|Budget Amount *help
¥5,800,000 (Direct Cost : ¥5,800,000)
Fiscal Year 1993 : ¥1,900,000 (Direct Cost : ¥1,900,000)
Fiscal Year 1992 : ¥3,900,000 (Direct Cost : ¥3,900,000)
Protein kinase FA(PKFA) is a serine/threonine kinase, which was originally discovered as the factor necessary to convert inactive phoshatase 1 to active form. Binding of insulin to its receptor stimulates the tyrosine kinase activity of the insulin receptor and leads to increased serine/threonine phosphorylation of many cellular proteins and decreased phosphorylation of other proteins. In order to clarify the mechanism of signal transduction of insulin, I studied PKFA, expecting that PKFA is a key enzyme which connects the activation of tyrosine kinase with increased serine/threonine phosphorylation and dephosphorylation of proteins in the cells stimulated by insulin. First, I tried to establish an assay system for PKFA activity and then, examined activation of PKFA and translocation of the enzyme from the plasma membrane to the cytosol after stimulation by insulin. However, I could not observe activation of the enzyme nor translocation of the enzyme in all systems that I examined. Thu
s, I changed the target from PKFA to mitogen activated protein (MAP) kinase which is converted to active from by phosphorylation of tyrosine and threonine, and studied the mechanism of signal transduction leading to the activation of MAP kinase in Swiss 3T3 cells.
Treatment of quiescent Swiss 3T3 cells with EGF resulted in the increase in the protein kinase activity that phosphorylated myelin basic protein, making a peak 2.5 min after the treatment. This kinase activity was identified as MAP kinase by FPLC Mono Q column chromatography. The increase in MAP kinase activity was also observed with phorbolester, thrombin, and bradykinin, and the increase in the kinase activity correlated well with the induction of DNA synthesis. I examined the mechanism of signal transduction from treatment of thrombin to the activation of MAP kinase in detail and confirmed the involvement of GTP binding protein, PI-3 kinase, and C kinase in the pathway to the activation of MAP kinase, In addition, the phosphorylation of DNA topoisomerase II was studied as one of final target of phosphorylation cascade.
増殖を停止したSwiss3T3細胞の培養系にEGFを添加すると、添加後2、5分をピークとするリン酸化活性の上昇が観察され、FPLC MonoQカラムにかけることによりこの活性の上昇はMAPキナーゼの活性化によるものであることが確認された。EGFの他、ホルボールエステル、トロンビン、ブラジキニン等でもMAPキナーゼの活性の上昇が観察され、この上昇とDNA合成の誘導とに相関がみられた。このうちトロンビンによるMAPキナーゼの活性化を詳細に検討し、この活性化の経路には、GTP結合蛋白質、PI-3キナーゼ、Cキナーゼが関与することを明らかにした。また、この研究と平行して、リン酸化カスケードの最終標的の一つとしてDNAトポイソメラーゼIIに着目し、そのリン酸化を詳細に解析した。 Less