CLONING OF A PHOSPHATIDYLINOSITOL KINASE

磷脂酰肌醇(PI)及其磷酸化的周转 导数对于信号的传递至关重要。 各种激素受体。PI周转率可能不重要 不仅在控制细胞生长方面，而且在调节 细胞分化。一种磷脂酰肌醇激酶催化 磷脂酰肌醇-4-单磷脂的磷酸化 磷酸盐酸盐。由于它在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 手机。最近从A431细胞中纯化了一种PI激酶，一种 人表皮癌细胞系。建议的目标是 研究是为了阐明这种PI激酶的一级结构 通过cDNA克隆和测序，并开始确定其作用 这种酶在控制细胞生长和分化中的作用。 PI激酶将从人胎盘cDNA文库中克隆出来 在lambda gt11表达载体中 含有PI激酶抗体的文库以及 人工合成的寡核苷酸探针。CDNA测序后，克隆 鉴定将通过表位选择以及 推导的氨基酸序列与已知pI的比较 激酶蛋白序列。核酸及其衍生蛋白 序列将通过以下方式分析与其他蛋白质的同源性 计算机分析。将利用Northern印迹来确定 PI激酶基因(S)的大小和组织分布。 PI激酶基因将被克隆到适当的表达载体中 并在NIH3T3细胞和小鼠BC3H1细胞中表达 分化为肌样细胞的成纤维细胞。这个 转基因细胞将被检查生长特性的变化。 这将与PI水平的变化相关 代谢产物和蛋白激酶C活性。在BC3H1细胞中， 磷脂酰肌醇激酶表达对细胞功能的影响 此外，还将考察差异化。这些数据将提供 磷脂酰肌醇激酶的结构信息以及它的 与其他蛋白质的关系，并将阐明 PI激酶在调节PI代谢和细胞生长中的作用 差异化。

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.