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 language | English (US) |
|---|---|
| Pages (from-to) | 21816-21822 |
| Number of pages | 7 |
| Journal | Journal of Biological Chemistry |
| Volume | 267 |
| Issue number | 30 |
| State | Published - Oct 25 1992 |
| Externally published | Yes |
Fingerprint
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 journal › Article
}
TY - JOUR
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.
AU - Jarpe, Matt B.
AU - Adams, Lonnie D.
AU - Fabbro, Doriano
AU - Raben, Daniel M.
PY - 1992/10/25
Y1 - 1992/10/25
N2 - 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.
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.
UR - http://www.scopus.com/inward/record.url?scp=0026801172&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0026801172&partnerID=8YFLogxK
M3 - Article
C2 - 1400491
AN - SCOPUS:0026801172
VL - 267
SP - 21816
EP - 21822
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 30
ER -