Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.11.1 (protein kinase)
 81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Butyrolactone I is a selective inhibitor of the cyclin-dependent kinase (cdk) family. It inhibits both cdk2 and cdc2 kinase, but scarcely affects C-kinase, A-kinase, casein kinases, MAP kinase or EGF receptor-tyrosine kinase (Kitagawa et al., 1993, Oncogene, 8, 2425-2432). We studied the effects of butyrolactone I on the cell cycle as well as on phosphorylation of retinoblastoma protein (pRB). Butyrolactone I inhibited phosphorylation of pRB catalyzed by cyclin A-cdk2 produced by baculovirus in vitro. Furthermore, it inhibited phosphorylation of pRB and cell cycle progression from G1 to S phase in WI38 cell cultures. WI38 cells arrested at the G0 phase by serum starvation progressed in the cell cycle after serum stimulation. pRB was phosphorylated after 10 h serum stimulation. Incorporation of [3H]thymidine into the cells began to increase after 16 h serum stimulation. These processes were inhibited by butyrolactone I. Flow cytometric analysis showed that exposure to butyrolactone I inhibited progression of the cell cycle from G1 to S phase. These data suggested that initiation of DNA synthesis was inhibited by butyrolactone I and that the cell cycle was arrested in the G1 phase. Butyrolactone I also inhibited H1 histone phosphorylation in human WI38 cells and their G2/M progression. tsFT210 cells, a temperature-sensitive cdc2 mutant cell line, were synchronized at G2/M at a nonpermissive temperature, butyrolactone I inhibited the cell cycle progression of these cells at G2/M at the permissive temperature. Thus butyrolactone I, a cyclin-dependent kinase family inhibitor, which prevented the phosphorylations of the cell cycle-regulating proteins pRB and H1 histone, inhibited the cell cycle at G1/S and G2/M, respectively. These results suggest that the phosphorylations of pRB and H1 histone may play crucial roles in G1/S and G2/M progression, respectively, although it is possible that phosphorylations of other proteins by cdks are involved in G1/S and G2/M progression.
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PMID:A cyclin-dependent kinase inhibitor, butyrolactone I, inhibits phosphorylation of RB protein and cell cycle progression. 805 18

The eukaryotic cell cycle is regulated by the sequential activation of cyclin-dependent kinases (CDKs). CDK activation is dependent on cyclin binding and phosphorylation of a conserved threonine (T161 in Cdc2) mediated by the CDK-activating kinase CAK. A CDK-related kinase, MO15 (ref. 10), has been identified as the catalytic subunit of CAK (refs 11-13). Here we use a yeast two-hybrid screen to show that a new human cyclin (cyclin H) is a MO15-associated protein. Cyclin H is a major MO15 partner in vivo and enhances the kinase activity of MO15 towards Cdk2/cyclin A. These findings demonstrate that a cyclin/kinase complex can function as a regulator of other cyclin/kinase complexes, and suggest that cyclin/kinase cascades may exist.
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PMID:A cyclin associated with the CDK-activating kinase MO15. 807 87

The human single-stranded-DNA-binding protein (HSSB, also called RP-A) is a trimeric complex (p70, p34, and p14) required for multiple functions in DNA transactions. We report here that the p34 subunit of HSSB was hyperphosphorylated by kinase activities present in G1 extract (obtained from HeLa cells in G1 phase) preincubated with human cyclin A. This hyperphosphorylated HSSB product included at least four species of p34 that migrated more slowly through denaturing polyacrylamide gels than the hypophosphorylated form. Fractionation of cyclin A-activated G1 extract identified two kinases involved in the hyperphosphorylation of HSSB p34: cdk-cyclin A complex and DNA-dependent p350 protein kinase (DNA-PK). Kinetic analysis revealed that in cyclin A-activated G1 extract, p34 was first phosphorylated by cdk-cyclin A prior to the action of DNA-PK. Addition of p21cip1, a specific inhibitor of cdk-cyclin A but not DNA-PK, nearly abolished the hyperphosphorylation of HSSB p34 in G1 extract preincubated with cyclin A. This suggests a requirement of the cdk-cyclin A activity for the phosphorylation of p34 by DNA-PK in G1 extract.
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PMID:Phosphorylation of the p34 subunit of human single-stranded-DNA-binding protein in cyclin A-activated G1 extracts is catalyzed by cdk-cyclin A complex and DNA-dependent protein kinase. 807 85

In normal human diploid fibroblasts, cyclins of the A, B, and D classes each associate with cyclin-dependent kinases (CDKs), proliferating cell nuclear antigen (PCNA), and p21, thereby forming multiple independent quaternary complexes. Upon transformation of diploid fibroblasts with the DNA tumor virus SV40, or its transforming tumor antigen (T), the cyclin D/p21/CDK/PCNA complexes are disrupted. In transformed cells, CDK4 totally dissociates from cyclin D, PCNA, and p21 and, instead, associates exclusively with a polypeptide of 16 kD (p16). Quaternary complexes containing cyclins A or B1 and p21/CDK/PCNA also undergo subunit rearrangement in transformed cells. Both PCNA and p21 are no longer associated with CDC2-cyclin B1 binary complexes. Cyclin A complexes no longer contain p21, and a new 19-kD polypeptide (p19) is found in association with cyclin A. The pattern of subunit rearrangement of cyclin-CDK complexes in SV40-transformed cells is also shared in those containing adeno- or papilloma viral oncoproteins. Rearrangement also occurs in p53-deficient cells derived from Li-Fraumeni patients that carry no known DNA tumor virus. These findings suggest a mechanism by which oncogenic proteins alter the cell cycle of transformed cells.
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PMID:Subunit rearrangement of the cyclin-dependent kinases is associated with cellular transformation. 810 26

The immunosuppressant rapamycin (RAP) is a potent inhibitor of the entry of interleukin (IL)-2-stimulated T cells into S-phase. Earlier results indicated that RAP treatment arrested the growth of the murine IL-2-dependent T cell line CTLL-2 in late G1-phase. To explore further the interactions of RAP with the cell cycle control machinery in T cells, we examined the effects of RAP treatment on the activation of the cyclin-dependent kinases p34cdc2 and p33cdk2 in G1-phase CTLL-2 cells. Stimulation of factor-deprived cells with IL-2 led to the assembly of high molecular weight complexes containing active p34cdc2 and p33cdk2. The appearance of these complexes was explained, at least in part, by the association of both cyclin-dependent kinases with IL-2-induced cyclin A. RAP treatment profoundly inhibited both cyclin A expression and the appearance of active cyclin A-cyclin-dependent kinase complexes in IL-2-stimulated, late G1-phase CTLL-2 cells. Although p34cdc2 activation was largely dependent on association with cyclin A, a significant proportion of the active p33cdk2 pool was complexed with cyclin E. In contrast to cyclin A, the IL-2-induced accumulation of cyclin E in G1-phase cells was only partially suppressed by RAP, and cyclin E-p33cdk2 complexes were readily detected in drug-treated cells. These cyclin E-cyclin-dependent kinase complexes were nonetheless devoid of histone H1 kinase activity. The inhibitory effects of RAP on the activation of cyclin E- and cyclin A-associated cyclin-dependent kinases suggest that one or both events participate in the regulation of T cell entry into S-phase.
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PMID:Rapamycin inhibition of interleukin-2-dependent p33cdk2 and p34cdc2 kinase activation in T lymphocytes. 822 84

The cyclin-dependent kinase Cdk2 associates with cyclins A, D, and E and has been implicated in the control of the G1 to S phase transition in mammals. To identify potential Cdk2 regulators, we have employed an improved two-hybrid system to isolate human genes encoding Cdk-interacting proteins (Cips). CIP1 encodes a novel 21 kd protein that is found in cyclin A, cyclin D1, cyclin E, and Cdk2 immunoprecipitates. p21CIP1 is a potent, tight-binding inhibitor of Cdks and can inhibit the phosphorylation of Rb by cyclin A-Cdk2, cyclin E-Cdk2, cyclin D1-Cdk4, and cyclin D2-Cdk4 complexes. Cotransfection experiments indicate that CIP1 and SV40 T antigen function in a mutually antagonistic manner to control cell cycle progression.
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PMID:The p21 Cdk-interacting protein Cip1 is a potent inhibitor of G1 cyclin-dependent kinases. 824 51

Rapamycin (RAP) inhibits several biologic responses in the YAC-1 T cell lymphoma, including the serum-driven proliferation and cyclin A mRNA expression, the induction of Ly-6E Ag expression by IFN, and the induction of IFN-gamma production by IL-1. RAP also suppresses the enzymatic activity of the 70 kDa S6 protein kinase (pp70s6k). To define the mechanistic relationship between these multiple effects of RAP, we have generated stable somatic mutants with altered sensitivities to this drug. A first series of mutants, represented by the R19, 4R16, and 10R13 clones, showed markedly reduced sensitivity to the inhibitory effect of RAP on all biologic responses tested and on pp70s6k activity. Two other mutant types, R103 and R125, were both highly sensitive to RAP-mediated suppression of proliferation, of IL-1-induced IFN-gamma production, and of pp70s6k activity but differed in their Ly-6E response. This response was not affected by RAP in the R125 clone and was enhanced in the R103 clone. Therefore, the inhibitory effects of RAP on proliferation and IL-1-mediated IFN-gamma induction both appear associated with the inhibition of pp70s6k activity, whereas the modulation of Ly-6E induction is independent from the latter. Moreover, the cellular binding of [3H]dihydro-FK-506 was found to be blocked by RAP in all mutant types to the same extent as in wild-type YAC-1 cells, suggesting that the altered sensitivity to the effects of RAP in these mutants is not due to an inability of the drug to enter the cells or to interact with FKBP. Further biochemical characterization of the mutant cells described here is expected to help clarify the mechanisms of RAP action.
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PMID:Relationship between multiple biologic effects of rapamycin and the inhibition of pp70S6 protein kinase activity. Analysis in mutant clones of a T cell lymphoma. 830 Nov 50

The Cdc2 protein kinase requires cyclin binding for activity and also binds to a small protein, Suc1. Charged-to-alanine scanning mutagenesis of Cdc2 was used previously to localize cyclin A- and B- and Suc1-binding sites (B. Ducommun, P. Brambilla, and G. Draetta, Mol. Cell. Biol. 11:6177-6184, 1991). Those sites were mapped by building a Cdc2 model based on the crystallographic coordinates of the catalytic subunit of cyclic AMP-dependent protein kinase (cAPK) (D. R. Knighton, J. Zheng, L. F. Ten Eyck, V. A. Ashford, N.-H. Xuong, S. S. Taylor, and J. M. Sowadski, Science 253:407-414, 1991). On the basis of this model, additional mutations were made and tested for cyclin A and Suc1 binding and for kinase activity. Mutations that interfere with cyclin A binding are localized primarily on the small lobe near its interface with the cleft and include an acidic patch on the B helix and R-50 in the highly conserved PSTAIRE sequence. Two residues in the large lobe, R-151 and T-161, influence cyclin binding, and both are at the surface of the cleft near its interface with the PSTAIRE motif. Cyclin-dependent phosphorylation of T-161 in Cdc2 is essential for activation, and the model provides insights into the importance of this site. T-161 is equivalent to T-197, a stable phosphorylation site in cAPK. On the basis of the model, cyclin binding very likely alters the surface surrounding T-161 to allow for T-161 phosphorylation. The two major ligands to T-197 in cAPK are conserved as R-127 and R-151 in Cdc2. The equivalent of the third ligand, H-87, is T-47 in the PSTAIRE sequence motif. Once phosphorylated, T-161 is predicted to play a major structural role in Cdc2, comparable to that of T-197 in cAPK, by assembling the active conformation required for peptide recognition. The inhibitory phosphorylation at Y-15 also comes close to the cleft interface and on the basis of this model would disrupt the cleft interface and the adjacent peptide recognition site rather than prevent ATP binding. In contrast to cyclin A, both lobes influence Suc1 binding; however, the Suc1-binding sites are far from the active site. Several mutants map to the surface in cAPK, which is masked in part by the N-terminal 40 residues that lie outside the conserved catalytic core. The other Suc1-binding site maps to the large lobe near a 25-residue insert and includes R-215.
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PMID:A three-dimensional model of the Cdc2 protein kinase: localization of cyclin- and Suc1-binding regions and phosphorylation sites. 833 38

The mature adult alveolar epithelial cell (AEC) is a highly differentiated phenotype that does not readily divide and exhibits numerous specialized functions. Yet, transformed AEC proliferate aggressively in certain forms of lung cancer. Normal AEC also proliferate but in a coordinated manner during embryonic growth and fetal development as well as during lung repair. Therefore, biochemical mechanisms regulating the cell cycle in AEC are clearly of fundamental significance for understanding lung development, lung injury, and cancer. Cyclin A is a protein that varies in abundance during the cell cycle and regulates critical transition points through its association with cyclin-dependent protein kinase subunits. We postulated that high expression of cyclin A might be associated with rapid proliferation in transformed AEC. We compared the expression of cyclin A mRNA and protein in primary cultures of fetal and adult rat AEC, in the E1A-T2 neonatal rat AEC, and in the malignant A549 human AEC. We used pharmacologic blockades with mimosine, aphidicolin, and nocodazole for cell cycle synchronization, which was verified by fluorescence-activated cell sorter (FACS) analysis of cellular DNA content. Transformed cells (A549 and E1A-T2) exhibited a much higher level of expression for both cyclin A mRNA and protein than did normal rat AEC. Induction of cyclin A mRNA expression in A549 human AEC and E1A-T2 rat AEC occurred in late G1, prior to the onset of S phase. Fetal and adult rat AEC and rat E1A-T2 AEC expressed two cyclin A mRNA transcripts, whereas human A549 cells in S phase and M phase expressed three cyclin A mRNA transcripts. We conclude that transformed AEC overexpress cyclin A in comparison with primary AEC cultures, while retaining cell cycle-dependent differences in cyclin A expression. We speculate that cyclin A expression is regulated both at the transcriptional and post-transcriptional levels, and that cyclin A may play a key role in the increased proliferation of transformed AEC that is associated with the pathogenesis of lung cancer.
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PMID:Cyclin A expression in normal and transformed alveolar epithelial cells. 833 81

The mitotic inducer p34cdc2 requires association with a cyclin and phosphorylation on Thr161 for its activity as a protein kinase. CAK, the p34cdc2 activating kinase, was previously identified as an enzyme necessary for this activating phosphorylation. We confirm here that CAK is a protein kinase and describe its purification over 13,000-fold from Xenopus egg extracts. We further show that CAK contains a protein identical or closely related to the previously identified Xenopus MO15 gene: p40MO15 copurifies with CAK, and an antiserum to p40MO15 specifically depletes cAK activity. CAK appears to be the only protein in Xenopus egg extracts that can activate complexes of either p34cdc2 or the closely related protein kinase, p33cdk2, with either cyclin A or cyclin B. The sequence similarity between p40MO15 and p34cdc2, and the approximately 200 kDa size of CAK, suggest that p40MO15 may itself be regulated by subunit association and by protein phosphorylations.
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PMID:CAK, the p34cdc2 activating kinase, contains a protein identical or closely related to p40MO15. 834 52


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