Effects Of Mutations P203L And V395I On NOK Induced Tumorigenesis And Metastasis

Protein tyrosine kinases are a part of signal generation enzymes in living organisms. They precisely mediated multiple cellular physiological activities including cell proliferation, differentiation, cell cycle control, embryogenesis, angiogenesis, and metabolisms. NOK was a lately identified oncogene and thought to be pseudokinase because of lacking of intact extracellular domain and the DFG motif which is highly conserved among receptor protein tyrosine kinases (RTKs). However, previous studies in our lab found that NOK could enhance tumor and metastasis in nude mice. So far, the mechanism of NOK induced tumorigenesis and metastasis has not been fully understood. In this study, we aim to find out the relationship between NOK’s structure and function, to elucidate the signaling pathways responsible for tumor formation and metastasis. We hope to uncover its specific role in tumorigenesis and metastasis so as to provide fundamental theoretic basis for cancer diagnosis and treatment.In this study, we used site directed mutagenesis which has been applied widely in gene modification to generate mutants. Using these mutants, we constructed eukaryotic expression plasmids which include pCDNA3.0-NOK-HA/NOK-Mutant-HA plasmids and pCDH-CMV-MCS-EF1-GFP- CD511B-NOK-HA/NOK-Mutant-HA plasmids as well as BaF3 stable cell lines.Firstly, we examined whether STYK1 (the mutant P203L) and V395I could affect NOK autophosphorylation and kinase activity. We found that although V395I mutation inhibited the NOK autophosphorylation, the kinase activity of these two mutants were not altered, indicating the phosphorylation status of NOK may not be the prerequisite for its kinase activation. Subsequently, we investigated the signaling transductions. The results in HEK293T cells showed that NOK was able to enhance phosphorylation levels of both ERK and Akt. However, mutant V395I did not impair the phosphorylation levels of NOK mediated activations on these two molecules, and STYK1 inhibited the activation of these two signaling molecules in HEK293T cells. As for the phosphorylated levels of STAT1,3 and 5, it did not change in the two mutations. In HeLa cells, STYK1 down-regulated ERK and Akt’s phosphorylation levels, while V395I only inhibited the activation of ERK but not Akt. In addition, both mutants negatively regulated the acitivation of STAT1 and STAT3, while the phosphorylation levels of STAT5 were inert to be changed in HeLa cells. In BaF3 cells, both STYK1 and V395I not only markedly inhibited STATl, STAT3 and STAT5 activations but also repressed ERK phosphorylation, while only STYK1 significantly decreased the phosphorylation level of NOK. The results indicate that the effects of STYK1 and V395I on NOK-mediated signaling transductions may have slight differences as the tested tissues different. We also detected there was an obvious decrease of cell proliferation in STYK1 and V395I group in HEK293T, HeLa and BaF3 cells. Therefore, both mutants have reduced proliferation potentials as compared with the wild type NOK. To directly evaluate the effect of STYK1 and V395I on NOK mediated tumorigenic potential in vitro, we employed the colony formation assay. Quantitative analysis indicates that both STYK1 and V395I could dramatically inhibited NOK induced anchor-independent growth in BaF3 stable cells.Animal studies were also utilized to investigate the effects of the two mutations on NOK mediated tumorigenesis and metastasis. We found that compared with the negative group, the time of tumor formation in NOK group is much shorter and the survival time of the nude mice is about 1.5 months, while the time in mutant groups STYK1 and V395I is postponed and the survival time is about 2.5-3.0 months. To further consolidate the results, different organs including liver, spleen, lung, kidney, lymphnode and bone marrow were collected for pathogenic analysis. Visable nodal metastases presented in the surface of livers in NOK group other than STYK1 and V395I group. The size of liver and spleen is significantly enlarged in NOK group compared with that in STYK1 and V395I groups. These results were further comfirmed by hematoxylin-eosin staining.Ultimately, using immunoprecipitation combined with mass spectrum technique, we analized the potential interacting proteins with NOK. About 40 potential proteins were revealed. These proteins are involed in a serial of physiological and biological processes and may play significant roles in NOK-induced tumorigenesis and metastasis.In this study, we investigated the relationship between NOK’s structure and function based on cellular and animal levels. Using site-directed mutagenesis, we built a flat for further research of NOK. This study also contributes to the better understanding of NOK-induced tumorigenesis and metastasis.