The Cytoplasmic Tyrosines of Integrin Subunit β1 Are Involved in Focal Adhesion Kinase Activation

Abstract

We have previously shown that mutation of the two tyrosines in the cytoplasmic domain of integrin subunit β1 (Y783 and Y795) to phenylalanines markedly reduces the capability of β1A integrins to mediate directed cell migration. In this study, β1-dependent cell spreading was found to be delayed in GD25 cells expressing β1A_Y783/795F compared to that in wild-type GD25-β1A. Focal adhesion kinase (FAK) tyrosine phosphorylation and activation were severely impaired in response to β1-dependent adhesion in GD25-β1A_Y783/795F cells compared to that in wild-type GD25-β1A or mutants in which only a single tyrosine was altered (β1A_Y783F or β1A_Y795F). Phosphorylation site-specific antibodies selective for FAK phosphotyrosine 397 indicated that the defect in FAK phosphorylation via β1A_Y783/795F lies at the level of the initial autophosphorylation step. Indeed, β1A-dependent tyrosine phosphorylation of tensin and paxillin was lost in the β1A_Y783/795F cells, consistent with the impairment in FAK activation. In contrast, p130^CAS overall tyrosine phosphorylation was unaffected by the β1 mutations. Despite the defect in β1-mediated FAK activation, FAK was still localized to focal adhesions. Taken together, the phenotype of the GD25-β1A_Y783/795F cells resembles, but is distinct from, the phenotype observed in FAK-null cells. These observations argue that tyrosines 783 and 795 within the cytoplasmic tail of integrin subunit β1A are critical mediators of FAK activation and cell spreading in GD25 cells.

ACKNOWLEDGMENTS

We thank Helena Larsson and Ivan Dikic, Uppsala, Sweden, for support and helpful discussions.

This work was supported by grants from Polysackaridforskning AB, Uppsala, Sweden (K.W.), the Swedish Medical Research Council (no. 7147) (S.J.), Swedish Cancer Foundation (no. 4158) (S.J.), King Gustaf V:s 80-års Fond (S.J. and R.F.), the National Institutes of Health (HL 21644 and HL 56396) (D.M.), and the Swedish Natural Science Research Council (R.F.).