Csk-homologous kinase interacts with SHPS-1 and enhances neurite outgrowth of PC12 cells Hiroaki Mitsuhashi,* Eugene Futai,* Noboru Sasagawa,* Yukiko Hayashi, Ichizo Nishino and Shoichi Ishiura* *Department of Life Sciences, Graduate School of Arts and Sciences, University of Tokyo, Tokyo, Japan  National Institute of Neuroscience, NCNP, Kodaira, Tokyo, Japan The transduction of intracellular signals following cell adhe- sion is important for brain development. Various neuronal functions are regulated by cell adhesion molecules, including axon guidance, cell migration, and synaptogenesis (Murase and Schuman 1999; Ronn et al. 2000; Scheiffele et al. 2000; Yamagata et al. 2003). Most of these cell adhesion molecules interact with intracellular signaling proteins. Accumulating evidence indicates that a tyrosine phosphorylation signal cascade is involved in neurite outgrowth, synapse formation, and synaptic plasticity (Flanagan and Vanderhaeghen 1998; Kullander and Klein 2002; Huang and Reichardt 2003). Src homology 2 (SH2) domain-containing protein tyrosine phosphatase substrate-1 (SHPS-1), also known as signal regulatory protein alpha (Kharitonenkov et al. 1997), P84 (Chen et al. 2004), brain immunoglobulin-like molecule with tyrosine-based activation motifs (Ohnishi et al. 1996), and gp93 (Wang et al. 2003), is a receptor-like transmembrane glycoprotein. SHPS-1 contains three immunoglobulin-like domains in its extracellular region, and its cytoplasmic region contains four tyrosine residues that are phosphorylated in response to various stimuli (Fujioka et al. 1996; Tsuda et al. 1998; Ohnishi et al. 1999; Maile and Clemmons 2002; Ruhul Amin et al. 2002). SHPS-1 is abundant in the central nervous system and in the immune system (Fujioka et al. 1996; Sano et al. 1999); it is expressed in synapse-rich areas, such as the molecular layer and synaptic glomeruli of the cerebellum and in the plexiform layers of the retina (Chuang and Lagenaur 1990; Comu et al. 1997). We previously showed that SHPS-1 is present at neuromuscular junctions and that its expression in skeletal muscle is regulated in a nerve-dependent manner (Mitsuhashi et al. 2005). These Received October 3, 2007; accepted October 29, 2007. Address correspondence and reprint requests to Shoichi Ishiura, Department of Life Sciences, Graduate School of Arts and Sciences, University of Tokyo, 3-8-1 Komaba, Meguro-ku, Tokyo 153-8902, Japan. E-mail: cishiura@mail.ecc.u-tokyo.ac.jp Abbreviations used: 3-AT, 3-amino-1,2,4-triazole; CHK, Csk-homolo- gous kinase; ECFP, enhanced cyan fluorescent protein; EYFP, enhanced yellow fluorescent protein; Grb2, growth factor receptor–bound protein 2; GFP, green fluorescent protein; GST, glutathione S-transferase; MAPK, mitogen-activated protein kinase; NGF, nerve growth factor; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; SH2, Src homology 2; SH3, Src homology 3; SHP-2, Src homology 2 domain- containing protein tyrosine phosphatase-2; SHPS-1, Src homology 2 domain-containing protein tyrosine phosphatase substrate 1. Abstract 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-inter- acting protein. Immunoprecipitation and pull-down assays using glutathione S-transferase (GST) fusion proteins con- taining 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 indi- cated 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 phos- phorylates and associates with SHPS-1 and is involved in neural differentiation via SHP-2 activation. Keywords: CHK, neurite, PC12, SHP-2, SHPS-1, tyrosine phosphorylation. J. Neurochem. (2008) 105, 101–112. d JOURNAL OF NEUROCHEMISTRY | 2008 | 105 | 101–112 doi: 10.1111/j.1471-4159.2007.05121.x Ó 2007 The Authors Journal Compilation Ó 2007 International Society for Neurochemistry, J. Neurochem. (2008) 105, 101–112 101