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

蛋白激酶FA（Protein Kinase FA，PKFA）是一种丝氨酸/苏氨酸激酶，最初被发现是将失活的磷酸酶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胰岛素受体的酪氨酸激酶活性，并导致许多细胞蛋白的丝氨酸/苏氨酸磷酸化增加和其他蛋白的磷酸化减少。为了阐明胰岛素的信号转导机制，本研究对PKFA进行了研究，期望PKFA是胰岛素刺激下酪氨酸激酶活化与蛋白质丝氨酸/苏氨酸磷酸化和去磷酸化之间的关键酶。首先，我试图建立一个测定系统的PKFA活性，然后，检查激活的PKFA和易位的酶从质膜到细胞质后，胰岛素刺激。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移位。Thu ...更多信息 本实验将PKFA的作用靶点改为丝裂原活化蛋白激酶（MAP），通过酪氨酸和苏氨酸的磷酸化将其转化为活性激酶，并研究了MAP激酶激活的信号转导机制：EGF处理静止期Swiss 3 T3细胞后，使髓鞘碱性蛋白磷酸化的蛋白激酶活性增加，在处理后2.5min达到峰值。通过FPLC Mono Q柱层析鉴定该激酶活性为MAP激酶。MAP激酶活性的增加，也观察到与佛波酯，凝血酶，缓激肽，和激酶活性的增加与DNA合成的诱导相关。本论文详细研究了凝血酶作用到MAP激酶激活的信号转导机制，证实了GTP结合蛋白、PI-3激酶和C激酶参与了MAP激酶的激活过程。另外，还研究了磷酸化级联反应的最终靶点之一DNA拓扑异构酶II的磷酸化。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