title: Alpha-thrombin stimulates nuclear diglyceride levels and differential nuclear localization of protein kinase C isozymes in IIC9 cells.
abstract: The mechanism by which an agonist, binding to a cell surface receptor, exerts an effect on events in the nucleus is not known. We have previously shown (Leach, K. L., Ruff, V. A., Wright, T. M., Pessin, M. S., and Raben, D. M. (1991) J. Biol. Chem. 266, 3215-3221) that alpha-thrombin treatment of IIC9 cells results in increased levels of cellular 1,2-diacylglycerol (DAG) and activation of protein kinase C (PKC). Here, we have examined whether changes in nuclear PKC and nuclear DAG also are induced following alpha-thrombin treatment. IIC9 cells were treated with 500 ng/ml alpha-thrombin, and nuclei were then isolated. Western blot analysis using isozyme-specific antibodies demonstrated the presence of PKC alpha, but not PKC epsilon or zeta in the nuclei of cells treated with either phorbol 12-myristate 13-acetate or alpha-thrombin. The increase in nuclear PKC alpha levels was accompanied by a 10-fold increase in nuclear PKC specific activity and stimulated phosphorylation of at least six nuclear proteins. The rise in nuclear PKC levels occurred rapidly and reached a maximum at 30-60 s, which was followed by a decline back to the control level over the next 15 min. In addition, alpha-thrombin treatment resulted in an immediate rise in DAG mass levels in the nuclear fractions. Kinetic analysis indicated that a maximum increase in DAG levels occurred 2.5-5 min after the addition of alpha-thrombin and remained elevated for at least 30 min. In cells labeled with [3H]myristic acid, alpha-thrombin treatment induced an increase in radiolabeled nuclear diglycerides, suggesting that the stimulated nuclear DAGs are derived, at least in part, from phosphatidylcholine. Our results suggest that increases in both nuclear DAG levels and PKC activity following alpha-thrombin treatment may play a role in mediating thrombin-induced nuclear responses such as changes in gene expression and cellular proliferation.
authors.affiliation: Department of Cell Biology, Upjohn Company, Kalamazoo, Michigan 49001.
authors.forename: K L
authors.initials: KL
authors.lastname: Leach
authors.forename: V A
authors.initials: VA
authors.lastname: Ruff
authors.forename: M B
authors.initials: MB
authors.lastname: Jarpe
authors.forename: L D
authors.initials: LD
authors.lastname: Adams
authors.forename: D
authors.initials: D
authors.lastname: Fabbro
authors.forename: D M
authors.initials: DM
authors.lastname: Raben
mesh_terms: D000818:Animals; D002467:Cell Nucleus / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism / Q000648:ultrastructure; D002478:Cells, Cultured; D006224:Cricetinae; D004075:Diglycerides / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005455:Fluorescent Antibody Technique; D007527:Isoenzymes / Q000378:metabolism*; D008854:Microscopy, Electron; D009687:Nuclear Proteins / Q000378:metabolism; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013917:Thrombin / Q000494:pharmacology*
chemical_list: C011439:1,2-diacylglycerol; D004075:Diglycerides; D007527:Isoenzymes; D009687:Nuclear Proteins; D011493:Protein Kinase C; D013917:Thrombin; D013755:Tetradecanoylphorbol Acetate