The Regulation of TAX1 Binding Protein 2 by P21-activated Protein Kinase 4 in Liver Cancer Metastasis

Abstract

Introduction: P21 activated protein kinases (PAK) are the main downstream effectors of small Rho GTPases Cdc42 and Rac1, which regulate various cellular processes including cell migration, cell cycle progression, and cell survival. Previous studies have shown that PAK4, one of the PAK family members, is highly cell-transforming and is overexpressed in many primary tumors including liver cancer (hepatocellular carcinoma, HCC). PAK4 was also proved to be associated with more aggressive behavior such as liver and venous invasion, suggesting the involvement of PAK4 in HCC metastasis. TAX1 binding protein 2 (TAX1BP2), which is an alternative splicing variant of ciliary rootlet coiled-coil protein has been suggested to be a tumor suppressor in HCC. TAX1BP2 can significantly suppress HCC cell tumorigenicity through the activation of the p38/p53/p21 pathway. Interestingly, our preliminary data showed that both PAK4 and TAX1BP2 were centrosomal proteins and TAX1BP2 was a phosphorylation substrate of PAK4. Hence, we hypothesize that PAK4 promote HCC formation via inhibitory phosphorylation of TAX1BP2. Method: In vitro kinase assay is performed to figure out whether TAX1BP2 is a phosphorylation substrate of PAK4. Trans-well migration assay is used to compare the migratory suppression activity of TAX1BP2 and PAK4 non-phosphorylatable and phosphorylation mimic TAX1BP2 mutants in HCC cells. Confocal immunofluorescence staining is used to map out the centrosome localization signal of PAK4 by examining centrosome localization of different truncation forms of PAK4, in order to figure out whether PAK4 promotes tumorigenesis through the perturbation of centrosome function. Results: TAX1BP2 is proved to be a good phosphorylation substrate of PAK4. Moreover, PAK4 phosphorylation significantly reduces the migratory suppression activity of TAX1BP2 on HCC cells. And the centrosome localization signal of PAK4 is observed within the p21 binding domain of PAK4. Conclusion: PAK4 is proved to promote HCC migration via inhibitory phosphorylation of TAX1BP2. The involvement of TAX1BP2 in PAK4-mediated hepatocarcinogenesis and the PAK4/TAX1BP2 signaling in HCC metastasis will be further investigated.