Hic-5-Reduced Cell Spreading on Fibronectin: Competitive Effects between Paxillin and Hic-5 through Interaction with Focal Adhesion Kinase

Abstract

Hic-5 is a paxillin homologue that is localized to focal adhesion complexes. Hic-5 and paxillin share structural homology and interacting factors such as focal adhesion kinase (FAK), Pyk2/CAKβ/RAFTK, and PTP-PEST. Here, we showed that Hic-5 inhibits integrin-mediated cell spreading on fibronectin in a competitive manner with paxillin in NIH 3T3 cells. The overexpression of Hic-5 sequestered FAK from paxillin, reduced tyrosine phosphorylation of paxillin and FAK, and prevented paxillin-Crk complex formation. In addition, Hic-5-mediated inhibition of spreading was not observed in mouse embryo fibroblasts (MEFs) derived from FAK^−/− mice. The activity of c-Src following fibronectin stimulation was decreased by about 30% in Hic-5-expressing cells, and the effect of Hic-5 was restored by the overexpression of FAK and the constitutively active forms of Rho-family GTPases, Rac1 V12 and Cdc42 V12, but not RhoA V14. These observations suggested that Hic-5 inhibits cell spreading through competition with paxillin for FAK and subsequent prevention of downstream signal transduction. Moreover, expression of antisense Hic-5 increased spreading in primary MEFs. These results suggested that the counterbalance of paxillin and Hic-5 expression may be a novel mechanism regulating integrin-mediated signal transduction.

ACKNOWLEDGMENTS

We are grateful to Kohzoh Kaibuchi (Nara Institute of Science and Technology, Ikoma, Japan) for providing Rho-family GTPase expression vectors, Michel L. Tremblay (McGill University, Montreal, Canada) for GST fusion Crk SH2 expression vectors, and Tadashi Yamamoto (Institute of Medical Science, University of Tokyo, Minato-ku, Japan) for MEFs from FAK-null mice. Anti-Cas antibody was kindly provided by Tatsuya Nakamoto, University of Tokyo Medical School. We also thank Takeshi Shirai, Momoko Fujisaki, Wataru Suzuki, and Tomoko Kanome for technical assistance.

This work was supported in part by grants-in-aid for Scientific Research, a grant-in-aid for Cancer Research, and the High-Technology Research Center Project from the ministry of Education, Science, Sports, and Culture of Japan and by a grant-in-aid from the Takeda Science Foundation.