Identification and Verification of Cdk5 Phosphorylated Deubiquitinating Enzyme BRCC3

Abstract

\n Background: It is known that the expression of the deubiquitinating enzyme BRCA1-BRCA2-containing complex subunit 3(BRCC3) is increased, as well as Cyclin-dependent protein kinase 5(Cdk5) in Parkinson’s Disease(PD), and both of them to be involved in neuroinflammatory response. But the regulatory mechanism of Cdk5 on the post-translational modification of BRCC3 remains unclear. This study aimed to investigate the phosphorylation of Cdk5 on the BRCC3. Methods and Results: Cdk5 phosphorylation of BRCC3 was predicted by GPS5.0 software. His-BRCC3 plasmid was constructed by cloning the BRCC3 gene into pGEX-6P-1 vector, and then His-BRCC3 fusion protein was induced by IPTG and purified by His-tag purification agarose. The BRCC3 fusion protein was reacted with Cdk5 in vitro, and the phosphorylation was detected by mass spectrometry and Western blot. The results showed that multiple phosphorylation sites were predicted by GPS5.0, and the His-BRCC3 fusion protein was successfully induced and purified. In vitro kinase assay, Western blot and mass spectrometry identified that Cdk5 can phosphorylate BRCC3. Conclusions: It has been demonstrated that the protein kinase Cdk5 can phosphorylate the deubiquitinating enzyme BRCC3 in vitro, which provide evidences for further study on the mechanism of neurodegeneration.