Expression and purification of Cdk5/p25 by the baculovirus system.

• Main Authors: Ya-Lin Hung, 洪雅琳
• Other Authors: Hsien-Bin Huang
• Format: Others
• Language: zh-TW
• Published: 2009
• Online Access: http://ndltd.ncl.edu.tw/handle/52845444378730122245

碩士 === 國立中正大學 === 生命科學系暨分子生物研究所暨生物醫學研究 === 97 === Cyclin dependent kinase 5(Cdk5) is a serine/threonine kinase with close homology to other Cdks. Cdk5 kinase activity is detected only in the post-mitotic neurons and it is required for proper development of the mammalian central nervous system. p35 is a neuronal-specific Cdk5 regulator that activates cdk5 kinase activity upon association. When p35 is cleaved by the protease calpain, which results in the generation of a truncated product termed p25. Unlike p35, p25 is more resistant to degradation. Binding of p25 to Cdk5 constitutively activates Cdk5, changes its cellular location and alter its substrate specificity. DARPP-32(dopamine and cAMP-regulated phosphoprotein, 32 kD) is an excellent substrate of PKA and Cdk5. When Thr-34 of DARPP-32 is phosphorylated by PKA, the protein will be converted into an inhibitor of PP1(protein phosphatase 1), while Thr-75 of DARPP-32 is phosphorylated by Cdk5, the protein will be converted into an inhibitor of PKA. Thus, DARPP-32 can regulate both an important protein kinase and an important protein phosphatase through phosphorylation of distinct sites. This dual function is believed to be important for the integration of the effects of various neurotransmitters on signaling pathway. The mechanism for Cdk5/p25 phosphorylated DARPP-32 to inhibited PKA remains unknown. In this study, the first is to purify Cdk5 and p25. Then we will employ the purified Cdk5/p25 to phosphorylate DARPP-32 and analyze the mechanism for Cdk5/p25-phosphorylated DARPP-32 in inhibition of PKA.