Analysis of signal transduction of insulin with special reference to protein kinase FA

蛋白激酶FA(PKFA)是一种丝氨酸/苏氨酸激酶，最初被发现是将失活的光分解酶1转化为活性形式所必需的因子。胰岛素与其受体的结合刺激了胰岛素受体的酪氨酸激酶活性，导致许多细胞蛋白质的丝氨酸/苏氨酸磷酸化增加，而其他蛋白质的磷酸化降低。为了阐明胰岛素的信号转导机制，我对PKFA进行了研究，期望PKFA是一个关键的酶，它将胰岛素刺激的细胞中酪氨酸激酶的激活与丝氨酸/苏氨酸磷酸化和蛋白质去磷酸化联系起来。首先建立了PKFA活性的检测体系，然后检测了胰岛素刺激后PKFA的活性以及酶从质膜到胞浆的移位。然而，在我检查的所有系统中，我都没有观察到酶的激活或酶的易位。清华…S等将靶标从PKFA转变为丝裂原活化蛋白(MAP)激酶，通过酪氨酸和苏氨酸的磷酸化转化为活性形式，并研究了信号转导导致瑞士3T3细胞MAP激酶激活的机制。经FPLC Mono Q柱层析鉴定为MAP激酶。磷酸酯、凝血酶和缓激肽也使MAP激酶活性增加，并且该活性的增加与DNA合成的诱导有很好的相关性。详细研究了凝血酶处理到MAP激活的信号转导机制，证实了GTP结合蛋白、PI-3和C激酶参与了MAP激活的通路，并研究了DNA拓扑异构酶II的磷酸化作为磷酸化级联反应的最终靶点之一。较少

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 … More 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. Less