CLONING OF A PHOSPHATIDYLINOSITOL KINASE

磷脂酰肌醇（PI）的周转及其磷酸化

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.