α-thrombin stimulates nuclear diglyceride levels and differential nuclear localization of protein kinase C isozymes in IIC9 cells

Karen L. Leach, Valerie A. Ruff, Matt B. Jarpe, Lonnie D. Adams, Doriano Fabbro, Daniel M. Raben

Research output: Contribution to journalArticle

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 α-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 α-thrombin treatment. IIC9 cells were treated with 500 ng/ml α-thrombin, and nuclei were then isolated. Western blot analysis using isozyme-specific antibodies demonstrated the presence of PKC α, but not PKC ∈ or ζ in the nuclei of cells treated with either phorbol 12-myristate 13-acetate or α-thrombin. The increase in nuclear PKC α 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, α-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 α-thrombin and remained elevated for at least 30 min. In cells labeled with [3H]myristic acid, α-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 α-thrombin treatment may play a role in mediating thrombin-induced nuclear responses such as changes in gene expression and cellular proliferation.

Original languageEnglish (US)
Pages (from-to)21816-21822
Number of pages7
JournalJournal of Biological Chemistry
Volume267
Issue number30
StatePublished - Oct 25 1992
Externally publishedYes

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Diglycerides
Nuclear Proteins
Thrombin
Protein Kinase C
Isoenzymes
Diacylglycerol Kinase
Phosphorylation
Level control
Myristic Acid
Cell Surface Receptors
Cell Nucleus
Phosphatidylcholines
Gene expression
Acetates
Western Blotting
Chemical activation
Cell Proliferation
Gene Expression
Kinetics
Antibodies

ASJC Scopus subject areas

  • Biochemistry

Cite this

α-thrombin stimulates nuclear diglyceride levels and differential nuclear localization of protein kinase C isozymes in IIC9 cells. / Leach, Karen L.; Ruff, Valerie A.; Jarpe, Matt B.; Adams, Lonnie D.; Fabbro, Doriano; Raben, Daniel M.

In: Journal of Biological Chemistry, Vol. 267, No. 30, 25.10.1992, p. 21816-21822.

Research output: Contribution to journalArticle

Leach, Karen L. ; Ruff, Valerie A. ; Jarpe, Matt B. ; Adams, Lonnie D. ; Fabbro, Doriano ; Raben, Daniel M. / α-thrombin stimulates nuclear diglyceride levels and differential nuclear localization of protein kinase C isozymes in IIC9 cells. In: Journal of Biological Chemistry. 1992 ; Vol. 267, No. 30. pp. 21816-21822.
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T1 - α-thrombin stimulates nuclear diglyceride levels and differential nuclear localization of protein kinase C isozymes in IIC9 cells

AU - Leach, Karen L.

AU - Ruff, Valerie A.

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AU - Fabbro, Doriano

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AB - 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 α-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 α-thrombin treatment. IIC9 cells were treated with 500 ng/ml α-thrombin, and nuclei were then isolated. Western blot analysis using isozyme-specific antibodies demonstrated the presence of PKC α, but not PKC ∈ or ζ in the nuclei of cells treated with either phorbol 12-myristate 13-acetate or α-thrombin. The increase in nuclear PKC α 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, α-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 α-thrombin and remained elevated for at least 30 min. In cells labeled with [3H]myristic acid, α-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 α-thrombin treatment may play a role in mediating thrombin-induced nuclear responses such as changes in gene expression and cellular proliferation.

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