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Molecular and Cellular Biology, April 2005, p. 3324-3337, Vol. 25, No. 8
0270-7306/05/$08.00+0     doi:10.1128/MCB.25.8.3324-3337.2005

p75-Ras-GRF1 Is a c-Jun/AP-1 Target Protein: Its Up Regulation Results in Increased Ras Activity and Is Necessary for c-Jun-Induced Nonadherent Growth of Rat1a Cells

Virna D. Leaner,^, Howard Donninger, Chad A. Ellis, Geoffrey J. Clark, and Michael J. Birrer^*

Cell and Cancer Biology Department, Center for Cancer Research, National Cancer Institute, Rockville, Maryland

Received 27 July 2004/ Returned for modification 8 September 2004/ Accepted 14 January 2005

The c-Jun/AP-1 transcription complex is associated with diverse cellular processes such as differentiation, proliferation, transformation, and apoptosis. These different biological endpoints are likely achieved by the regulation of specific target gene expression. We describe the identification of Ras guanine nucleotide exchange factor 1, Ras-GRF1, by microarray analysis as a c-Jun/AP-1 regulated gene essential for anchorage-independent growth of immortalized rat fibroblasts. Increased Ras-GRF1 expression, in response to inducible c-Jun expression in Rat1a fibroblasts, was confirmed by both real-time PCR and Northern blot analysis. We show that c-Jun/AP-1 can bind and activate the Ras-GRF1 promoter in vivo. A 75-kDa c-Jun/AP-1-inducible protein, p75-Ras-GRF1, was detected, and the inhibition of its expression with antisense oligomers significantly blocked c-Jun-regulated anchorage-independent cell growth. p75-Ras-GRF1 expression occurred with a concomitant increase in activated Ras (GTP bound), and the activation of Ras was significantly inhibited by antisense Ras-GRF1 oligomers. Moreover, p75-Ras-GRF1 could be coprecipitated with a Ras dominant-negative glutathione S-transferase (GST) construct, GST-Ras15A, demonstrating an interaction between p75-Ras-GRF1 and Ras. A downstream target of Ras activation, Elk-1, had increased transcriptional activity in c-Jun-expressing cells, and this activation was inhibited by dominant-negative Ras. In addition, c-Jun overexpression resulted in an increase in phospho-AKT while phosphorylation of ERK1/2 remained largely unaffected. The inhibition of phosphatidylinositol 3-kinase (PI3K)-AKT signal transduction by Ly294002 and wortmannin significantly blocked c-Jun-regulated morphological transformation, while inhibition of basal MEK-ERK activity with PD98059 and U0126 had little effect. We conclude that c-Jun/AP-1 regulates endogenous p75-Ras-GRF1 expression and that c-Jun/AP-1-regulated anchorage-independent cell growth requires activation of Ras-PI3K-AKT signal transduction.

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* Corresponding author. Mailing address: National Cancer Institute, 9610 Medical Center Dr., Room 300, Rockville, MD 20850-3300. Phone: (301) 402-9586. Fax: (301) 402-4422. E-mail: birrerm{at}mail.nih.gov.

Present address: Division of Medical Biochemistry, Institute for Infectious Disease and Molecular Medicine, Faculty of Health Sciences, University of Cape Town, Cape Town, South Africa.

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Molecular and Cellular Biology, April 2005, p. 3324-3337, Vol. 25, No. 8
0022-538X/05/$08.00+0     doi:10.1128/MCB.25.8.3324-3337.2005

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