Recent advances on cyclins, CDKs and CDK inhibitors

doi:10.1016/S0962-8924(96)10055-6

Trends in Cell Biology

Volume 7, Issue 3, March 1997, Pages 95-98

https://doi.org/10.1016/S0962-8924(96)10055-6 Get rights and content

In eukaryotes, cell division is controlled by cyclin-dependent kinases (CDKs). Here we summarize a few new developments on the regulation of the cell cycle by CDK—cyclin complexes. We have focused on three aspects in which there has been recent progress: the structural analysis of these complexes, the phenotypes of mice carrying knockouts of CDK inhibitors and the role of proteolysis in the regulation of the cell cycle.

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It has been found that mammalian Cdk2-cyclin A complexes can phosphorylate B-Myb to enhance its transactivation activity (39, 44–47). We searched the G. lamblia genome database and identified a putative Cyclin A. Like human Cyclin A1, the giardial Cyclin A has two putative cyclin domains that may form α-fold to provide interaction surfaces for Cdks or regulatory molecules (86, 87). We found that Cyclin A is also highly expressed during encystation and there is an interaction between Cdk2 and Cyclin A-associated complexes (Fig. 8).
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Cyclin L is an RS domain protein involved in pre-mRNA splicing
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We report the cDNA cloning and functional characterization of human cyclin L, a novel cyclin related to the C-type cyclins that are involved in regulation of RNA polymerase II (pol II) transcription. Cyclin L also contains a COOH-terminal dipeptide repeat of alternating arginines and serines, a hallmark of the SR family of splicing factors. We show that recombinant cyclin L interacts with p110 PITSLRE kinase, and that cyclin L antibody co-immunoprecipitates a kinase activity from HeLa nuclear extracts that phosphorylates the carboxyl-terminal domain (CTD) of pol II and splicing factor SC35, and is inhibited by the cdk inhibitor p21. Cyclin L antibody inhibits the second step of RNA splicing in vitro, and recombinant cyclin L protein stimulates splicing under suboptimal conditions. Significantly, the IC₅₀ for splicing inhibition by p21 is similar to the IC₅₀ for inhibition of the cyclin L-associated kinase activity. Cyclin L and its associated kinase are thus new members of the pre-mRNA processing machinery.
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cdk2·cyclin E and cdk5·p25 are two members of the cyclin-dependent kinase family that are potential therapeutic targets for oncology and Alzheimer’s disease, respectively. In this study we have investigated the mechanism for these enzymes. Kinases catalyze the transfer of phosphate from ATP to a protein acceptor, thus utilizing two substrates, ATP and the target protein. For a two-substrate reaction, possible kinetic mechanisms include: ping-pong, sequential random, or sequential ordered. To determine the kinetic mechanism of cdk2·GST-cyclin E and cdk5·GST-p25, kinase activity was measured in experiments in which concentrations of peptide and ATP substrates were varied in the presence of dead-end inhibitors. A peptide identical to the peptide substrate, but with a substitution of valine for the phosphoacceptor threonine, competed with substrate with a K _i value of 0.6 mm. An aminopyrimidine, PNU 112455A, was identified in a screen for inhibitors of cdk2. Nonlinear least squares and Lineweaver-Burk analyses demonstrated that the inhibitor PNU 112455A was competitive with ATP with a K _i value of 2 μm. In addition, a co-crystal of PNU 112455A with cdk2 showed that the inhibitor binds in the ATP binding pocket of the enzyme. Analysis of the inhibitor data demonstrated that both kinases use a sequential random mechanism, in which either ATP or peptide may bind first to the enzyme active site. For both kinases, the binding of the second substrate was shown to be anticooperative, in that the binding of the first substrate decreases the affinity of the second substrate. For cdk2·GST-cyclin E the kinetic parameters were determined to be K _m_{, ATP} = 3.6 ± 1.0 μm, K _m_{, peptide} = 4.6 ± 1.4 μm, and the anticooperativity factor, α = 130 ± 44. For cdk5·GST-p25, theK _m_{, ATP} = 3.2 ± 0.7 μm, K _m_{, peptide} = 1.6 ± 0.3 μm, and α = 7.2 ± 1.8.
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Although nearly 60% of mesotheliomas contain SV40 early region DNA sequences, the role of T/t antigens in initiating and maintaining the transformed state of mesothelioma cells remains unclear. The majority of mesothelioma cells which contain SV40 early region sequences exhibit extremely low basal expression of SV40 oncoproteins; however, T/t antigen expression can be induced under conditions of cellular stress. Abrogation of SV40 T/t expression by antisense techniques induces apoptosis in part via restoration of p53 function, and enhances chemosensitivity in SV40 (+) MPM cells by mechanisms which have not been fully elucidated. This review briefly summarizes our ongoing efforts to define the role of SV40 oncoproteins in modulating the malignant phenotype of mesothelioma cells, and highlights strategies which may prove efficacious in vivo for circumventing SV40 T/t antigen expression in mesotheliomas.

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Trends in Cell Biology

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Review
Recent advances on cyclins, CDKs and CDK inhibitors