Integrin and cytoskeletal regulation of growth factor signaling to the MAP kinase pathway.
Integrin-mediated anchorage of NIH3T3 fibroblasts to the extracellular matrix component fibronectin permits efficient growth factor signaling to the p42 and p44 forms of mitogen-activated protein kinase (MAPK). Since integrins bridge the extracellular matrix to focal adhesion sites and to the actin cytoskeleton, we analyzed the role of these integrin-associated structures in efficient growth factor activation of p42 and p44-MAPKs. Use of specific reagents that disrupt actin stress fiber and focal adhesion formation demonstrated that upon readhesion of NIH3T3 cells to fibronectin, cells that were poorly spread and lacked prominent focal adhesions but that formed cortical actin structures, efficiently signaled to p42 and p44-MAPKs upon EGF stimulation. In contrast, failure to form the cortical actin structures, despite attachment to fibronectin, precluded effective EGF signaling to p42 and p44-MAPKs. Actin cytoskeletal changes induced by expression of dominant-negative and constitutively active forms of Rho GTPases did not alter EGF activation of MAPK in adherent cells. However, active Cdc42, but not active Rac1 or RhoA, partially rescued EGF signaling to p44-MAPK in cells maintained in suspension. These data indicate that a limited degree of adhesion-mediated cytoskeletal organization and focal adhesion complex formation are required for efficient EGF activation of p42 and p44-MAPKs. Our studies exclude a major role for the GTPases RhoA and Rac1 in the formation of cytoskeletal structures relevant for signaling, but indicate that structures regulated by Cdc42 enhance the ability of suspension cells to activate MAPK in response to growth factors.
Aplin
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AE
Department of Pharmacology, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
Juliano
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RL
D016475:3T3 Cells; D000199:Actins / Q000378:metabolism; D000818:Animals; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000494:pharmacology; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D002448:Cell Adhesion; D018797:Cell Cycle Proteins / Q000378:metabolism; D002455:Cell Division; D015638:Cytochalasin D / Q000494:pharmacology; D003599:Cytoskeleton / Q000378:metabolism*; D004789:Enzyme Activation; D004815:Epidermal Growth Factor / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism; D006133:Growth Substances / Q000378:metabolism*; D016023:Integrins / Q000378:metabolism*; D008156:Luciferases / Q000235:genetics; D051379:Mice; D015398:Signal Transduction / Q000187:drug effects; D013844:Thiazoles / Q000494:pharmacology; D053778:Thiazolidines; D014162:Transfection; D020764:cdc42 GTP-Binding Protein; D020742:rhoA GTP-Binding Protein