|Budget Amount *help
¥3,100,000 (Direct Cost : ¥3,100,000)
Fiscal Year 1999 : ¥1,400,000 (Direct Cost : ¥1,400,000)
Fiscal Year 1998 : ¥1,700,000 (Direct Cost : ¥1,700,000)
Hic-5 is a CAKβ-binding protein localized at focal adhesions. We showed that overexpression of CAKβ or Fyn, but not FAK, enhanced the tyrosine-phosphorylation of coexpressed Hic-5. These phosphoorylations were further augmented by stimulating cells with osmotic stress. The Y60F mutant of Hic-5 was not phosphorylated, and Hic-5 phosphorylated on tyrosine 60 was bound specifically to the SH2 domain of Csk. Coexpression experiments revealed that the phosphorylation of Hic-5 by CAKβ required the kinase activation of CAKβ and binding of Hic-5 by CAKβ. Specific phosphorylation of Hic-5 by CAKβ and Fyn may activate a signaling pathway mediated by Hic-5.
CAKβ/PYK2 is a protein-tyrosine kinase of the focal adhesion kinase (FAK) family. Whereas FAK predominantly localizes at focal adhesions, CAKβ localizes at the perinuclear region. Here we expressed in cultured cells two point mutants of CAKβ, P717A and P859A, which each lost one of the two PXXP motifs, the ligand sequenc3 for SH3 domains, found at the CAKβC-terminal region. We found a remarkable change in the subcellular distribution of the P859A mutant; that of the P717A mutant was the same as the wild type. The P859A mutant localized exclusively in the cell nucleus in all cell lines examined. Wild-type CAKβ also accumulated in the nucleus when cells were treated with an inhibitor of the nuclear export of proteins. These results indicate that CAKβ shuttles between the cytoplasm and the nucleus. On nuclear accumulation of P859A-CAKβ, a CAKβ-binding protein, Hic-5, also accumulated in the nucleus. P859A-CAKβ and co-expressed Hic-5 formed nuclear speckles, in which one other CAKβ-binding proteins, p130ィイD1CasィエD1, was also concentrated. These findings on nuclear translocation of CAKβ imply that CAKβmay regulate nuclear processes such as transcription, particularly because Hic-5 was recently shown to be a coactivator of nuclear receptors.