, distributed across two exons. Potential phosphorylation sites include the sequence PSPTSPT, which resembles an RNA polymerase II repeat reported to be a target of the cell cycle control kinase cdc2. Comparison of the derived protein sequence with those of rodent homologs allows classification into three groups. Group 1 contains G0S24 and the rat and mouse TIS11 genes (also known as TTP, Nup475, and Zfp36). Members of this group have three tetraproline repeats. Groups 1 and 2 have a serine-rich region and an "arginine element" (RRLPIF) at the carboxyl terminus. All groups contain cysteine- and histidine-rich putative zinc finger domains and a serine-phenylalanine "SFS" domain similar to part of the large subunit of eukaryotic RNA polymerase II. Comparison of group 1 human and mouse genomic sequences shows high conservation in the 5' flank and exons. A CpG island suggests expression in the germ line. G0S24 has potential sites for transcription factors in the 5' flank and intron; these include a serum response element. Protein and genomic sequences show similarities with those of a variety of proteins involved in transcription, suggesting that the G0S24 product has a similar role.
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PMID:A human putative lymphocyte G0/G1 switch gene homologous to a rodent gene encoding a zinc-binding potential transcription factor. 842 74

We previously presented evidence that a Z-Phe-Ser-argininal-susceptible protease which is involved in oocyte maturation of the starfish, Asterina pectinifera is the proteasome (Takagi Sawada et al, Dev. Biol. 150, 414-418 (1992)). In the present study, we investigated the timing of the function of and the role of the protease in oocyte maturation using Z-Phe-Ser-argininal. By adding the inhibitor in maturing oocytes at various times after 1-methyladenine treatment, the inhibitory ability was markedly reduced in half the time required for germinal vesicle breakdown. Furthermore, the inhibitor potently blocked the activation of histone H1 kinase and the dephosphorylation of cdc2 kinase during oocyte maturation. These results indicate that the Z-Phe-Ser-argininal-susceptible protease, probably the proteasome, plays a key role in the step of the signal transduction pathway that triggers the dephosphorylation of cdc2 kinase in response to the maturation-inducing hormone.
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PMID:Protease triggers dephosphorylation of cdc2 kinase during starfish oocyte maturation. 913 20

The carboxy terminus of protein phosphatase 2A (PP2A) catalytic subunit is highly conserved. Seven out of the last nine residues, including two potential in vivo phosphorylation sites, threonine 304 and tyrosine 307, are completely invariant in all known PP2As. Mutational analysis of the carboxy terminus in vivo was facilitated by efficient immunoprecipitation of trimeric PP2A holoenzyme via an epitope-tagged catalytic subunit. The results indicate that the catalytic subunit carboxy terminus is important for complex formation with the PP2A 55 kDa regulatory B subunit, but not with polyomavirus oncogene, middle tumor antigen (MT), a viral B-type regulatory subunit. Replacing catalytic subunit threonine 304 or tyrosine 307 with a negatively charged amino acid abolished binding of the B subunit to the dimeric enzyme core and altered substrate specificity. Certain other amino acid substitutions of different size and/or charge also abolished or greatly reduced B subunit binding. Substitution of alanine at position 304 or phenylalanine at position 307 did not dramatically reduce B subunit binding or phosphatase activity in vitro, yet the latter substitutions are not found in naturally occurring PP2As. Thus, the wild-type residues are important for a yet unknown function in vivo. Additionally, deleting the carboxy terminal nine amino acids inhibited binding of the B subunit to the dimeric enzyme core, indicating a requirement for one or more of these amino acids for complex formation. MT interaction with the dimeric PP2A enzyme core was not inhibited by any of these mutations. Finally, unlike B subunit, MT does not activate the phosphatase activity of the PP2A heterodimer towards cdc2-phosphorylated histone H1.
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PMID:Protein phosphatase 2A subunit assembly: the catalytic subunit carboxy terminus is important for binding cellular B subunit but not polyomavirus middle tumor antigen. 928 86

Tyr-Phe and Met limitation in vitro inhibited cell proliferation and proliferating cell nuclear antigen (PCNA) expression to a greater extent than serum limitation. Tyr-Phe and serum limitation arrested cells in the G0/G1 phase; Met limitation blocked cells in the G0/G1 and S phases. Tyr-Phe limitation progressively decreased cyclin D1 expression to 30% of control within four days and did not affect expression of cyclin D3 or cyclin-dependent kinase (CDK2, CDK4, and CDK5) expression, Met limitation decreased cyclin D3 expression to 25% of control and CDK2 expression to 32% of control by Day 4 and did not affect expression of cyclin D1, CDK4, and CDK5. Serum limitation inhibited cyclin D1 and cyclin D3 expression to 24% of control after four days and did not effect CDK expression. Expression of two CDK inhibitors, p21WAF1/Cip1 and p27Kip1, was not changed by amino acid or serum limitation. Dietary restriction of Tyr-Phe in mice bearing subcutaneous B16BL6 melanoma tumors decreased tumor growth rate compared with mice fed a normal diet. Tumors from Tyr-Phe-restricted mice exhibited decreased PCNA expression, G0/G1 phase cell cycle arrest, and reduced cyclin D1 expression. These data indicate that decreased tumor growth in vivo associated with dietary restriction of Tyr and Phe is cell cycle specific.
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PMID:Tyrosine and phenylalanine restriction induces G0/G1 cell cycle arrest in murine melanoma in vitro and in vivo. 942 72

A cdk2 binding domain on p27Kip1 located within the sequence of amino acids 53-85 was further characterized by generating a series of point mutations within amino acid residues 62-75. Two regions, FDF (residues 62-64) and GXY (residues 72 and 74), were identified within the beta hairpin region of p27Kip1. Mutations within these regions essentially completely inhibited the binding to in vitro translated cdk2 and cdk2/cyclin E complexes formed in vitro or in vivo. The p27Kip1 GST-fusion protein of the point mutation that replaces phenylalanine at residue 64 to alanine (F64A) showed approximately twofold less inhibition of cdk2 kinase activity. The cellular response to the introduction of the F64A mutant form of p27Kip1 was compared to that of p27Kip1 wild type by transfecting HeLa cells with constructs of full length sense and antisense coding sequences. Overexpression of the F64A mutant form of p27Kip1 bound significantly lower levels of cdk2 as compared to wild type and did not affect the cdk2 related kinase activity of the transfected HeLa cells. Overexpression of wild type p27Kip1 resulted in a reduction of the level of cdk2 kinase activity and effectively suppressed the growth of the transfected HeLa cells.
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PMID:Identification of cdk2 binding sites on the p27Kip1 cyclin-dependent kinase inhibitor. 948 39

HIV-1 Vpr induces cell cycle G2 arrest, morphological changes and cell death in human and fission yeast cells. The cellular targets for G2 arrest were expected to be the inhibitory phosphorylation sites of Cdc2, as G2 arrest correlates with hyperphosphorylation and decreased activity of Cdc2 in both human and fission yeast cells. In this study, we present direct evidence of genetic suppression of Vpr-induced G2 arrest by cdc2 mutations. Mutations in cdc2 (cdc2-1w and cdc2-3w) reduce the ability of Vpr to induce G2 arrest. A strain with a mutation changing the Tyr15 of Cdc2 to the non-phosphorylated Phe (Y15F) eliminated Vpr-induced G2 arrest indicating that Tyr15 of Cdc2 is the sole target for induction of G2 arrest by Vpr. Although the G2 arrest induced by DNA damage also proceeds through phosphorylation of Tyr15, the rad1, rad3, rad9 and rad17 mutations, which eliminate the G2 checkpoint for DNA damage, did not block the G2 arrest induced by Vpr. Furthermore, Vpr expression did not alter sensitivity of these rad mutants to UV radiation. Thus, the pathways for the induction of G2 arrest by DNA damage and Vpr are not identical. Interestingly, Vpr still induces cell death and morphological changes in the Y15F Cdc2 strain indicating that G2 arrest is not required for morphological changes and cell death. This conclusion was further supported by the observation that mutations in Vpr, which have lost their ability to induce G2 arrest, retained the ability to kill cells.
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PMID:Cell cycle G2 arrest induced by HIV-1 Vpr in fission yeast (Schizosaccharomyces pombe) is independent of cell death and early genes in the DNA damage checkpoint. 1095 88

P19(INK4d) is a tumor suppressing protein and belongs to a family of cyclin D-dependent kinase inhibitors of CDK4 and CDK6, which play a key role in human cell cycle control. P19 comprises ten alpha-helices arranged sequentially in five ankyrin repeats forming an elongated structure. This rather simple topology, combined with its physiological function, makes p19 an interesting model protein for folding studies. Urea-induced unfolding transitions monitored by far-UV CD and phenylalanine fluorescence coincide and suggest a two-state mechanism for equilibrium unfolding. Unfolding of p19 followed by 2D (1)H-(15)N HSQC spectra revealed a third species at moderate urea concentrations with a maximum population of about 30 % near 3.2 M urea. It shows poor chemical shift dispersion, but cross-peaks emerge for some residues that are distinct from the native or unfolded state. This equilibrium intermediate either arises only at high protein concentrations (as in the NMR experiment) or has similar optical properties to the unfolded state. Stopped-flow far-UV CD experiments at various urea concentrations revealed that alpha-helical structure is formed in three phases, of which only the fastest phase (10 s(-1)) depends upon the urea concentration. The kinetic of the slowest phase (0.017 s(-1)) can be resolved by 1D real-time NMR and accelerated by cyclophilin. It is limited in rate by prolyl isomerization, and native-like ordered structure cannot form prior to this isomerization. The two fast phases lead to 83 % native protein within the dead time of the NMR experiment. In contrast to p16(INK4a), which exhibits only a marginal stability and high unfolding rates, p19 shows the expected stability for a protein of this size with a clear kinetic barrier between the unfolded and folded state. Therefore, p19 might complement the function of less stable INK4 inhibitors in cell cycle control under unfavorable conditions.
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PMID:Protein folding and stability of human CDK inhibitor p19(INK4d). 1178 24


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