Several classes of RNA tumor viruses contain genes for transforming proteins that are tyrosine-specific protein kinases. The genome of normal cells contains DNA homolgous to the viral oncogene and uninfected cells express low levels of a tyrosine-specific kinase closely related to the one encoded by the virus. The function of this protein kinase in normal cells is not known. In addition, normal cells contain other tyrosine-specific protein kinases. Some are known to be receptors for growth factors and hormones. The function of others, most of which have not yet been characterized, is not known. In the course of studies of the structure and function of adenylate cyclase from bovine brain, we have highly purified, in relatively large quantities, two tyrosine phosphoproteins with molecular weights of 61,000 (p61) and 64,000 (p64). The former seems to be a tyrosine kinase related to the oncogene product of Rous sarcoma virus (pp60v-src). The latter is a substate for a tyrosine kinase and may, itself, also be an enzyme. We now propose to characterize the structure and function of these proteins from normal cells. We will characterize the kinetic properties of the enzymes. We will study their structure with peptide maps to determine whether they are similar to pp60v-src and to each other. We will determine whether p61 or p64 have phosphatidylinositol kinase activity. We will determine the functional sequences of phosphorlyation of p61 and p64 on serine and threonine as well as of phosphorylation on tyrosine. We will raise antibodies against the proteins to use for studies of localization of p61 and p64 to different brain regions and different tissues. We also propose to look for other classes of tyrosine protein kinases in detergent extracts of brain membranes. We will use angiotensis as a substrate to identify the tyrosine kinases. We will compare the kinetic and structural properties of these enzymes with the oncogene-related enzyme that we have already purified. We have recently developed specific antibodies against brain adenylate cyclase regulatory components. It is known that adenylate cyclase activity is altered in Rous sarcoma virus-infected cells which have elevated tyrosine kinase activity. We will use the antibodies to precipitate the adenylate cyclase components from [P32] radiolabeled intact brain cells to determine whether tyrosine phosphorylation is a mechanism for regulating the activity of this central enzyme.
| Denker, B M; Neer, E J; Schmidt, C J (1992) Mutagenesis of the amino terminus of the alpha subunit of the G protein Go. In vitro characterization of alpha o beta gamma interactions. J Biol Chem 267:6272-7 |
| Denker, B M; Schmidt, C J; Neer, E J (1992) Promotion of the GTP-liganded state of the Go alpha protein by deletion of the C terminus. J Biol Chem 267:9998-10002 |
| Denker, B M; Tempst, P; Neer, E J (1991) Characterization of a mastoparan-stimulated nucleotidase from bovine brain. Biochem J 278 ( Pt 2):341-5 |
| Silbert, S; Michel, T; Lee, R et al. (1990) Differential degradation rates of the G protein alpha o in cultured cardiac and pituitary cells. J Biol Chem 265:3102-5 |
