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The serum response factor is extensively modified by phosphorylation following its synthesis in serum-stimulated
R P Misra1, V M Rivera, J M Wang
1Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts 02115.
Abstract:
Growth factor regulation of c-fos proto-oncogene transcription is mediated by a 20-bp region of dyad symmetry, termed the serum response element. The inner core of this element binds a 67-kDa phosphoprotein, the serum response factor (SRF), that is thought to play a pivotal role in the c-fos transcriptional response. To investigate the mechanism by which SRF regulates c-fos expression, we generated polyclonal anti-SRF antibodies and used these antibodies to analyze the biochemical properties of SRF. These studies indicate that the synthesis of SRF is transient, occurring within 30 min to 4 h after serum stimulation of quiescent fibroblasts. Newly synthesized SRF is transported to the nucleus, where it is increasingly modified by phosphorylation during progression through the cell cycle. Within 2 h of serum stimulation, differentially modified forms of SRF can be distinguished on the basis of the ability to bind a synthetic serum response element. SRF protein exhibits a half-life of greater than 12 h and is predominantly nuclear, with no change occurring in its localization upon serum stimulation. We find that the induction of SRF synthesis is regulated at the transcriptional level and that cytoplasmic SRF mRNA is transiently expressed with somewhat delayed kinetics compared with c-fos mRNA expression. These features of SRF expression suggest a model whereby newly synthesized SRF functions in the shutoff of c-fos transcription.
Insights
Newly synthesized serum response factor (SRF) is crucial for regulating c-fos transcription. This transiently expressed protein, phosphorylated in the nucleus, suggests a role in shutting off c-fos gene expression.
Area of Science:
- Molecular Biology
- Cellular Biology
- Gene Regulation
Background:
- Growth factors regulate c-fos proto-oncogene transcription via the serum response element (SRE).
- The SRE binds serum response factor (SRF), a phosphoprotein critical for c-fos transcriptional response.
Purpose of the Study:
- To investigate the mechanism by which SRF regulates c-fos expression.
- To analyze the biochemical properties of SRF using polyclonal anti-SRF antibodies.
Main Methods:
- Generation of polyclonal anti-SRF antibodies.
- Analysis of SRF biochemical properties, including synthesis, phosphorylation, nuclear transport, and half-life.
- Comparison of SRF and c-fos mRNA kinetics.
Main Results:
- SRF synthesis is transient (30 min to 4 h) after serum stimulation of quiescent fibroblasts.
- Newly synthesized SRF is phosphorylated in the nucleus during cell cycle progression.
- SRF protein has a half-life >12 h, is predominantly nuclear, and its localization is unaffected by serum stimulation.
- SRF synthesis induction is transcriptionally regulated; SRF mRNA expression is transient and slightly delayed compared to c-fos mRNA.
Conclusions:
- Newly synthesized SRF plays a role in the shutoff of c-fos transcription.
- Differential phosphorylation of SRF affects its DNA-binding ability.
- SRF expression kinetics suggest a feedback mechanism for c-fos regulation.