CYCLIC NUCLEOTIDE INDEPENDENT PHOSPHORYLATION

当今的主要问题之一是确定所涉及的控制机制 在细胞周期的调节中。一些蛋白激酶已经被 被证明参与了细胞周期的激活，在荷尔蒙 水平和在整个细胞周期的进程中。我们的研究已经 重点研究了一种独特的蛋白激酶PAK I，它参与维持 处于非分裂状态的细胞。在拟议的研究中，我们将继续 蛋白激酶的特性及其在蛋白激酶I中的作用 对细胞周期的调节。这种蛋白激酶似乎是 在细胞周期调控中具有相当重要的作用。 A.完成PAK I基因的克隆、测序和分析。 量化细胞和组织中PAK I的mRNA水平。表达重组 在大肠杆菌和/或昆虫细胞中表达Pak I。 B.用合成肽确定PAK I的识别序列 并确定PAK的蛋白质底物。检查 磷酸化对底物活性的调节。特定于站点 受调控蛋白质中PAK I磷酸化位点的突变 通过PAK I将为活动的变化做准备和分析。 C.分析PAK I在调节细胞周期利用中的作用 哺乳动物细胞。鉴定3T3-L1细胞中PAK I的活性形式 检查体内激活的要求。 D.确定在体外磷酸化PAK I的蛋白激酶和 磷酸化部位。检查磷酸化和磷酸化的影响 自磷酸化对纯化酶活性的影响。特定于站点 将准备PAK I上的磷酸化位点的突变，并 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的特性，分析PAK I在维持中的作用 体细胞在一种非底物参与的生长控制和检测 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.