1. Academic Validation
  2. Identification and Verification of the Ability of Cdk5 to Phosphorylate Deubiquitinating Enzyme BRCC3 In Vitro

Identification and Verification of the Ability of Cdk5 to Phosphorylate Deubiquitinating Enzyme BRCC3 In Vitro

  • Bull Exp Biol Med. 2022 Apr;172(6):701-708. doi: 10.1007/s10517-022-05460-z.
C H Zhang 1 K Qin 1 S L Tian 1 Y Han 1 S Y Xu 2 X Y Shao 3
Affiliations

Affiliations

  • 1 Department of Human Anatomy, Laboratory Animal Center, Guilin Medical College, Guilin, Guangxi, China.
  • 2 Laboratory Animal Center, Guilin Medical College, Guilin, Guangxi, China. shaoyexu@glmc.edu.cn.
  • 3 Department of Human Anatomy, Laboratory Animal Center, Guilin Medical College, Guilin, Guangxi, China. xyshao@glmc.edu.cn.
Abstract

It is known that the expression of the deubiquitinating Enzyme BRCA1-BRCA2-containing complex subunit 3 (BRCC3) and cyclin-dependent protein kinase 5 (CDK5) is increased in Parkinson's disease (both are involved in neuroinflammatory response). However, the regulatory mechanism of CDK5 on the post-translational modification of BRCC3 remains unclear. Here we studied whether CDK5 phosphorylates BRCC3. Phosphorylation of BRCC3 by CDK5 was predicted by GPS 5.0 software. His-BRCC3 plasmid was constructed by cloning the BRCC3 gene into pGEX-6P-1 vector, and then His-BRCC3 fusion protein was induced with isopropyl β-d-1-thiogalactopyranoside and purified using His-Tag affinity chromatography purification Agarose. Phosphorylation of BRCC3 fusion protein by CDK5 in vitro was detected by mass spectrometry and Western blotting. The results showed that multiple phosphorylation sites were predicted by GPS 5.0, and the His-BRCC3 fusion protein was successfully induced and purified. In vitro kinase assay, Western blotting, and mass spectrometry showed that CDK5 can phosphorylate BRCC3. It has been demonstrated that protein kinase CDK5 can phosphorylate the deubiquitinating Enzyme BRCC3 in vitro, which provides new data for further study on the mechanism of neurodegeneration.

Keywords

BRCA1-BRCA2-containing complex subunit 3 (BRCC3); cyclin-dependent kinase 5 (Cdk5); kinase reaction; phosphorylation; phosphorylation mass spectrometry.

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