Csk-homologous kinase interacts with SHPS-1 and enhances neurite outgrowth of PC12 cells.
SHPS-1 is an immunoglobulin superfamily protein with four immunoreceptor tyrosine-based inhibitory motifs (ITIMs) in its cytoplasmic region. Various neurotrophic factors induce the tyrosine phosphorylation of SHPS-1 and the association of SHPS-1 with the protein tyrosine phosphatase SHP-2. Using a yeast two-hybrid screen, we identified a protein tyrosine kinase, Csk-homologous kinase (CHK), as an SHPS-1-interacting protein. Immunoprecipitation and pull-down assays using glutathione S-transferase (GST) fusion proteins containing the Src homology 2 (SH2) domain of CHK revealed that CHK associates with tyrosine-phosphorylated SHPS-1 via its SH2 domain. HIS3 assay in a yeast two-hybrid system using the tyrosine-to-phenylalanine mutants of SHPS-1 indicated that the first and second ITIMs of SHPS-1 are required to bind CHK. Over-expression of wild-type CHK, but not a kinase-inactive CHK mutant, enhanced the phosphorylation of SHPS-1 and its subsequent association with SHP-2. CHK phosphorylated each of four tyrosines in the cytoplasmic region of SHPS-1 in vitro. Co-expression of SHPS-1 and CHK enhanced neurite outgrowth in PC12 cells. Thus, CHK phosphorylates and associates with SHPS-1 and is involved in neural differentiation via SHP-2 activation.
Mitsuhashi
Hiroaki
H
Department of Life Sciences, Graduate School of Arts and Sciences, University of Tokyo, Tokyo, Japan.
Futai
Eugene
E
Sasagawa
Noboru
N
Hayashi
Yukiko
Y
Nishino
Ichizo
I
Ishiura
Shoichi
S
D000818:Animals; D001426:Bacterial Proteins / Q000096:biosynthesis; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000166:cytology; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D004622:Embryo, Mammalian; D015870:Gene Expression / Q000502:physiology; D005982:Glutathione Transferase / Q000502:physiology; D006801:Humans; D008164:Luminescent Proteins / Q000096:biosynthesis; D009154:Mutation / Q000502:physiology; D016501:Neurites / Q000502:physiology*; D009474:Neurons / Q000166:cytology; D016716:PC12 Cells / Q000166:cytology*; D010766:Phosphorylation; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D011971:Receptors, Immunologic / Q000378:metabolism*; D014162:Transfection; D020798:Two-Hybrid System Techniques; D014443:Tyrosine / Q000378:metabolism; D018909:src Homology Domains / Q000502:physiology
D001426:Bacterial Proteins; D008164:Luminescent Proteins; D011971:Receptors, Immunologic; C501924:Sirpa protein, rat; C054562:yellow fluorescent protein, Bacteria; D014443:Tyrosine; D005982:Glutathione Transferase; C085377:MATK protein, human; D016392:Proto-Oncogene Proteins pp60(c-src)