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.
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.
Leaner
Virna D
VD
National Cancer Institute, 9610 Medical Center Dr., Room 300, Rockville, MD 20850-3300, USA.
Donninger
Howard
H
Ellis
Chad A
CA
Clark
Geoffrey J
GJ
Birrer
Michael J
MJ
D000818:Animals; D001483:Base Sequence; D002448:Cell Adhesion / Q000235:genetics / Q000502:physiology; D002478:Cells, Cultured; D004318:Doxycycline / Q000494:pharmacology; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism; D015870:Gene Expression; D051379:Mice; D008969:Molecular Sequence Data; D020411:Oligonucleotide Array Sequence Analysis; D016376:Oligonucleotides, Antisense; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D000081082:Phosphoinositide-3 Kinase Inhibitors; D011401:Promoter Regions, Genetic / Q000235:genetics / Q000502:physiology; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt; D016755:Proto-Oncogene Proteins c-jun / Q000378:metabolism / Q000502:physiology*; D051381:Rats; D015398:Signal Transduction; D018808:Transcription Factor AP-1 / Q000378:metabolism / Q000502:physiology*; D015854:Up-Regulation; D018631:ras Proteins / Q000378:metabolism*; D020666:ras-GRF1 / Q000235:genetics / Q000378:metabolism*
D004318:Doxycycline; D016376:Oligonucleotides, Antisense; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D047428:Protein Kinase Inhibitors; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins; D051057:Proto-Oncogene Proteins c-akt; D016755:Proto-Oncogene Proteins c-jun; D018808:Transcription Factor AP-1; D018631:ras Proteins; D020666:ras-GRF1; C494920:Akt1 protein, rat