The N-terminal domains of cyclin-dependent kinase inhibitory proteins block the phosphorylation of cdk2/Cyclin E by the CDK-activating kinase.
It has been suggested that binding of p27 and p21 kinase inhibitory proteins (KIPs) to cyclin-dependent kinases (cdks) render them inaccessible to cdk-activating kinase (CAK), presumably by steric hindrance by the C-terminal residues. However, this common mechanism of inhibition is inconsistent with the known structural divergence in the p27 and p21 C-terminal domains. Therefore, we studied the direct binding of N-terminal minimal domain of p27 (amino acids 28-81) to cdk2/cyclin E. An unlabeled p27 minimal domain, mutated in the N-terminal LFG motif, was unable to compete with a labeled minimal domain for binding to cdk2/cyclin E. The p27 and its minimal domain inhibited CAK-mediated phosphorylation of cdk2/cyclin E. This inhibitory effect was significantly diminished with p27 minimal domain mutated in the LFG motif. A synthetic peptide, ACRRLFGPVDSE, from the N-terminal residues 17-28 of p21, was also a potent inhibitor of CAK-mediated cdk2/cyclin E phosphorylation. Taken together, these results show that anchoring of p27 or p21 KIPs to cyclin E via the N-terminal LFG-containing motif can block CAK access to its cdk2/cyclin E substrate.
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Protein Science, Pharmacia and Upjohn, Michigan, 49007, Kalamazoo.
Evans
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Sharma
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D000818:Animals; D042846:CDC2-CDC28 Kinases; D018797:Cell Cycle Proteins; D002460:Cell Line; D002845:Chromatography; D019927:Cyclin E / Q000378:metabolism*; D051357:Cyclin-Dependent Kinase 2; D050760:Cyclin-Dependent Kinase Inhibitor p27; D018844:Cyclin-Dependent Kinases / Q000378:metabolism*; D006801:Humans; D007313:Insecta; D008869:Microtubule-Associated Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D010455:Peptides / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012588:Scintillation Counting; D019809:Streptavidin / Q000378:metabolism; D025521:Tumor Suppressor Proteins; D000095043:Cyclin-Dependent Kinase-Activating Kinase
D018797:Cell Cycle Proteins; D019927:Cyclin E; D008869:Microtubule-Associated Proteins; D010455:Peptides; D011994:Recombinant Proteins; D025521:Tumor Suppressor Proteins; D050760:Cyclin-Dependent Kinase Inhibitor p27; D019809:Streptavidin; D017346:Protein Serine-Threonine Kinases; D042846:CDC2-CDC28 Kinases; C495894:CDK2 protein, human; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases; D000095043:Cyclin-Dependent Kinase-Activating Kinase