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J Neurochem ; 102(5): 1477-1487, 2007 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-17394551

RESUMO

Cyclin-dependent kinase 5 (Cdk5) is a proline-directed Ser/Thr kinase that plays important roles in various neuronal activities, including neuronal migration, synaptic activity, and neuronal cell death. Cdk5 is activated by association with a neuron-specific activator, p35 or its isoform p39, but little is known about the kinase activity of Cdk5--p39. In fact, kinase-active Cdk5--p39 was not prepared from rat brain extracts nor from HEK293 cells expressing Cdk5 and p39 by immunoprecipitation in the presence of non-ionic detergent, under conditions with which active Cdk5--p35 could be isolated. p39 dissociated from Cdk5 in the presence of detergent, indicating that p39 has a lower binding affinity for Cdk5 than p35. We developed a method for purifying kinase-active Cdk5--p39 from Sf9 cells infected with baculovirus encoding Cdk5 and p39. The purified Cdk5--p39 complex showed similar substrate specificity to that of Cdk5--p35, but with opposite sensitivity to detergent. Cdk5--p39 was inactivated by Triton X-100, whereas Cdk5--p35 was activated. The N-terminal deletion from p35 and p39, the amino acid sequences of which are different, did not change the stability or substrate specificity of either Cdk5 complex. The different stability between Cdk5--p35 and Cdk5--p39 suggests their distinct roles under different regulation mechanisms in neurons.


Assuntos
Quinase 5 Dependente de Ciclina/metabolismo , Complexos Multienzimáticos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Animais , Animais Recém-Nascidos , Linhagem Celular Transformada , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/metabolismo , Ácidos Cólicos/farmacologia , Quinase 5 Dependente de Ciclina/genética , Relação Dose-Resposta a Droga , Ativação Enzimática , Expressão Gênica/efeitos dos fármacos , Humanos , Imunoprecipitação , Insetos , Masculino , Octoxinol/farmacologia , Ratos , Ratos Wistar , Tensoativos/farmacologia , Transfecção/métodos
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