Point Mutation At Single Tyrosine Residue Of NOK Abrogates Tumorigenesis

Receptor protein-tyrosine kinases (RPTKs) are tightly regulated during normal cellular signal transduction processes including cell growth, differentiation, and metabolism. Aberrant expression of RPTK usually results in developmental disorders and cancer. Recently, a RPTK like molecule named Novel Oncogene with Kinase-domain (NOK) has been identified and characterized. Over-expression of NOK gene severely caused cellular transformation as well as tumorigenesis and metastasis in nude mice.In current study, we generated two point mutations (Y327F and Y356F) within the endodomain of NOK that are the potential tyrosine phosphorylation sites controlling major intracellular signaling. Using BaF3 cells stably expressing the fusion protein with ectodomain of mouse erythropoietin receptor (mEPOR) and transmembrane/endodomain of NOK (BaF3-E/N), we were able to show that point mutations at Y327F and Y356F completely blockaded cellular transformation by NOK gene as examined by colony formation, cellular DNA synthesis and FACS. Moreover, tumorigenesis and metastasis induced by BaF3-E/N were completely abrogated upon the introduction of either single mutation. Importantly, signaling studies revealed that the activation of ERK pathway was completely inhibited by Y356F and significantly reduced by Y327F. Both mutations significantly prevented Akt and STAT5 activation. In addition that NOK inhibiting E-cadherin, which is an important metastasis marker gene, was also renewed by the two mutations.Taken together, our studies both in vitro and in vivo demonstrate that these two tyrosine phosphorylation sites Y327F and Y356F may play critical role in NOK mediated tumorigenesis. Our study suggested a potential mechanism of tumorigenesis induced by PTKs, resulting from the conservation of these two tyrosine residues in most of the PTKs subfamilies.