| Project/Area Number |
09670137
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
General medical chemistry
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| Research Institution | Sapporo Medical University |
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Principal Investigator |
SASAKI Terukatsu Sapporo Medical University, Cancer Research Institute, Professor, 医学部・附属がん研究所, 教授 (00045494)
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| Co-Investigator(Kenkyū-buntansha) |
SASAKI Hiroko Sapporo Medical University, Cancer Research Institute, Assistant Professor, 医学部・附属がん研究所, 講師 (60045424)
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| Project Period (FY) |
1997 – 1998
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| Project Status |
Completed (Fiscal Year 1998)
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| Budget Amount *help |
¥3,000,000 (Direct Cost: ¥3,000,000)
Fiscal Year 1998: ¥1,200,000 (Direct Cost: ¥1,200,000)
Fiscal Year 1997: ¥1,800,000 (Direct Cost: ¥1,800,000)
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| Keywords | CAKbeta / PYK2 / protein-tyrosine kinase / cDNA expression cloning / Hic-5 / SH2 domain / tyrosine-phosphorylation / nuclear translocation / FAK / パキシリン / 焦点接着 |
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| Research Abstract |
1. A cDNA for a CAKbeta/PYK2-binding protein (CBP-1) was cloned by screening a human brain cDNA library with the CAKbeta C-domain as a probe. CBP-1 was the human homologue of Hic-5. Our CBP-1 cDNA provided an evidence that Hic-5 has more than 16 additional amino acid residues at its N-terminal region in addition to the sequence previously described for mouse Hic-5. Hic-5 localizedatfocal adhesions. Hic-5 has 4 LIM domains and 5 LD motifs and is most closely related to paxillin. Hic-5 bound to the C-terminal half of the CAKbeta C-domain with its N-domain. CAKbeta was coimmunoprecipitated with Hic-5 from the WFB cell lysate. When WFB cells were stimulated to enhance the tyrosine-phosphorylation of CAKbeta, the tyrosine-phosphorylation of Hic-5 was also enhanced.
2. In COS-7 cells, the tyrosine-phosphorylation of Hic-5 was enhanced by co-expression of CAKbeta and was further enhanced by stimulating the coexpressed cells with osmotic-stress. The tyrosine-60 residue of Hic-5 was the major site of the tyrosine-phosphorylation because the Y6OF mutant of Hic-5 was nottyrosine-phosphorylated. The CAKbeta mutant deleted at the Hic-5 binding site was defective in the tyrosine-phosphorylation of Hic-5. The tyrosine-phosphorylated Hic-5 was bound to the SH2 domain of Csk but not to the SH2 domains of Fyn, Src, and Crk.
3. The wild type CAKbeta localized in cytoplasm. A single amino acidsubstituted mutant of CAKbeta designated "A mutant" was found to localize exclusively in nucleus. A translocation of wild type CAKbeta to nucleus was observed under certain culture conditions. A portion of Hic-5 was also translocated to nucleus when coexpressed with the A mutant of CAKbeta.
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