Distinct mechanisms mediate the initial and sustained phases of integrin-mediated activation of the Raf/MEK/mitogen-activated protein kinase cascade.
Integrin-mediated adhesion to the extracellular matrix activates the canonical mitogen-activated protein kinase cascade, although the exact mechanism is not fully resolved. We show that integrin-mediated activation of Raf-1, an upstream regulator of mitogen-activated protein kinase, occurs in two phases. Efficient early activation of Raf required Raf-Ras interaction but was not affected by protein kinase C (PKC) inhibitors, while a lower, sustained level of activity was independent of Raf-Ras interaction but was reduced by PKC inhibitors. The combination of PKC inhibition and lack of Ras binding completely blocked integrin-mediated Raf activity. The activity of a membrane-bound Raf mutant that is deficient in Ras binding (Raf-R89L-CAAX) was also regulated by adhesion. Raf-R89L-CAAX activity was low in nonadherent cells, was rapidly stimulated to wild-type levels by cell adhesion, and remained at nearly maximal levels longer than wild-type activity. The activation of wild-type and mutant Raf proteins was ablated by cytochalasin D, demonstrating that cytoskeletal organization is required for activation of Raf, even when targeted to the membrane. These data suggest distinct initial and sustained phases of integrin-mediated Raf activation that require Raf membrane localization and possibly PKC activity, respectively, and that integrin-mediated adhesion may regulate a cytoskeleton-associated factor(s) responsible for Raf activation.
Department of Pharmacology and The Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill School of Medicine, Chapel Hill, North Carolina 27599-7365, USA.
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Howe
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Juliano
D016475:3T3 Cells; D000199:Actins; D000818:Animals; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D002448:Cell Adhesion; D003599:Cytoskeleton; D004789:Enzyme Activation; D016023:Integrins / Q000378:metabolism*; D048369:MAP Kinase Kinase 1; D051379:Mice; D020929:Mitogen-Activated Protein Kinase Kinases; D009154:Mutation; D011485:Protein Binding; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D019908:Proto-Oncogene Proteins c-raf / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction; D018631:ras Proteins / Q000378:metabolism
D000199:Actins; D016023:Integrins; D011505:Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases; D019908:Proto-Oncogene Proteins c-raf; D011493:Protein Kinase C; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D048369:MAP Kinase Kinase 1; C482127:Map2k1 protein, mouse; D020929:Mitogen-Activated Protein Kinase Kinases; D018631:ras Proteins