Dependence of Activated Gα12-induced G1 to S Phase Cell Cycle Progression on Both Ras/Mitogen-activated Protein Kinase and Ras/Rac1/Jun N-terminal Kinase Cascades in NIH3T3 Fibroblasts

doi:10.1074/jbc.272.8.4904

Dependence of Activated Gα12-induced G₁ to S Phase Cell Cycle Progression on Both Ras/Mitogen-activated Protein Kinase and Ras/Rac1/Jun N-terminal Kinase Cascades in NIH3T3 Fibroblasts*

From the Departments of ^‡ Cardiovascular Biology
^¶ Physiology, and
^§ Internal Medicine, Faculty of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113, Japan and
the ** Howard Hughes Medical Institute, Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0295

^‡‡ To whom all correspondence should be addressed:
Dept. of Cardiovascular Biology, Faculty of Medicine, University of Tokyo, 7-3-1 Hongo Bunkyo-ku, Tokyo 113, Japan.
Tel: 81-3-3812-2111 (ext. 3469); Fax: 81-3-5800-6845.

↵^∥ Present address: Tokai University School of Medicine, Bouseidai, Isehara, Kanagawa, 259-11, Japan.

Abstract

We evaluated the roles of mitogen-activated protein kinase (MAPK) and Jun N-terminal kinase (JNK) signaling cascades in Gα12-induced G₁ to S phase cell cycle progression in NIH3T3(M17) fibroblasts. Transient expression of a constitutively active mutant of Gα12, Gα12(R203C), resulted in a 2-fold increase in the number of bromodeoxyuridine-positive S phase cells over vector control level under serum-deprived conditions. Consistent with the ability of Gα12(R203C) to induce G₁/S transition, its expression led to a 2-fold increase in cyclin A promoter activity, which showed a marked synergism with a low concentration of serum, resulting in up to a 15-fold elevation over the basal level. In addition, Gα12(R203C) caused a 2-fold stimulation in E2F-mediated transactivation. Wild type Gα12 showed similar stimulatory effects on cyclin A promoter activity and E2F-mediated transactivation, although of lesser magnitude. We observed a modest but constitutive activation of MAPK in cells transfected with Gα12(R203C), which was abolished by a dominant negative form of Ras. Gα12(R203C) also induced a 3-fold increase in JNK activity, which was abolished by dominant negative forms of either Rac1 or Ras. The expression of dominant negative forms of Ras, MAPK, Rac1, or JNK inhibited Gα12(R203C)-induced increases in bromodeoxyuridine-positive cells. Also, the dominant negative forms of Ras, MAPK, and JNK strongly inhibited Gα12(R203C)-induced stimulation of cyclin A promoter activity. These results demonstrate that both the Ras/MAPK and Ras/Rac1/JNK pathways convey necessary, if not sufficient, mitogenic signals induced by Gα12 activation.

Footnotes

↵* This work was supported by grants from the Ministry of Education, Science and Culture of Japan, funds for cardiovascular research from Tsumura Co., and funds from the Japan Heart Foundation and Japan Research Foundation for Clinical Pharmacology. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

MAPK

mitogen-activated protein kinase

JNK

Jun N-terminal kinase

pRb

retinoblastoma protein

DMEM

Dulbecco's modified Eagle's medium

PCR

polymerase chain reaction

BrdUrd

bromodeoxyuridine

bp

base pair(s)

MEK

mitogen-activated protein kinase kinase.
- Received June 27, 1996.
- Revision received November 19, 1996.