Vaccinia-related kinase 2 modulates role of dysbindin by regulating protein stability


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Shin Wonsik1,Jeong Young-Hun1,Choi Jung-Hyun1,Lee Dohyun1,Kin Sangjun1,Kim Kyoung-Tai1
1POHANG UNIVERSITY OF SCIENCE AND TECHNOLOGY

Abstract

Background

Vaccinia-related kinase 2 (VRK2) is a serine/threonine kinase that acts as an effector of signaling pathways that regulate apoptosis, tumor cell growth and tumor cell invasion. Although some proteins, such as histone H3, USP25, and NFAT-1, are known to be phosphorylated by VRK2, the role of VRK2 is still not well-known. 

Dysbindin is known as one of the strong risk factors for schizophrenia. The expression of dysbindin is indeed significantly reduced in schizophrenia patients. In the brains of schizophrenic patients, the protein and messenger RNA (mRNA) expression levels of dysbindin were lowered. 

Method

Immunoprecipitation and GST-pulldown assay were conducted to identify an interaction between VRK2 and dysbindin. in vitro kinase assay was conducted to investigate phosphorylation of dysbindin by VRK2. Ubiquitylation assay was used performed to find if VRK2 regulates stability of dysbindin. Neurite outgrowth was induced by retinoic acid and neurons were measured by ImageJ software to identify if VRK2 has a role in neuronal growth.

Results

  1. We identified dysbindin as a novel interacting protein of VRK2 through immunoprecipitation and showed that VRK2 phosphorylates Ser 297 and Ser 299 of dysbindin using in vitro kinase assay. In addition, We found that VRK2-mediated phosphorylation of dysbindin enhanced ubiquitination of dysbindin and consequently resulted in the decrease of its protein stability through Western blotting. Overexpression of VRK2 in human neuroblastoma (SH-SY5Y) cells reduced neurite outgrowth induced by retinoic acid. Furthermore, a phosphomimetic mutant of dysbindin alleviated neurite outgrowth and affected surface expression of N-methyl D-aspartate 2A (NR2A), a subunit of NMDAR in mouse hippocampal neurons.

Conclusion

In conclusion, These findings suggest that VRK2 regulates dysbindin, thus revealing the relationship between the two proteins, implicating that VRK2 plays an important role not only in tumor growth and tumor invasion but also in Schizophrenia.