p35, the neuronal-specific activator of cyclin-dependent kinase 5 (Cdk5) is degraded by the ubiquitin-proteasome pathway.
Cyclin-dependent kinase 5 (Cdk5) was originally isolated by its close homology to the human CDC2 gene, which is a key regulator of cell cycle progression. However, unlike other Cdks, the activity of Cdk5 is required in post-mitotic neurons. The neuronal-specific p35 protein, which shares no homology to cyclins, was identified by virtue of its association and activation of Cdk5. Gene targeting studies in mice have shown that the p35/Cdk5 kinase is required for the proper neuronal migration and development of the mammalian cortex. We have investigated the regulation of the p35/Cdk5 kinase. Here we show that p35, the activator of Cdk5, is a short-lived protein with a half-life (t1/2) of 20 to 30 min. Specific proteasome inhibitors such as lactacystin greatly stabilize p35 in vivo. Ubiquitination of p35 can be readily demonstrated in vitro and in vivo. Inhibition of Cdk5 activity by a specific Cdk inhibitor, roscovitine, or by overexpression of a dominant negative mutant of Cdk5 increases the stability of p35 by 2- to 3-fold. Furthermore, phosphorylation mutants of p35 also stabilize p35 2- to 3-fold. Together, these observations demonstrate that the p35/Cdk5 kinase can be subject to rapid turnover in vivo and suggest that phosphorylation of p35 upon Cdk5 kinase activation plays a autoregulatory role in p35 degradation mediated by ubiquitin-mediated proteolysis.
Patrick
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Howard Hughes Medical Institute, Department of Pathology, Harvard Medical School, Boston, Massachusetts 02115, USA. gpatrick@fas.harvard.edu
Zhou
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P
Kwon
Y T
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Howley
P M
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Tsai
L H
LH
D000111:Acetylcysteine / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D019556:COS Cells; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000378:metabolism; D051360:Cyclin-Dependent Kinase 5; D018844:Cyclin-Dependent Kinases; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D004622:Embryo, Mammalian; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006207:Half-Life; D006801:Humans; D007700:Kinetics; D008322:Mammals; D051379:Mice; D009097:Multienzyme Complexes / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D009474:Neurons / Q000166:cytology / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D046988:Proteasome Endopeptidase Complex; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D011687:Purines / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000378:metabolism; D000077546:Roscovitine; D017384:Sequence Deletion; D014162:Transfection; D014452:Ubiquitins / Q000378:metabolism*
D004791:Enzyme Inhibitors; D009097:Multienzyme Complexes; D009419:Nerve Tissue Proteins; D011687:Purines; D011994:Recombinant Proteins; D014452:Ubiquitins; C089495:neuronal Cdk5 activator (p25-p35); D000077546:Roscovitine; C067713:lactacystin; D051360:Cyclin-Dependent Kinase 5; D017346:Protein Serine-Threonine Kinases; C495905:CDK5 protein, human; C495906:Cdk5 protein, mouse; C495907:Cdk5 protein, rat; D018844:Cyclin-Dependent Kinases; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; D000111:Acetylcysteine