CYCLIC NUCLEOTIDE INDEPENDENT PHOSPHORYLATION

目前的主要问题之一是确定所涉及的控制机制 调节细胞周期。 许多蛋白激酶已经被 显示参与细胞周期的激活，无论是在激素 在细胞周期中的水平和进程。 我们的研究 集中在一个独特的蛋白激酶，PAK I，这是参与维持 细胞处于非分裂状态。 在拟议的研究中，我们将继续 蛋白激酶的表征，并检查PAK I在 调节细胞周期。 这种蛋白激酶似乎是 在细胞周期调控中具有重要意义。 A. 完成PAK Ⅰ cDNA的克隆、测序和分析。 定量细胞和组织中PAK I的mRNA水平。 表达重组 PAK I in E.大肠杆菌和/或昆虫细胞。 B。 用合成肽确定PAK I的识别序列 并鉴定PAK I的蛋白质底物。 审查的影响 磷酸化对底物活性的调节。 位点特异 蛋白质中PAK I磷酸化位点的突变被证明是受调节的 通过PAK I制备并分析活性变化。 C. 分析PAK I在细胞周期调控中的作用 哺乳动物细胞 表征3 T3-L1细胞中PAK I的活性形式， 检查体内激活的要求。 D. 鉴定在体外使PAK I磷酸化的蛋白激酶， 磷酸化位点。 检查磷酸化的影响， 自磷酸化对纯化酶活性的影响。 位点特异 将制备PAK I上磷酸化位点的突变， 将分析这些突变对PAK I活性的影响。 拟议研究的结果将使我们能够完成 PAK I的表征，以分析PAK I在维持 体细胞在一个非底物参与生长控制和检查 PAK I活性的调节。

One of the major problems today is defining the control mechanisms involved in regulation of the cell cycle. A number of protein kinases have been shown to be involved in activation of the cell cycle, both at the hormonal level and during progression through the cell cycle. Our studies have focused on a unique protein kinase, PAK I, which is involved in maintaining cells in a non-dividing state. In the proposed studies we will continue characterization of the protein kinase and examine the role of PAK I in regulation of the cell cycle. This protein kinase appears to be of considerable importance in cell cycle regulation. A. Complete the cloning, sequencing and analysis of the cDNA for PAK I. Quantify mRNA levels for PAK I in cells and tissues. Express recombinant PAK I in E. coli and/or insect cells. B. Determine the recognition sequence for PAK I with synthetic peptides and identify protein substrates for PAK I. Examine the effects of phosphorylation on regulation of substrate activity. Site-specific mutations of PAK I phosphorylation sites in proteins shown to be regulated by PAK I will be prepared and analyzed for changes in activity. C. Analyze the role of PAK I in regulation of the cell cycle utilizing mammalian cells. Characterize the active form of PAK I in 3T3-L1 cells and examine the requirements for activation in vivo. D. Identify the protein kinases which phosphorylate PAK I in vitro and the sites of phosphorylation. Examine the effects of phosphorylation and autophosphorylation on the activity of the purified enzyme. Site-specific mutations of the phosphorylation sites on PAK I will be prepared and the effects of these mutations on PAK I activity will be analyzed. Results from the proposed research will enable us to complete the characterization of PAK I, to analyze the role of PAK I in maintaining somatic cells in a non-substrates involved in growth control and to examine the regulation of PAK I activity.