Characterization of upstream and downstream signaling pathway of P21-activated kinase 4 through TAX1 binding protein 2 and INKA1 in cancer metastasis

Abstract

P21 activated protein kinase 4 (PAK4) regulates various cellular processes including cell migration, cell cycle progression, and cell survival. PAK4 overexpression, which has been reported in diverse cancer cell lines and various clinical samples, including hepatocellular carcinoma (HCC) and pancreatic cancer, is associated with poor survival and more aggressive tumor behaviors in patients. TAX1 binding protein 2 (TAX1BP2), which is an alternative splicing variant of ciliary rootlet coiled-coil protein, was first identified as a novel interacting protein of human T-lymphocyte virus I (HTLV-1) coded oncoprotein TAX. Further studies revealed that TAX1BP2 was frequently underexpressed in HCC, and its underexpression was associated with a poorer prognosis. In terms of tumor-suppressive activity, TAX1BP2 was found to upregulate p53 tumor suppressor and p21wa1f/cip1 cell cycle inhibitor in a p38 MAPK dependent manner. In the current study, I characterized the interaction of PAK4 and TAX1BP2 both in vitro and in cells. PAK4 phosphorylated TAX1BP2 at serine 387 promoted the protein degradation of TAX1BP2 through the ubiquitin-proteasome pathway, which resulted in attenuation of the proliferation and migration inhibition functions of TAX1BP2. Meanwhile, the centrosome targeting domain of PAK4 was mapped to the 100-300 a.a. region of PAK4, which was crucial for the interaction between PAK4 and TAX1BP2. Because of the important roles of PAK4 in tumorigenesis and cancer metastasis, PAK4 can be a good target for cancer therapy. However, no safe PAK4 inhibitor is available clinically so far. Excitingly, an endogenous PAK4 inhibitor, INKA1 was identified recently. It was reported that the functional domains of INKA1 inhibited the kinase activity of PAK4 in vitro. It is still unclear whether INKA1 could suppress PAK4 activity in cancer cells. Therefore, in this study, I characterized the PAK4 kinase inhibition functions of INKA1. Firstly, the interaction of PAK4 and INKA1 was confirmed in HCC and pancreatic cancer cells. Secondly, the kinase activity of PAK4 was inhibited by full-length INKA1 and the functional domains of INKA1 in vitro and in cells. Based on the amino acid sequence of the PAK4 inhibitory domain of INKA1, I designed a small peptide, which efficiently inhibited the PAK4 activity in cells. With the clinical data analysis, I found that INKA1 underexpression was associated with patients’ survival in pancreatic cancer. More importantly, the tumorigenic activity of pancreatic cancer cells, including proliferation, anchorage-independent growth, migration, and invasion, were suppressed by INKA1. Meanwhile, matrix metalloproteinase-2 (MMP2) was significantly suppressed by INKA1 in pancreatic cells via inhibiting the activation of NF-κB signaling. Interestingly, INKA1 was also a phosphorylation substrate of PAK4, and the PAK4 phosphorylation attenuated the PAK4 inhibitory function of INKA1. Finally, I identified four novel INKA1-interacting proteins, and these proteins may cooperate with INKA1 to suppress PAK4 activity. In summary, the present study characterized the interactions of PAK4 with its downstream effector TAX1BP2 and endogenous inhibitor INKA1. The findings advance our understanding of PAK4 and provide important insight on the development of PAK4 as a therapeutic target for cancers.