The serum response factor is extensively modified by phosphorylation following its synthesis in serum-stimulated fibroblasts.

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.

Expression of the SRF gene occurs through a Ras/Sp/SRF-mediated-mechanism in response to serum growth signals

“…Maximal message accumulation (eightfold) occurs between 60 ± 120 min after stimulation, and begins to decline by 180 min after stimulation. The time course of SRF message accumulation mimics that of SRF message accumulation seen after serum-starved ®broblasts are stimulated with 20% fetal bovine serum (Misra et al, 1991), or bFGF (Spencer et al, 1999).…”

“…Previously, we (Misra et al, 1991;Spencer and Misra, 1996) and others (Norman et al, 1988) have demonstrated that in response to serum stimulation the SRF gene is rapidly induced at the transcriptional level in a protein synthesis independent manner. In murine ®broblasts SRF gene expression is transient.…”

“…Previously, we have shown that stimulation of quiescent mouse ®broblasts with serum induces expression of the endogenous SRF gene (Misra et al, 1991). To determine whether LPA activates SRF gene expression, a Northern blot analysis of SRF message levels was performed on RNA isolated from NIH3T3 cells before and after LPA treatment.…”

“…A time course analysis of serum stimulated expression of the 7322SRF/c-fos construct revealed that in unstimulated cells the reporter message is virtually undetectable, but that by 30 min after serum stimulation message levels peak, returning to basal levels by 120 ± 240 min after stimulation (Spencer et al, 1999). Maximal accumulation of message from the chimeric construct occurred signi®cantly earlier than maximal accumulation of message from the endogenous SRF gene (Misra et al, 1991), and more closely parallels accumulation of message from the endogenous c-fos gene. The transient and robust nature of stimulation from the SRF/c-fos reporters suggested that this system would allow us to very sensitively measure activity of the SRF promoter.…”

“…In murine ®broblasts SRF gene expression is transient. SRF message levels peak at approximately 90 ± 120 min after serum stimulation and return to nearly basal levels by 4 ± 6 h after stimulation (Misra et al, 1991). In recent studies, we have shown that maximal responsiveness of the SRF promoter to serum and basic ®broblast growth factor (bFGF) requires two SRF binding sites located within the ®rst 63 nucleotides upstream of the start site of transcriptional initiation, a GC box, containing overlapping Sp1/Egr-1 binding sites, located at 83 nucleotides upstream of the start site, and in the case of bFGF an ETS binding sequence located 103 nucleotides upstream of the start site of initiation (Spencer et al, 1999;Spencer and Misra, 1996).…”