title: Dissection of pathways implicated in integrin-mediated actin cytoskeleton assembly. Involvement of protein kinase C, Rho GTPase, and tyrosine phosphorylation.
abstract: A panel of antibodies to the alphaIIbbeta3 integrin was used to promote adhesion of Chinese hamster ovary cells transfected with the alphaIIbbeta3 fibrinogen receptor. While some alphaIIbbeta3 antibodies were not able to induce p125 focal adhesion kinase (p125FAK) tyrosine phosphorylation, all the antibodies equally support cell adhesion but not spreading and assembly of actin stress fibers. Absence of stress fibers was also obtained by plating on antibodies directed to the hamster beta1 integrin. In contrast, cells plated on matrix proteins spread organizing actin stress fibers. Treatment with phorbol esters phorbol 12-myristate 13-acetate (PMA) induced cells to spread on antibodies-coated dishes but not to organize actin in stress fibers. The combination of PMA and cytotoxic necrotizing factor 1 (CNF1), a specific Rho activator, induced cell spreading and organization of stress fibers. PMA or the combination of PMA and CNF1 also increases tyrosine phosphorylation of p125FAK in response to antibodies that were otherwise unable to trigger this response. These data show that: 1) matrix proteins and antibodies differ in their ability to induce integrin-dependent actin cytoskeleton organization (while matrix induced stress fibers formation, antibodies did not); 2) p125FAK tyrosine phosphorylation is insufficient per se to trigger actin stress fibers formation since antibodies that activate p125FAK tyrosine phosphorylation did not lead to actin stress fibers assembly; and 3) the inability of anti-integrin antibodies to trigger stress fibers organization is overcome by concomitant activation of the protein kinase C (PKC) and Rho pathways; PKC activation leads to cell spreading and Rho activation is required to organize actin stress fibers.
authors.affiliation: Dipartimento di Genetica, Biologia e Chimica Medica, Universita' di Torino, 10126 Torino, Italy. defilip@golgi.molinette.unito.it
authors.forename: P
authors.initials: P
authors.lastname: Defilippi
authors.forename: M
authors.initials: M
authors.lastname: Venturino
authors.forename: D
authors.initials: D
authors.lastname: Gulino
authors.forename: A
authors.initials: A
authors.lastname: Duperray
authors.forename: P
authors.initials: P
authors.lastname: Boquet
authors.forename: C
authors.initials: C
authors.lastname: Fiorentini
authors.forename: G
authors.initials: G
authors.lastname: Volpe
authors.forename: M
authors.initials: M
authors.lastname: Palmieri
authors.forename: L
authors.initials: L
authors.lastname: Silengo
authors.forename: G
authors.initials: G
authors.lastname: Tarone
mesh_terms: D000199:Actins / Q000378:metabolism*; D000818:Animals; D001427:Bacterial Toxins / Q000494:pharmacology; D016466:CHO Cells; D002448:Cell Adhesion; D015815:Cell Adhesion Molecules / Q000378:metabolism; D006224:Cricetinae; D003599:Cytoskeleton / Q000378:metabolism* / Q000648:ultrastructure; D003603:Cytotoxins / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D029968:Escherichia coli Proteins; D005340:Fibrinogen / Q000378:metabolism; D019084:Fluorescent Antibody Technique, Indirect; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; D019204:GTP-Binding Proteins / Q000378:metabolism*; D020690:GTPase-Activating Proteins; D016023:Integrins / Q000378:metabolism*; D051379:Mice; D010766:Phosphorylation; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000235:genetics / Q000378:metabolism*; D011493:Protein Kinase C / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011817:Rabbits; D011972:Receptor, Insulin / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014162:Transfection; D014443:Tyrosine / Q000378:metabolism*
chemical_list: D000199:Actins; D001427:Bacterial Toxins; D015815:Cell Adhesion Molecules; D003603:Cytotoxins; D029968:Escherichia coli Proteins; D020690:GTPase-Activating Proteins; D016023:Integrins; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; C085379:rho GTPase-activating protein; C060296:cytotoxic necrotizing factor type 1; D014443:Tyrosine; D005340:Fibrinogen; D011505:Protein-Tyrosine Kinases; D011972:Receptor, Insulin; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; C496051:Ptk2 protein, mouse; D011493:Protein Kinase C; D019204:GTP-Binding Proteins; D013755:Tetradecanoylphorbol Acetate