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v-Raf activates transcription of growth-responsive promoters via GC-rich sequences that bind the transcription factor
R J Miltenberger1, P J Farnham, D E Smith
1McArdle Laboratory for Cancer Research, University of Wisconsin Medical School, Madison 53706, USA.
Abstract:
The serine/threonine kinase, Raf-1, is a component of intracellular signaling pathways that control responses to extracellular stimuli. Previously, we have shown that serum-induced transcription from the murine rep-3b and human mdr1 promoters is Raf-dependent and that the activated Raf kinase, v-Raf, induces transcription of mdr1 via a GC-rich element. We now demonstrate that GC-rich sequences in the rep-3b promoter are both necessary and sufficient for induction by v-Raf. The GC-rich, v-Raf-responsive elements of rep-3b and mdr1 bind the general transcription factor Sp1 in electromobility shift assays. Mutation of a minimal GC-rich element abolished inducibility by v-Raf and eliminated binding by the transcription factor Sp1. However, Sp1 binding activity following serum stimulation of quiescent NIH 3T3 cells was unchanged, suggesting that mitogenic signals may stimulate the transactivation potential of prebound Sp1.
Insights
The serine/threonine kinase Raf-1 regulates gene transcription. This study shows Raf-1 activates specific gene promoters by interacting with the transcription factor Sp1, influencing cellular responses to external signals.
Area of Science:
- Molecular Biology
- Cell Signaling
- Gene Regulation
Background:
- Raf-1 is a key kinase in intracellular signaling pathways controlling cellular responses.
- Previous work demonstrated Raf-dependent transcription of murine rep-3b and human mdr1 promoters.
- Activated v-Raf kinase was shown to induce mdr1 transcription through a GC-rich element.
Purpose of the Study:
- To investigate the role of GC-rich sequences in the rep-3b promoter for v-Raf-mediated induction.
- To identify the transcription factors involved in v-Raf-responsive elements of rep-3b and mdr1 promoters.
- To elucidate the mechanism by which mitogenic signals affect Sp1 activity in response to Raf signaling.
Main Methods:
- Electromobility shift assays (EMSA) to assess transcription factor binding.
- Site-directed mutagenesis of GC-rich elements in promoter regions.
- Analysis of promoter activity and transcription factor binding in response to serum stimulation.
Main Results:
- GC-rich sequences in the rep-3b promoter were found to be necessary and sufficient for v-Raf induction.
- These GC-rich elements in both rep-3b and mdr1 promoters bind the general transcription factor Sp1.
- Mutation of a critical GC-rich element abolished v-Raf inducibility and Sp1 binding.
- Sp1 binding activity remained unchanged after serum stimulation, suggesting altered transactivation potential.
Conclusions:
- GC-rich elements are crucial for Raf-mediated transcriptional regulation.
- The transcription factor Sp1 is a key mediator of v-Raf-induced gene expression.
- Mitogenic signals may enhance gene transcription by modulating the activity of pre-bound Sp1, rather than altering Sp1 binding itself.