| Summary: | 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所 === 98 === DARPP-32 (dopamine and cAMP-regulated phosphoprotein, 32 kD ) is an excellent substrate of PKA and Cdk5. When Thr-34 is phosphorylated by PKA, DARPP-32 will be converted into an inhibitor of PP1 (protein phosphatase 1); while Thr-75 is phosphorylated by Cdk5, the protein will be converted into an inhibitor of PKA. Thus, DARPP-32 can regulate both PKA and PP1 through phosphorylation of distinct sites. This dual function is believed to be important for the integration of various neurotransmitter-mediated signaling pathways. Previous studies in our lab have established a baculovirus expression system to prepare the recombinant Cdk5/p25. In my study, I will verify whether Cdk5 only phosphorylates Thr75 of DARPP-32. Therefore, I prepared DARPP-32-T75A, DARPP-32-T75D, and DARPP-32-T75E for in vitro kinase assay. The result of the kinase activity assay found that Cdk5 will still phosphrylate DARPP-32-T75A. Hence, the Cdk5 phosphorylation sites on DARPP-32 remained to be identified. In the meantime, I also prepared 15N-PKA (Protein kinase A) for structural analysis by NMR.
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