Roles of Necl-5/poliovirus receptor and rho-associated kinase (ROCK) in the regulation of transformation of integrin αVβ3- based focal complexes into focal adhesions

Abstract

Focal complexes are continuously formed and transformed into focal adhesions during cell movement. We previously demonstrated that Necl-5 co-localizes with integrin αVβ3 at focal complexes, whereas Necl-5 does not localize at focal adhesions in moving NIH3T3 cells, suggesting that Necl-5 may be dissociated from integrin αVβ3 during the transformation of focal complexes into focal adhesions, but the underlying mechanism remains unknown. Here, we explore the roles of Necl-5 and Rho-associated kinase (ROCK) in the regulation of the transformation of focal complexes into focal adhesions. We found that inhibition of Necl-5expression and expression of a constitutively active mutant of ROCK1 enhanced, whereas treatment with a ROCK inhibitor Y-27632 inhibited the transformation of focal complexes into focal adhesions. In HEK293 cells ectopically expressing Necl-5 and integrin αVβ 3, treatment of cells with Y-27632 increased the binding of Necl-5 to clustered integrin αVβ3. The experiments using inhibitors of myosin ATPase and actin polymerization revealed that actomyosin-driven contractility exerts a similar function as ROCK. The phosphorylation of integrin β3 at Tyr747, which is known to be critical for the formation of focal adhesions, plays a pivotal role for the interaction between Necl-5 and integrin αVβ 3. These results indicate that the transformation of focal complexes into focal adhesions is negatively and positively regulated by Necl-5 and ROCK, respectively, and that ROCK-dependent actomyosin-driven contractility is a critical determinant for the regulation of the interaction between Necl-5 and integrin αVβ3. © 2008 by The American Society for Biochemistry and Molecular Biology, Inc.