The Ras and protein kinase C signaling pathways are functionally antagonistic in GH4 neuroendocrine cells.
Oncogenic Ras appears to act via protein kinase C (PKC)-dependent and PKC-independent pathways. In several systems, oncogenic Ras cooperates with c-Jun to activate gene transcription from promoters containing an AP-1 site by augmenting phosphorylation of the transcriptional activation domain of c-Jun. We have previously shown that oncogenic valine 12 Ras and PKA each separately activate the rat PRL (rPRL) promoter but together are mutually antagonistic. The goal of this study was to determine whether oncogenic Ras acts through PKC and c-Jun to activate transcription of an rPRL-luciferase reporter construct transiently transfected into GH4 rat pituitary cells. Our results show that phorbol 12-myristate 13-acetate (TPA) activates rPRL promoter activity through PKC, and that TPA activation of PKC diminishes the Ras response in a dose-dependent manner. Additionally, inhibition of PKC with staurosporine does not block the oncogenic Ras effect. Similarly, rPRL promoter activity in GH4 cells expressing oncogenic Ras fails to respond to TPA activation of PKC. Finally, cotransfection of a c-Jun expression vector results in inhibition of basal, TPA, and oncogenic Ras-stimulated activity of the rPRL promoter. Thus, we show that the mechanism of Ras signaling does not involve PKC, and that PKC does not signal via Ras. Taken together, these results verify that the Ras and PKC signaling pathways are separate and mutually antagonistic, and that c-Jun is not the nuclear mediator of either the Ras or PKC signal. These findings emphasize the possibility that the roles and/or functions of specific components in signaling pathways may be different in distinct cell types.
Oberwetter
J M
JM
Department of Medicine, University of Colorado Health Sciences Center, Denver 80262.
Conrad
K E
KE
Gutierrez-Hartmann
A
A
D000818:Animals; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D015870:Gene Expression / Q000235:genetics; D010902:Pituitary Gland / Q000166:cytology* / Q000378:metabolism / Q000502:physiology*; D011388:Prolactin / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics; D011493:Protein Kinase C / Q000502:physiology*; D016755:Proto-Oncogene Proteins c-jun / Q000502:physiology; D016283:Proto-Oncogene Proteins p21(ras) / Q000502:physiology*; D051381:Rats; D015398:Signal Transduction / Q000502:physiology*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014158:Transcription, Genetic / Q000235:genetics
D016755:Proto-Oncogene Proteins c-jun; D011388:Prolactin; D011493:Protein Kinase C; D016283:Proto-Oncogene Proteins p21(ras); D013755:Tetradecanoylphorbol Acetate