guanosine-diphosphate and Astrocytoma

Site- and phosphorylation state-specific antibodies are useful to analyze spatiotemporal distribution of site-specific phosphorylation of target proteins in vivo. Using several polyclonal and monoclonal antibodies that can specifically recognize four phosphorylated sites on glial fibrillary acidic protein (GFAP), we have previously reported that Thr-7, Ser-13, and Ser-34 on this intermediate filament protein are phosphorylated at the cleavage furrow during cytokinesis. This observation suggests that there exists a protein kinase named cleavage furrow kinase specifically activated at metaphase-anaphase transition (Matsuoka, Y., Nishizawa, K., Yano, T., Shibata, M., Ando, S., Takahashi, T., and Inagaki, M. (1992) EMBO J. 11, 2895-2902; Sekimata, M., Tsujimura, K., Tanaka, J., Takeuchi, Y., Inagaki, N., and Inagaki, M. (1996) J. Cell Biol. 132, 635-641). Here we report that GFAP is phosphorylated specifically at Thr-7, Ser-13, and Ser-34 by Rho-associated kinase (Rho-kinase), which binds to the small GTPase Rho in its GTP-bound active form. The kinase activity of Rho-kinase toward GFAP is dramatically stimulated by guanosine 5'-(3-O-thio)-triphosphate-bound RhoA. Furthermore, the phosphorylation of GFAP by Rho-kinase results in a nearly complete inhibition of its filament formation in vitro. The possibility that Rho-kinase is a candidate for cleavage furrow kinase is discussed.

Topics: Antibodies; Antibodies, Monoclonal; Astrocytoma; Glial Fibrillary Acidic Protein; Glutathione Transferase; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Diphosphate; Humans; Intracellular Signaling Peptides and Proteins; Kinetics; Phosphopeptides; Phosphorylation; Protein Kinases; Protein Serine-Threonine Kinases; Recombinant Fusion Proteins; Recombinant Proteins; rho-Associated Kinases; Substrate Specificity; Tumor Cells, Cultured

Thrombin stimulation of 1321N1 astrocytoma cells results in polyphosphoinositide hydrolysis, Ca2+ mobilization, AP-1-mediated transcriptional activation, and DNA replication. Thrombin stimulation also activates Ras as assessed by an increase in the proportion of Ras in a GTP bound state. We examined the functional requirement for endogenous Ras protein in mediating thrombin-induced responses. Microinjection of a dominant interfering mutant of H-Ras into 1321N1 cells inhibited DNA synthesis in response to thrombin as did microinjection of an inhibitory antibody to Ras. Stimulation of AP-1-mediated transcriptional activity was also reduced by the expression of interfering Ras mutants. However, neither the stimulation of polyphosphoinositide hydrolysis nor the mobilization of intracellular Ca2+ was dependent on endogenous Ras function. These observations indicate that thrombin stimulation of mitogenesis requires Ras protein function. Our data suggest that the G-protein-coupled thrombin receptor stimulates pathways, which in part are convergent with those stimulated by tyrosine kinase growth factor receptors.

Topics: Astrocytoma; Cell Division; DNA Replication; Genes, ras; Growth Substances; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Diphosphate; Guanosine Triphosphate; Humans; Kinetics; Mutagenesis, Site-Directed; Phosphatidylinositols; Proto-Oncogene Proteins c-jun; Proto-Oncogene Proteins p21(ras); Thrombin; Transfection; Tumor Cells, Cultured

Epinephrine increased adenylate cyclase activity 10 to 15 fold in lysates of the cultured human astrocytoma cell line 132-1N1. GTP had little effect on adenylate cyclase activity of lysed cell preparations either with or without added epinephrine. However, the epinephrine stimulation of adenylate cyclase was essentially lost (less than 90%) when a washed nuclei-free membrane preparation of the cyclase was assayed. A 10 to 15 fold epinephrine stimulation of the membrane adenylate cyclase could be demonstrated if cytosol of GTP were added to the assay with the hormone. The criteria of anion exchange, cation exchange, gel exclusion and paper chromatography indicated that the cytosolic agents which acted synergistically with hormones were GTP and GDP. The apparent Kact's for the synergistic action of GDP and GTP were essentially identical (1.0 muM) and of all the other nucleotides examined only GDP had a potency similar to GTP. However, the effect of GDP was apparently due to its rapid conversion to GTP even in the absence of a regenerating system. With epinephrine pretreatment of the intact 132-1N1 cells there was a specific loss of epinephrine stimulation of adenylate cyclase activity. The hormone pretreatment did not alter the capacity of the cytosol from these desensitized cells to potentiate epinephrine stimulation of the cyclase. Rather, the alteration was in the particulate fraction of the lysate. The desensitization of the membranous cyclase was stable and not reversed by GTP.

Topics: Adenylyl Cyclases; Astrocytoma; Cell Membrane; Cells, Cultured; Cytosol; Epinephrine; Guanosine Diphosphate; Guanosine Monophosphate; Guanosine Triphosphate; Humans; Prostaglandins E