title: IGF-I stimulates cooperative interaction between the IGF-I receptor and CSK homologous kinase that regulates SHPS-1 phosphorylation in vascular smooth muscle cells.
abstract: IGF-I plays an important role in smooth muscle cell proliferation and migration. In vascular smooth muscle cells cultured in 25 mm glucose, IGF-I stimulated a significant increase in Src homology 2 domain containing protein tyrosine phosphatase substrate-1 (SHPS-1) phosphorylation compared with 5 mm glucose and this increase was required for smooth muscle cell proliferation. A proteome-wide screen revealed that carboxyl-terminal SRC kinase homologous kinase (CTK) bound directly to phosphotyrosines in the SHPS-1 cytoplasmic domain. Because the kinase(s) that phosphorylates these tyrosines in response to IGF-I is unknown, we determined the roles of IGF-I receptor (IGF-IR) and CTK in mediating SHPS-1 phosphorylation. After IGF-I stimulation, CTK was recruited to IGF-IR and subsequently to phospho-SHPS-1. Expression of an IGF-IR mutant that eliminated CTK binding reduced CTK transfer to SHPS-1, SHPS-1 phosphorylation, and cell proliferation. IGF-IR phosphorylated SHPS-1, which provided a binding site for CTK. CTK recruitment to SHPS-1 resulted in a further enhancement of SHPS-1 phosphorylation. CTK knockdown also impaired IGF-I-stimulated SHPS-1 phosphorylation and downstream signaling. Analysis of specific tyrosines showed that mutation of tyrosines 428/452 in SHPS-1 to phenylalanine reduced SHPS-1 phosphorylation but allowed CTK binding. In contrast, the mutation of tyrosines 469/495 inhibited IGF-IR-mediated the phosphorylation of SHPS-1 and CTK binding, suggesting that IGF-IR phosphorylated Y469/495, allowing CTK binding, and that CTK subsequently phosphorylated Y428/452. Based on the above findings, we conclude that after IGF-I stimulation, CTK is recruited to IGF-IR and its recruitment facilitates CTK's subsequent association with phospho-SHPS-1. This results in the enhanced CTK transfer to SHPS-1, and the two kinases then fully phosphorylate SHPS-1, which is necessary for IGF-I stimulated cellular proliferation.
authors.affiliation: Department of Medicine, University of North Carolina, School of Medicine, Chapel Hill, North Carolina 27599, USA.
authors.forename: Yashwanth
authors.initials: Y
authors.lastname: Radhakrishnan
authors.forename: Xinchun
authors.initials: X
authors.lastname: Shen
authors.forename: Laura A
authors.initials: LA
authors.lastname: Maile
authors.forename: Gang
authors.initials: G
authors.lastname: Xi
authors.forename: David R
authors.initials: DR
authors.lastname: Clemmons
mesh_terms: D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D020868:Gene Silencing / Q000187:drug effects; D005947:Glucose / Q000494:pharmacology; D006801:Humans; D007334:Insulin-Like Growth Factor I / Q000494:pharmacology*; D051379:Mice; D009131:Muscle, Smooth, Vascular / Q000166:cytology*; D032389:Myocytes, Smooth Muscle / Q000187:drug effects / Q000201:enzymology*; D010766:Phosphorylation / Q000187:drug effects; D019000:Phosphotyrosine / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000378:metabolism*; D051381:Rats; D017526:Receptor, IGF Type 1 / Q000378:metabolism*; D011971:Receptors, Immunologic / Q000378:metabolism*; D034421:Sus scrofa
chemical_list: D011971:Receptors, Immunologic; D019000:Phosphotyrosine; D007334:Insulin-Like Growth Factor I; D017526:Receptor, IGF Type 1; C085377:MATK protein, human; D016392:Proto-Oncogene Proteins pp60(c-src); D005947:Glucose