Cell Adhesion Kinase β Forms a Complex with a New Member, Hic-5, of Proteins Localized at Focal Adhesions*
- Manabu Matsuya‡§,
- Hiroko Sasaki‡§,
- Hiroshi Aoto‡,
- Toshihiro Mitaka¶,
- Kazuko Nagura‡,
- Takeaki Ohba‡,
- Masaho Ishino‡,
- Shuji Takahashi‖,
- Rumiko Suzuki‡ and
- Terukatsu Sasaki‡**
- From the Departments of ‡Biochemistry and¶Pathology, Cancer Research Institute, and the ‖Department of Pathology, Sapporo Medical University School of Medicine, South-1, West-17, Chuo-Ku, Sapporo 060, Japan
Abstract
Cell adhesion kinase β (CAKβ/PYK2) is the second protein-tyrosine kinase of the focal adhesion kinase subfamily. We identified a cDNA that encodes a CAKβ-binding protein. This cDNA clone encodes the human homologue of Hic-5, the cDNA of which was cloned in 1994 as transforming growth factor β1- 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 vitrowith the extreme C-terminal region (residue 801 to the end) of CAKβ. CAKβ was coimmunoprecipitated with Hic-5 from the WFB cell lysate. The coimmunoprecipitation of CAKβ with Hic-5 was markedly inhibited by the addition of the extreme C-terminal region of CAKβ. Coimmunoprecipitation of Hic-5 with CAKβ, which was shown in COS-7 cells doubly transfected with cDNA constructs of CAKβ and Myc-tagged Hic-5, was lost when the CAKβ 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 CAKβ 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 CAKβ, implying possible involvement of Hic-5 in the downstream signaling of CAKβ.
Footnotes
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↵* This work was supported in part by grants-in-aid from the Ministry of Education, Science, Sports and Culture, Japan.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank™/EMBL Data Bank with accession number(s) AB007836.
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↵§ The first two authors contributed equally to this work.
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↵** To whom correspondence should be addressed: Dept. of Biochemistry, Cancer Research Institute, Sapporo Medical University School of Medicine, South-1, West-17, Chuo-Ku, Sapporo 060, Japan. Tel.: 81-11-611-2111 (ext. 2380); Fax: 81-11-612-5861; E-mail:sasaki{at}cc.sapmed.ac.jp.
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↵1 The abbreviations used are: FAK, focal adhesion kinase; CAKβ, cell adhesion kinase β; N-domain, N-terminal domain; C-domain, C-terminal domain; SH2, Src homology domain 2; SH3, Src homology domain 3; PAGE, polyacrylamide gel electrophoresis; PVDF, polyvinylidene difluoride; ECL, enzyme-linked chemiluminescence; PBS, phosphate-buffered saline; GST, glutathione S-transferase; PBS1 and PBS2, paxillin-binding subdomains 1 and 2; bp, base pair(s); HSV, herpes simplex virus.
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- Received May 16, 1997.
- Revision received October 20, 1997.
- The American Society for Biochemistry and Molecular Biology, Inc.
