Point mutation at single tyrosine residue of novel oncogene NOK abrogates tumorigenesis in nude mice.
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 tyrosine-->phenylalanine (Y-->F) 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.
Chen
Yue
Y
Department of Biological Sciences and Biotechnology, State Key Laboratory of Biomembrane and Membrane Biotechnology, Tsinghua Institute of Genome Research, Institute of Biomedicine, Tsinghua University, Beijing, China.
Li
Ying-Hua
YH
Chen
Xi-Ping
XP
Gong
Li-Min
LM
Zhang
Shu-Ping
SP
Chang
Zhi-Jie
ZJ
Zhang
Xiu-Fang
XF
Fu
Xin-Yuan
XY
Liu
Li
L
D000818:Animals; D051379:Mice; D000595:Amino Acid Sequence; D017209:Apoptosis / Q000235:genetics; D001665:Binding Sites; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D048708:Cell Growth Processes / Q000235:genetics; D002471:Cell Transformation, Neoplastic / Q000235:genetics / Q000378:metabolism; D017124:Conserved Sequence; D004789:Enzyme Activation; D008819:Mice, Nude; D008969:Molecular Sequence Data; D015513:Oncogene Proteins / Q000235:genetics / Q000378:metabolism; D010649:Phenylalanine / Q000235:genetics; D010766:Phosphorylation; D017354:Point Mutation; D020794:Receptor Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D014162:Transfection; D014443:Tyrosine / Q000235:genetics
C505409:Casp3 protein, mouse; D053148:Caspase 3; D020169:Caspases; D015513:Oncogene Proteins; D010649:Phenylalanine; D020794:Receptor Protein-Tyrosine Kinases; D014443:Tyrosine; C000731233:Styk1 protein, mouse