CLONING OF A PHOSPHATIDYLINOSITOL KINASE

磷脂酰肌醇（PI）的周转及其磷酸化 衍生物是至关重要的信号转导通过一个 各种激素受体。 PI周转可能很重要， 不仅控制细胞生长， 细胞分化 PI激酶催化 PI的磷酸化产生磷脂酰肌醇-4-磷酰肌醇， phate。 由于其在PI代谢途径中的位置， PI激酶具有调节信号转导的潜力 因此，它可以在全球范围内发挥广泛的调节作用， cell. 最近从A431细胞中纯化了PI激酶， 人表皮癌细胞系 建议的目标 研究的目的是阐明这种PI激酶的一级结构 并开始确定其作用 这种酶在细胞生长和分化的控制。 PI激酶将从人胎盘cDNA文库中克隆 在允许筛选λ> 11表达载体中， 用PI激酶的抗体以及用 合成寡核苷酸探针。 cDNA测序后，克隆 鉴定将通过表位选择以及 通过将衍生的氨基酸序列与已知的PI进行比较， 激酶蛋白质序列。 核酸和衍生蛋白质 将通过以下方法分析序列与其他蛋白质的同源性： 计算机分析 将采用北方印迹法测定 PI激酶mRNA的大小和组织分布。 将PI激酶cDNA克隆到适当的表达载体中， 载体并在小鼠NIH 3 T3细胞和BC 3 H1细胞中表达 分化成肌细胞样细胞的成纤维细胞。 的 将检查转染细胞的生长特性的变化 这将与PI水平的变化相关 代谢产物和蛋白激酶C活性。 在BC 3 H1细胞中， PI激酶的表达对细胞的能力的影响， 也将进行区分。 这些数据将提供 关于PI激酶的结构以及其 与其他蛋白质的关系，并将阐明的作用， PI激酶在PI代谢和细胞生长调节中的作用， 分化

The turnover of phosphatidylinositol (PI) and its phosphorylated derivatives is crucial for the transduction of signals through a variety of hormone receptors. PI turnover may be important not only in the control of cell growth but also in the regulation of cellular differentiation. A PI kinase catalyzes the phosphorylation of PI to produce phosphatidylinositol-4-monophos- phate. Because of its position in the pathway of PI metabolism, the PI kinase has the potential to regulate signal transduction and thus could have a widespread regulatory function within the cell. A PI kinase has recently been purified from A431 cells, a human epidermal carcinoma cell line. The goal of the proposed research is to elucidate the primary structure of this PI kinase through cDNA cloning and sequencing and to begin to define the role of this enzyme in the control of cell growth and differentiation. The PI kinase will be cloned from a human placental cDNA library in a lambda gt11 expression vector which will permit screening of the library with antibodies to the PI kinase as well as with synthetic oligonucleotide probes. Following cDNA sequencing, clone identification will be carried out by epitope selection as well as by the comparison of derived amino acid sequence with known PI kinase protein sequence. The nucleic acid and derived protein sequence will be analyzed for homology with other proteins via computer analysis. Northern blotting will be utilized to determine the size and tissue distribution of the mRNA(s) for the PI kinase. The PI kinase cDNA will be cloned into an appropriate expression vector and expressed in NIH 3T3 cells and in BC3H1 cells, a mouse fibroblast that differentiates into a myocyte-like cell. The transfected cells will be examined for changes in growth properties and this will be correlated with alterations in the levels of PI metabolites and protein kinase C activity. In the BC3H1 cells, the effects of PI kinase expression on the ability of the cells to differentiate will also be examined. These data will provide information on the structure of the PI kinase as well as its relationship to other proteins and will elucidate the role of the PI kinase in the regulation of PI metabolism and cell growth and differentiation.