| Project/Area Number |
04807017
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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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 Hiroko Sapporo Medical University, Cancer Research Institute, Assistant Professor, 医学部・付属癌研究所, 講師 (60045424)
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| Co-Investigator(Kenkyū-buntansha) |
SASAKI Terukatsu Sapporo Medical University, Cancer Research Institute, Professor, 医学部・付属癌研究所, 教授 (00045494)
NAGURA Kazuko Sapporo Medical University, Cancer Research Institute, Research Associate, 医学部・付属癌研究所, 技師
ISHINO Masaho Sapporo Medical University, Cancer Research Institute, Instructor, 医学部・付属癌研究所, 助手 (30232325)
山下 由美 札幌医科大学, 附属癌研究所, 技師
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| Project Period (FY) |
1992 – 1993
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| Project Status |
Completed (Fiscal Year 1993)
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| Budget Amount *help |
¥1,900,000 (Direct Cost: ¥1,900,000)
Fiscal Year 1993: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 1992: ¥1,200,000 (Direct Cost: ¥1,200,000)
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| Keywords | protein-tyrosine kinase / focal adhesion kinase / FAK / CAKbeta / Focal Adhesion / レトロウイルスベクター / ポリメラーゼ連鎖反応 / フォーカル・アドヘジョン・キナーゼ / 組み換えレトロウィルス / 神経細胞株 |
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| Research Abstract |
Primary cultured cells from newborn rat brain were immortalized by infecting retroviruses containing either SV40 large T antigen gene or adenovirus E1A gene. cDNAs encoding protein-tyrosine kinases (PTK) expressed in the immortalized cells were cloned by the polymerase chain reaction-based approach. A second PTK of the focal adhesion kinase (FAK) subfamily, which we call CAKbeta (cell adhesion kinase beta), was identified as one of the PTKs encoded by the cDNAs. The entire rat CAKbeta amino acid sequence was deduced from cDNA clones. CAKbeta is a large (115.7-kDa) PTK that contains N-terminal and C-terminal domains of 418 and 330 amino acid residues in addition to the central kinase domain of 261 amino acids. The amino acid sequence of the catalytic domain of CAKbeta is 60% identical with that of mouse FAK.The two proteins have a homology over their entire lengths except for the extreme N-terminal 88 residues and share 45% overall sequence identity. The CAKbeta gene is less evenly expressed in a variety of rat organs than the FAK gene. Anti-CAKbeta antibody affinity-purified from rabbit antiserum specifically immunoprecipitated a 113-kDa protein from rat brain, 3Y1 cells, and from COS-7 cells transfected with CAKbeta cDNA.Phosphorylation of CAKbeta was shown in immune-complex kinase assay. The tyrosine-phosphorylated state of CAKb in 3Y1 cells was not reduced on trypsinization, nor enhanced in response to plating the cells onto fibronectin. Subcellular localization of CAKbeta was studied by confocal laser scanning microscopy in the immunostained COS-7 cells transfected with epitope-tagged-CAKbeta cDNA.CAKbeta localized to sites of cell-to-cell contact at the middle to upper portions of the cells. The localization was different from that of FAK,which was found at the bottom of the cells. Thus, CAKbeta is a PTK with a possibility to participate in the signal transduction regulated by cell-to-cell contacts.
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