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Cell adhesion kinase beta forms a complex with a new member, Hic-5, of proteins localized at focal adhesions
1Department of Biochemistry, Sapporo Medical University School of Medicine, South-1, West-17, Chuo-Ku, Sapporo 060, Japan.
Abstract:
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.
Insights
Hic-5, a protein similar to paxillin, binds to cell adhesion kinase beta (CAKbeta/PYK2) at focal adhesions. This interaction suggests Hic-5 may play a role in CAKbeta signaling pathways.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Cell adhesion kinase beta (CAKbeta/PYK2) is a key protein-tyrosine kinase in the focal adhesion kinase subfamily.
- Focal adhesions are critical cellular structures involved in cell adhesion and signaling.
Purpose of the Study:
- To identify and characterize CAKbeta-binding proteins.
- To investigate the interaction between Hic-5 and CAKbeta and its functional implications.
Main Methods:
- cDNA cloning and sequencing to identify Hic-5.
- Immunoprecipitation and co-immunoprecipitation assays to confirm protein interactions.
- Site-directed mutagenesis to map binding domains.
- Subcellular localization studies using immunofluorescence.
Main Results:
- Identified human Hic-5 as a CAKbeta-binding protein.
- Demonstrated that Hic-5 localizes to focal adhesions.
- Showed that the N-terminal domain of Hic-5 directly binds to the C-terminal region of CAKbeta.
- Confirmed that Hic-5 is tyrosine-phosphorylated and its phosphorylation is enhanced by hypertonic osmotic stress when associated with CAKbeta.
Conclusions:
- Hic-5 is a paxillin-related focal adhesion protein that interacts with CAKbeta.
- The interaction between Hic-5 and CAKbeta suggests a role for Hic-5 in CAKbeta-mediated downstream signaling pathways.