The role of Src kinase in insulin-like growth factor-dependent mitogenic signaling in vascular smooth muscle cells.
Activation of the MAPK pathway mediates insulin-like growth factor-I (IGF-I)-dependent proliferation in vascular smooth muscle cells (SMC). Our previous studies have shown that IGF-I-induced Shc phosphorylation is necessary for sustained activation of MAPK and increased cell proliferation of SMCs, and both Shc and the tyrosine phosphatase SHP-2 must be recruited to the membrane protein SHPS-1 in order for Shc to be phosphorylated. These studies were undertaken to determine whether Src kinase activity is required to phosphorylate Shc in response to IGF-I in SMC and because SHP-2 binds to Src whether their interaction was also required for IGF-I-stimulated mitogenesis. Our results show that IGF-I induces activation of Src kinase and that is required for Shc phosphorylation and for optimal MAPK activation. We tested whether Shc is a substrate of c-Src in SMC by disrupting Src/Shc association using a peptide containing a YXXL (Tyr328) motif sequence derived from Src. The peptide blocked the binding of Src and Shc in vitro and in vivo. Cells expressing a mutant Src (Src-FF) that had Tyr328/Tyr358 substituted with phenylalanines (Src-FF) showed defective Src/Shc binding, impaired IGF-I-dependent Shc phorylation, and impaired mitogenesis. This supports the conclusion that Src phosphorylates Shc. IGF-I induced both Src/SHP-2 and Src/SHPS-1 association. SMCs expressing an SHP-2 mutant that had the polyproline-rich region of SH2 deleted (SHP-2Delta10) had disrupted SHP-2/Src association, impaired IGF-I-dependent Shc phosphorylation, and an attenuated mitogenic response. IGF-I-induced association of Src and SHPS-1 was also impaired in SHP-2Delata10-expressing cells, although SHP-2/SHPS-1 association was unaffected. Upon IGF-I stimulation, a complex assembles on SHPS-1 that contains SHP-2, c-Src, and Shc wherein Src phosphorylates Shc, a signaling step that is necessary for an optimal mitogenic response.
Lieskovska
Jaroslava
J
Department of Medicine, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599-7170, USA.
Ling
Yan
Y
Badley-Clarke
Jane
J
Clemmons
David R
DR
D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D000943:Antigens, Differentiation / Q000502:physiology*; D006801:Humans; D066298:In Vitro Techniques; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism; D020935:MAP Kinase Signaling System; D008969:Molecular Sequence Data; D009131:Muscle, Smooth, Vascular / Q000166:cytology*; D054592:Protein Tyrosine Phosphatase, Non-Receptor Type 11; D017027:Protein Tyrosine Phosphatases / Q000378:metabolism; D011971:Receptors, Immunologic / Q000502:physiology*; D055765:Shc Signaling Adaptor Proteins; D015398:Signal Transduction; D013002:Somatomedins / Q000378:metabolism*; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; D013552:Swine; D019061:src-Family Kinases / Q000502:physiology*
D048868:Adaptor Proteins, Signal Transducing; D000943:Antigens, Differentiation; D047908:Intracellular Signaling Peptides and Proteins; D011971:Receptors, Immunologic; C528024:SHC1 protein, human; C501920:SIRPA protein, human; D055765:Shc Signaling Adaptor Proteins; D013002:Somatomedins; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; D019061:src-Family Kinases; C516724:PTPN11 protein, human; D054592:Protein Tyrosine Phosphatase, Non-Receptor Type 11; D017027:Protein Tyrosine Phosphatases