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Point Mutation at Single Tyrosine Residue of Novel Oncogene NOK Abrogates Tumorigenesis in Nude Mice

¹ Department of Biological Sciences and Biotechnology, State Key Laboratory of Biomembrane and Membrane Biotechnology, ² Tsinghua Institute of Genome Research, Institute of Biomedicine, Tsinghua University; Departments of ³ Microbiology and Etiology and ⁴ Pathology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China; ⁵ Department of Cardiology, the University of Texas M.D. Anderson Cancer Center, Houston, Texas; and ⁶ Department of Microbiology and Immunology, Walther Oncology Center, Indiana University School of Medicine, Indianapolis, Indiana

Requests for reprints: Li Liu, Department of Biological Sciences and Biotechnology, Institute of Biomedicine, Tsinghua Institute of Genome Research, Tsinghua University, Beijing 100084, China. Phone: 86-10-6277-3624; Fax: 86-10-6277-3624; E-mail: liu_li{at}mail.tsinghua.edu.cn; or Xin-Yuan Fu, Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, IN 46202-5120. E-mail: xfu{at}iupi.edu; or Xiu-Fang Zhang, Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing 100084, China. E-mail: zxf-dbs{at}mail.tsinghua.edu.cn.

Receptor protein-tyrosine kinases (RPTKs) are tightly regulated during normal cellular processes including cell growth, differentiation, and metabolism. Recently, a RPTK-like molecule named novel oncogene with kinase-domain (NOK) has been cloned and characterized. Overexpression of NOK caused severe cellular transformation as well as tumorigenesis and metastasis in nude mice. In the current study, we generated two tyrosinephenylalanine (YF) point mutations (Y327F and Y356F) within the endodomain of NOK that are well conserved in many RPTK subfamilies and are the potential tyrosine phosphorylation sites important for major intracellular signaling. Using BaF3 cells stably expressing the ectodomain of mouse erythropoietin receptor, and the transmembrane and endodomain of NOK (BaF3-E/N), we were able to show that point mutations at either Y327 or Y356 dramatically blocked cellular transformation by NOK as examined by colony formation and cellular DNA synthesis. In addition, tumorigenesis induced by BaF3-E/N was completely abrogated upon the introduction of either single mutation. Importantly, signaling studies revealed that the activation of extracellular signal-regulated kinase was inhibited by Y356F and was significantly reduced by Y327F. Both mutations significantly impaired Akt phosphorylation. Interestingly, both mutations did not affect the kinase activity of NOK. Moreover, apoptotic analysis revealed that both mutations accelerated cell death by activating caspase-3–mediated pathways. Thus, our study shows that these potential tyrosine phosphorylation sites may play critical roles in NOK-mediated tumorigenesis both in vitro and in vivo.