Phorbol Ester Inhibits the Phosphorylation of the Retinoblastoma Protein without Suppressing Cyclin D-associated Kinase in Vascular Smooth Muscle Cells

doi:10.1074/jbc.271.14.8345

Phorbol Ester Inhibits the Phosphorylation of the Retinoblastoma Protein without Suppressing Cyclin D-associated Kinase in Vascular Smooth Muscle Cells (*)

From the National Cardiovascular Center Research Institute and the
Research Institute for Microbial Diseases, Osaka University, Suita, Osaka 565, Japan

^§ To whom correspondence should be addressed:
Dept. of Bioscience, National Cardiovascular Center Research Institute, 5-7-1 Fujishiro-dai, Suita, Osaka 565, Japan.
Tel.: 81-6-833-5012; Fax: 81-6-872-7485; sasaguri{at}ri.ncvc.go.jp.

Abstract

To elucidate the role of protein kinase C in vascular smooth muscle cell proliferation, we examined the effects of phorbol 12-myristate 13-acetate (PMA) on G events in human arterial cells. About 15 h after G cells were stimulated with fetal bovine serum and basic fibroblast growth factor, [³H]thymidine incorporation started. PMA (10 nM) inhibited the incorporation over 90% when added earlier than 3 h after stimulation, but had no effect when added 12 h or later. PMA inhibited the phosphorylation of the retinoblastoma protein (pRb), which normally began at about 9 h. PMA did not inhibit the gene expression of Cdk2, Cdk3, Cdk4, Cdk5, and cyclins G, C, and D, all of which began at 0-3 h. However, PMA reduced the expression of cyclins E and A, which usually began at 3-9 h and about 15 h, respectively. PMA inhibited the histone H1 kinase activity of Cdk2, which increased from about 9 h, whereas PMA did not inhibit the pRb kinase activities of cyclin D-associated kinase(s) and Cdk4, detectable from 0-3 h. These results suggested that the PMA-induced inhibition of pRb phosphorylation is not mediated by suppressing cyclin D-associated kinase(s) including Cdk4, but involves the suppression of Cdk2 activity that results from the reduced expression of cyclins E and A.

Footnotes

↵* This study was supported in part by grants from the Ministry of Education, Science, and Culture (a grant-in-aid for scientific research), the Ministry of Health and Welfare (Research Grant for Cardiovascular Diseases 6B-1), the Science and Technology Agency (Special Coordination Funds for Promoting Science and Technology (Encouragement System of COE)), the Uehara Memorial Foundation, and the Yamanouchi Foundation for Research on Metabolic Disorders. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

VSMC

vascular smooth muscle cell

PKC

protein kinase C

pRb

the retinoblastoma protein

Cdk

cyclin-dependent kinase

PMA

phorbol 12-myristate 13-acetate

DAG

1,2-sn-diacylglycerol

TGF-β

transforming growth factor-β

DMEM

Dulbecco's modified Eagle's medium

PBS

phosphate-buffered saline

BSA

bovine serum albumin

TdR

thymidine

PAGE

polyacrylamide gel electrophoresis

GST

glutathione S-transferase.
↵²T. Sasaguri, unpublished data.
- Received July 28, 1995.
- Revision received January 22, 1996.