[[Frontiers in Bioscience, Landmark, 20, 1179-1189, June 1, 2015]

NOK mediated mitogenic signaling is altered by P203L and V395I mutations

Sheng-Qi Hou 1 , Li Liu 1

1Department of Microbiology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences & School of Basic Medicine, Peking Union Medical College, Beijing 100005, China

TABLE OF CONTENTS

1. Abstract
2. Introduction
3. Materials and methods
    3.1. Plasmid construction and site-directed mutagenesis
    3.2. Cell culture, transient transfection and antibodies
    3.3. Generation of BaF3 stable cells
    3.4. Western blot analysis
    3.5. Cell proliferation assay and anchor independent growth assay
    3.6. Immunoprecipitation
    3.7. Autophosphorylation assay
    3.8. In vitro tyrosine kinase activity assay
4. Results
    4.1. NOK and its two mutant derivatives STYK1 and V395I
    4.2. The effects of STYK1 and V395I on NOK autophosphorylation and kinase activity
    4.3. The effects of STYK1 and V395I on NOK mediated signaling pathways
    4.4. The effect of STYK1 and V395I on NOK mediated cell proliferation
    4.5. STYK1 and V395I fail to promote anchorage-independent growth of BaF3 stable cells
5. Discussion
6. Acknowledgments
7. References

1. ABSTRACT

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.

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Abbreviation: novel oncogene with kinase-domain (NOK); serine/threonine/tyrosine kinase 1 (STYK1); receptor protein tyrosine kinase (RPTK); Src homology2 (SH2); phosphortyrosine binding (PTB); amino acid (aa); extracellular regulated protein kinase (ERK); Akt (protein kinase B, PKB); signal transducer and activator of transcription (STAT); praline (P); leucine (L); valine (V); isoleucine (I); insertionally activated chicken erbB (IA c-erbB)

Key Words: point mutations, RPTK, NOK, Signal Transductions, Autophosphorylation, Kinase Activity, Review

Send correspondence to: Li Liu, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences & School of Basic Medicine, Peking Uion Medical College, Beijing 100005, China, Tel: 86 10 69156454, Fax: 86 10 65233768, E-mail: lliu@pumc.edu.cn