Roles of VRK3 in regulation of PARP1 stability and PARP inhibitor sensitivity


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Han Seong Hyun1
1Pohang University of Science and Technology

Abstract

Background

Poly(ADP-ribose) polymerase 1 is one of the most critical protein in DNA repair response. In a cancer therapy, PARP inhibitor is considered as a promising drug for BRCA mutated cancer. Recently, as PARP-DNA trapping model was introduced, PARP inhibitors were applied in many other cancer treatments. However, it has been reported that many tumors have resistance to PARP inhibitor. As emerging evidence indicates that level of PARP1 is critical determinants for PARP inhibitor sensitivity, identification of regulating mechanism of PARP1 is crucial in overcoming the resistance of PARP inhibitor used for cancer. 

VRK3 is a member of the Vaccinia Related Kinase family. It was reported that VRK3 negatively regulates ERK activity via VHR phosphatase. 

Method

Transfection

Transient transfections were performed by electroporation using a Microporator (Invitrogen)

Immunoprecipitation

IP was performed using 5*106 cells in lysis buffer. Cell lysates were incubated overnight with antibody and Protein G-sepharose beads at 4°C on a rotater. After incubation, beads were washed three times and boiled for 5 min at 95°C.

Colony formation assay

VRK3 expression vector was transfected by electroporation into MDA-MB-231 cells. 3*103 cells were plated with or without PARP1 inhibitor for 2 weeks. Survival colonies were stained with crystal violet.

Results

We found that VRK3 directly interacts with PARP1 and regulates its level.

Furthermore, VRK3 regulates PARP1 stability but not its expression.

We found that up-regulation of VRK3 leads to stabilization of PARP1 and makes cancer cells more sensitive to PARP inhibitor. Inversely, down-regulation of VRK3 results in loss of PARP1.

Finally, we figured out that RNF144a mediated PARP1 degradation is inhibited by VRK3.

Conclusion

VRK3, novel protein kinase, is a positive regulator of PARP1 by increasing its protein stability through interrupting the interaction between RNF144a and PARP1. These study indicates that VRK3 has important role in PARP inhibitor sensitivity via regulation of PARP1.