Cell adhesion kinase β forms a complex with a new member, Hic-5, of proteins localized at focal adhesions

Cell adhesion kinase beta (CAK beta/PYK2) is the second protein-tyrosine kinase of the focal adhesion kinase subfamily, We identified a cDNA that encodes a CAK beta-binding protein, This cDNA clone encodes the human homologue of Hic-5, the cDNA of which was cloned in 1994 as transforming growth factor beta 1- and hydrogen peroxide-inducible mRNA. We found that Hic-5 exclusively localized at focal adhesions in a rat fibroblast line, WFB., This localization of Hic-5 was confirmed in WFB cells expressing Myc-tagged Hic-5, The amino acid sequence of Hic-5 is highly similar to that of paxillin in the four LD motifs as well as in the four contiguous LIM domains. The Hic-5 N-terminal domain directly associated in vitro with the extreme C-terminal region (residue 801 to the end) of CAK beta., CAK beta was coimmunoprecipitated with Hic-5 from the WFB cell lysate, The coimmunoprecipitation of CAK beta with Hic-5 was markedly inhibited by the addition of the extreme C-terminal region of CAK beta., Coimmunoprecipitation of Hic-5 with CAK beta, which was shown in COS-7 cells doubly transfected with cDNA constructs of CAK beta and Myc-tagged Hic-5, was lost when the CAK beta amino acid residues 741-903 were deleted, Hic-5 was tyrosine-phosphorylated in Src-transformed 3Y1 cells and in cells treated with pervanadate. Hic-5 associated with CAK beta was selectively tyrosine-phosphorylated in WFB cells exposed to hypertonic osmotic stress, These results indicate that Hic-5 is a paxillin-related component of focal adhesions and binds to CAK beta, implying possible involvement of Hic-5 in the downstream signaling of CAK beta.