(alpha)v(beta)3 integrins and Pyk2 mediate insulin-like growth factor I activation of Src and mitogen-activated protein kinase in 3T3-L1 cells.
IGF-I stimulates cell growth through interaction of the IGF receptor with multiprotein signaling complexes. However, the mechanisms of IGF-I receptor-mediated signaling are not completely understood. We have previously shown that IGF-I-stimulated 3T3-L1 cell proliferation is dependent on Src activation of the ERK-1/2 MAPK pathway. We hypothesized that IGF-I activation of the MAPK pathway is mediated through integrin activation of Src-containing signaling complexes. The disintegrin echistatin decreased IGF-I phosphorylation of Src and MAPK, and blocking antibodies to (alpha)v and beta3 integrin subunits inhibited IGF-I activation of MAPK, suggesting that (alpha)v(beta)3 integrins mediate IGF-I mitogenic signaling. IGF-I increased ligand binding to (alpha)v(beta)3 as detected by immunofluorescent staining of ligand-induced binding site antibody and stimulated phosphorylation of the beta3 subunit, consistent with inside-out activation of (alpha)v(beta)3 integrins. IGF-I increased tyrosine phosphorylation of the focal adhesion kinase (FAK) Pyk2 (calcium-dependent proline-rich tyrosine kinase-2) to a much greater extent than FAK, and increased association of Src with Pyk2 but not FAK. The intracellular calcium chelator BAPTA prevented IGF-I phosphorylation of Pyk2, Src, and MAPK, suggesting that IGF-I activation of Pyk2 is calcium dependent. Transient transfection with a dominant-negative Pyk2, which lacks the autophosphorylation and Src binding site, decreased IGF-I activation of MAPK, but no inhibition was seen with transfected wild-type Pyk2. These results indicate that IGF-I signaling to MAPK is dependent on inside-out activation of (alpha)v(beta)3 integrins and integrin-facilitated multiprotein complex formation involving Pyk2 activation and association with Src.
Sekimoto
Hiroko
H
Rhode Island Hospital, Department of Pediatrics, 593 Eddy Street, MPS-2, Providence, RI 02903, USA.
Eipper-Mains
Jodi
J
Pond-Tor
Sunthorn
S
Boney
Charlotte M
CM
D041721:3T3-L1 Cells; D000818:Animals; D019138:Antibodies, Blocking / Q000494:pharmacology; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D004789:Enzyme Activation; D051417:Focal Adhesion Kinase 1; D051418:Focal Adhesion Kinase 2; D051416:Focal Adhesion Protein-Tyrosine Kinases; D007334:Insulin-Like Growth Factor I / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000502:physiology*; D039302:Integrin alphaVbeta3 / Q000276:immunology / Q000378:metabolism*; D051379:Mice; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction; D019061:src-Family Kinases / Q000378:metabolism*
D019138:Antibodies, Blocking; D039302:Integrin alphaVbeta3; D004533:Egtazic Acid; D007334:Insulin-Like Growth Factor I; D011505:Protein-Tyrosine Kinases; D051417:Focal Adhesion Kinase 1; D051418:Focal Adhesion Kinase 2; D051416:Focal Adhesion Protein-Tyrosine Kinases; C496051:Ptk2 protein, mouse; C496053:Ptk2b protein, mouse; D019061:src-Family Kinases; D020928:Mitogen-Activated Protein Kinases; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid