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.
Highlights
A panel of antibodies to the ␣IIb3 integrin was used to promote adhesion of Chinese hamster ovary cells transfected with the ␣IIb3 fibrinogen receptor
Phorbol 12-myristate 13-acetate (PMA) or the combination of PMA and Cytotoxic necrotizing factor 1 (CNF1) increases tyrosine phosphorylation of p125 focal adhesion kinase (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; 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
Tyrosine phosphorylation of the p125 focal adhesion kinase (p125FAK),1 which is localized in the focal contacts [8, 9], is an early event in integrin signaling in cells plated on matrix proteins (10 –13) and by clustering of 1 integrins on cells adherent to plastic dishes [14] or on cells in suspension [12]
Summary
A panel of antibodies to the ␣IIb3 integrin was used to promote adhesion of Chinese hamster ovary cells transfected with the ␣IIb3 fibrinogen receptor. While some ␣IIb3 antibodies were not able to induce p125 focal adhesion kinase (p125FAK) tyrosine phosphorylation, all the antibodies support cell adhesion but not spreading and assembly of actin stress fibers. Herbimycin A and genistein, two known inhibitors of kinase activity, prevent focal adhesion formation and actin stress fibers assembly in cells plated on fibronectin [10, 12]. Treatment of cells with phosphotyrosine phosphatase inhibitors leads to increased integrin-dependent organization of focal adhesion and actin stress fibers [16]. To assess the ability of integrins to regulate cell spreading and actin cytoskeleton organization, Chinese hamster ovary (CHO) cells transfected with the human ␣IIb3 receptor were plated on anti-human ␣IIb3 or anti-hamster ␣51 antibodies, and the roles of p125FAK kinase, protein kinase C and small GTPase Rho were analyzed. This paper is available on line at http://www.jbc.org show that p125FAK tyrosine phosphorylation is not sufficient for stress fibers organization
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