Vaccinia-related kinase3(VRK3)와 VRK3의 새로운 결합단백질인 RNF144a, PARP1, p53과의 상호조절에 관한 연구

Abstract

Vaccinia related kinase 3 is a member of the VRK family and is ubiquitously expressed in tissues. VRK3 has been reported to be a negative regulator of ERK that protects cells from excessive stress and inhibits ERK dependent apoptosis. VRK3 was originally known as pseudokinase, but our group revealed that VRK3 has kinase activity and phosphorylates Barrier-to-autointegration factor(BAF). Recently, it has been reported that VRK3 knockout mice exhibit autism-like behavior, suggesting that VRK3 plays an crucial role in neurodevelopmental disorder. However, the role of VRK3 in cells and its upstream regulator is not fully elucidated yet. In the first chapter, I demonstrate that RNF144a, an E3 ligase, promotes the degradation of VRK3 via poly-ubiquitination and thus affects VRK3-mediated ERK activity. Under oxidative stress, VRK3 migrates from the nucleus to the cytoplasm which increases its chance to interact with RNF144a, thereby promoting the degradation of VRK3. Overexpression of RNF144a increases ERK activity and promotes ERK-dependent apoptosis via downregulation of VRK3. In contrast, depletion of RNF144a increases the protein level of VRK3 and protects cells from excessive ERK activity. These findings suggest that VRK3 protects cells by suppressing oxidative stress-induced ERK, and while RNF144a sensitively regulates this process.

In the second chapter, I show that VRK3 regulates Poly(ADP-ribose)polymerase1 stability and thus the resistance to the PARP inhibitor. PARP1 is one of the most critical proteins in the DNA repair response and is highly expressed in breast and ovarian cancer cells. In cancer therapy, PARP inhibitors, which inhibit enzyme activity of PARP1, are considered to be promising drugs for cancers with BRCA mutations. Recently, a PARP-DNA trapping model was introduced, and PARP inhibitors have been applied to treat different cancers. However, in many cases, tumors have shown resistance to PARP inhibitors. Emerging evidence indicates that the amount of PARP1 on damaged DNA is a critical determinant of sensitivity toward a PARP inhibitor. Therefore, identifying the mechanism by which PARP1 is regulated is crucial in overcoming the cancer resistance to PARP inhibitors. Despite its importance, the molecular mechanisms that regulate PARP1 are not yet well known. In this work, I found that VRK3 positively regulates PARP1 stability by blocking the action of RNF144a. According to my data, VRK3 overexpression increases the stability of PARP1 and sensitivity toward PARP inhibitors. Inversely, loss of VRK3 results in the downregulation of PARP1. This study indicates that VRK3 plays an important role in PARP inhibitor sensitivity by regulating PARP1 stability.

In the last chapter, I demonstrate that VRK3 has a tumor suppressive role as it promotes ubiquitination of MDM2 and stabilizes p53. p53, the most well-known tumor suppressor protein, inhibits tumor progression by promoting cell cycle arrest and apoptosis. The stability of p53 is critical for the survival or death of cancer cells, but the mechanisms regulating p53 are not yet fully understood. Here, I introduce VRK3 as a novel tumor suppressor that increases the p53 stability. I observed that the p53 and p21 were significantly decreased in a VRK3-deficient cell line and in mouse embryonic fibroblasts from VRK3 knockout mice. I found that VRK3 directly binds to MDM2, E3 ligase of p53, and destabilizes MDM2 by promoting its ubiquitination. Interestingly, p53 also reduces VRK3 stability by regulating the expression of RNF144a, an E3 ligase of VRK3. This suggests that VRK3 forms a novel negative feedback loop with p53. Taken together, I demonstrate that VRK3 plays tumor suppressor roles by stabilizing p53 and inducing p21.