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Serum-, TPA-, and Ras-induced expression from Ap-1/Ets-driven promoters requires Raf-1 kinase
J T Bruder1, G Heidecker, U R Rapp
1Laboratory of Viral Carcinogenesis, National Cancer Institute, Frederick Cancer Research and Development Center, Maryland 21702-1201.
Abstract:
Raf-1 serine-threonine protein kinase has the hallmarks of a critical switch that connects growth factor receptor activation at the cell membrane with transcriptional events in the nucleus. We show by use of Raf-1 dominant-negative mutants that Raf-1 is required for serum-, TPA-, and Ras-induced expression from the oncogene-responsive element in the polyomavirus enhancer. The minimal region of Raf-1 that displays this dominant-negative phenotype (Raf-C4) contains a cysteine finger motif. Raf-C4 appears to function by titrating out a Raf-1-activating factor that is induced by Ras following serum or TPA treatment of NIH-3T3 cells. In addition, we show that Raf-1 and Ras cooperate in trans-activation through the oncogene-responsive element and that the cysteine-rich region is necessary for this effect.
Insights
Raf-1 protein kinase is essential for growth factor signaling, connecting cell surface receptors to nuclear gene expression. Dominant-negative mutants revealed Raf-1
Area of Science:
- Molecular Biology
- Cell Signaling
- Oncogenesis
Background:
- Raf-1 serine-threonine protein kinase acts as a crucial link between cell surface growth factor receptors and nuclear transcriptional events.
- Understanding the precise role of Raf-1 in signal transduction pathways is vital for comprehending cellular growth and proliferation.
Purpose of the Study:
- To investigate the requirement of Raf-1 in mediating transcriptional responses induced by growth factors and oncogenic signaling.
- To identify the minimal functional region of Raf-1 responsible for dominant-negative effects and its mechanism of action.
Main Methods:
- Utilized dominant-negative Raf-1 mutants to assess Raf-1's necessity in gene expression.
- Employed the oncogene-responsive element in the polyomavirus enhancer to measure transcriptional activity.
- Investigated the role of the cysteine-rich region and a cysteine finger motif in Raf-1 function.
Main Results:
- Raf-1 is indispensable for serum, TPA, and Ras-induced expression from the polyomavirus enhancer's oncogene-responsive element.
- A minimal Raf-1 region (Raf-C4), containing a cysteine finger motif, exhibited dominant-negative activity.
- Raf-C4 functions by sequestering a Ras-induced activating factor for Raf-1.
- Raf-1 and Ras collaborate in trans-activation via the oncogene-responsive element, with the cysteine-rich region being critical for this cooperation.
Conclusions:
- Raf-1 is a key mediator of signal transduction pathways initiated by growth factors and oncogenic mutations.
- The cysteine-rich domain of Raf-1, including the cysteine finger motif, plays a critical role in its function and interaction with Ras.
- These findings elucidate a mechanism by which Raf-1 integrates upstream signals to regulate gene expression.