The novel oncogene with kinase-domain (NOK), is an atypical receptor protein tyrosine kinase with potent oncogenic potential. In the current study, we generated two point mutations (P203L and V395I) on NOK gene. NOK(P203L) is identical to serine/threonine/tyrosine kinase 1 (STYK1), the aliases of NOK, while the V395I mutation was recovered from human glioblastoma. Both mutations did not impair NOK kinase activities, but V395I inhibited NOK autophosphorylation. Although with overall inhibition, both STYK1 and V395I affected the activities of extracellular regulated protein kinase (ERK), Akt and signal transducer and activator of transcription (STAT) differently in HEK293T cells versus HeLa and BaF3 stable cells The proliferation potentials for both STYK1 and V395I were significantly inhibited. Single mutation at either site was sufficient to abolish the IL-3 independent growth and the anchor-independent growth of of BaF3 stable cells. Overall, our data indicates that both P203 and V395 residues on NOK are important for NOK mediated mitogenic signaling, and the substitutions of P203L and V395I may selectively affect certain mitogenic signaling cascades in a tissue specific manner.