Cell adhesion kinase beta forms a complex with a new member, Hic-5, of proteins localized at focal adhesions.
Cell adhesion kinase beta (CAKbeta/PYK2) is the second protein-tyrosine kinase of the focal adhesion kinase subfamily. We identified a cDNA that encodes a CAKbeta-binding protein. This cDNA clone encodes the human homologue of Hic-5, the cDNA of which was cloned in 1994 as transforming growth factor beta1- 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 CAKbeta. CAKbeta was coimmunoprecipitated with Hic-5 from the WFB cell lysate. The coimmunoprecipitation of CAKbeta with Hic-5 was markedly inhibited by the addition of the extreme C-terminal region of CAKbeta. Coimmunoprecipitation of Hic-5 with CAKbeta, which was shown in COS-7 cells doubly transfected with cDNA constructs of CAKbeta and Myc-tagged Hic-5, was lost when the CAKbeta 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 CAKbeta 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 CAKbeta, implying possible involvement of Hic-5 in the downstream signaling of CAKbeta.
Matsuya
M
M
Department of Biochemistry, Sapporo Medical University School of Medicine, South-1, West-17, Chuo-Ku, Sapporo 060, Japan.
Sasaki
H
H
Aoto
H
H
Mitaka
T
T
Nagura
K
K
Ohba
T
T
Ishino
M
M
Takahashi
S
S
Suzuki
R
R
Sasaki
T
T
D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002460:Cell Line; D002675:Child, Preschool; D003001:Cloning, Molecular; D003598:Cytoskeletal Proteins / Q000737:chemistry; D018076:DNA, Complementary; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005260:Female; D051418:Focal Adhesion Kinase 2; D005982:Glutathione Transferase / Q000235:genetics; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D060588:LIM Domain Proteins; D008246:Lysophospholipids / Q000494:pharmacology; D008969:Molecular Sequence Data; D018384:Oxidative Stress; D051419:Paxillin; D010750:Phosphoproteins / Q000737:chemistry; D011233:Precipitin Tests; D011505:Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D003598:Cytoskeletal Proteins; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D047908:Intracellular Signaling Peptides and Proteins; D060588:LIM Domain Proteins; D008246:Lysophospholipids; C496061:PXN protein, human; D051419:Paxillin; D010750:Phosphoproteins; C496063:Pxn protein, rat; D011993:Recombinant Fusion Proteins; C490351:TGFB1I1 protein, human; C490352:Tgfb1i1 protein, rat; D005982:Glutathione Transferase; D011505:Protein-Tyrosine Kinases; D051418:Focal Adhesion Kinase 2; C496054:Ptk2b protein, rat