| Project/Area Number |
01570243
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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 |
細菌学
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| Research Institution | Jichi Medical School |
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Principal Investigator |
SHINOMIYA Hiroto Jichi Medical School, Dept. of Microbiology Assistant Professor, 医学部 (80162618)
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| Co-Investigator(Kenkyū-buntansha) |
NAKANO Masayasu Jichi Medical School, Dept. of Microbiology Professor, 医学部, 教授 (70048958)
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| Project Period (FY) |
1989 – 1990
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| Project Status |
Completed (Fiscal Year 1990)
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| Budget Amount *help |
¥1,800,000 (Direct Cost: ¥1,800,000)
Fiscal Year 1990: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 1989: ¥1,100,000 (Direct Cost: ¥1,100,000)
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| Keywords | Lipopolysaccharide / Macrophage / Phosphoprotein(pp) / C3H / HeJ mouse / pp65 / Plastin / Protein kinase / リン酸化蛋白(PP) / C3H / HeJマウス / 遺伝子クロ-ニング / プロテインキナ-ゼ / リン酸化蛋白質 / C3H@HeJマウス / 新しい蛋白質 |
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| Research Abstract |
Modification of cellular proteins via phosphorylation is known to be a major regulatory mechanism whereby external stimuli control intracellular events. We demonstrated that bacterial lipopolysaccharide (LPS) induced a distinct set of phosphorylated prtein (pp) in murine peritoneal macrophages, and that the LPS-induced pp were specifically located in cytosol and/or membrane fractions. One of the most heavily phosphorylated substrate proteins with a molecular mass of 65kDa (pp65) was purified to homogeneity via SDS-PAGE analysis and autoradiography by sequential chromatography on Sephacryl S-200, HPLC anion exchange and hydroxyapatite HPLC. Our pp65 is apparently the first purified LPS-induced pp. Analysis of the protein amino acid sequence of the pp65 revealed that pp65 is a novel protein having homology with human plastin. Serine residues on pp65 were found to be exclusively phosphorylated, indicating a contribution by LPS-inducible serine kinase(s). Interestingly, LPS-induced phosphorylation of pp65 was not observed in macrophages from a LPS-nonresponsive C3H/HeJ strain of mice, although their macrophages had about the same amounts of unphosphorylated p65 as normal macrophages whnn detected under Western blot analysis using polyclonal anti-pp65 antibodies. This suggests that the functional defect of C3H/HeJ macrophages exists somewhere in the process before the pp65 phosphorylation. Considering these observation, the pp65 seems to play a crucial role in macrophage activation, and the structure and function of the pp65 should lead to progress in our understanding of the mechanisms of macrophage activation by LPS.
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