Identification of multiple SRF N-terminal phosphorylation sites affecting DNA binding properties.

Abstract

Human serum response factor (SRF) bearing a histidine tag was expressed using vaccinia virus. The recombinant protein was purified and shown to be phosphorylated mainly in its N‐terminal part. The corresponding phosphorylation sites were mapped by microsequencing and also appear to be phosphorylated in endogenous serum response factor. Four phosphorylation sites are located on serines within amino acids 77–85, while another phosphorylation site has been identified at Ser103. Mutations that considerably reduced or abolished phosphorylation at amino acids 77–85 caused a decrease in binding to the c‐fos serum response element accompanied by markedly reduced association and dissociation rates. In contrast, replacing Ser103 by alanine decreased DNA binding activity without drastically affecting the on/off rates. The combination of abolishing phosphorylation at amino acids 77–85 and 103 displayed greatly reduced on/off rates of DNA binding, but the reduction of DNA binding activity was partially alleviated. None of these mutations affect either the ability to interact with p62TCF or stimulation of transcription in vitro. These findings imply possible roles for SRF phosphorylation in the regulation of c‐fos transcription.

Transcriptional repression mediated by the serum response factor

“…3B), indicating that this effect is not SRE-specific. Further mutating the phosphoacceptor S ~°3 [15,18] to alanine in the mutant M9/AC9 had no additional effect. The M6/AC9 mutant, differing from AC9 only by the mutation of S ~°3 to alanine, displayed a phenotype comparable to AC9.…”

“…We questioned whether phosphorylation was involved in the effects on binding site independent transcription mediated by the N-terminus. For this purpose, the four potential casein kinase II sites S 77, S 79, S 83 and S 85 [15,16,23] were mutated to alanine in the M4/AC9 mutant (Fig. 3A).…”

“…In vitro studies revealed that the core region of SRF (amino acids 133-222) is sufficient for DNA binding, dimerization and ternary complex factor recruitment [14]. However, modulation of the DNA-binding properties of SRF can be achieved by phosphorylation in the N-terminal region, where five phosphorylation sites have been mapped in vivo [15,16]. Additional sites might be phosphorylated in vivo in the C-terminal region of SRF [13,17].…”

SRF is a nuclear repressor of Smad3-mediated TGF-β signaling

“…To confirm the activation of SRF by the addition of 20% serum after 24 h serum starvation, we examined the phosphorylation of SRF at Ser-103. SRF contains a number of phosphorylation sites , and it is a target of several kinase pathways (Manak and Prywes, 1991;Janknecht et al, 1992;Liu et al, 1993;Rivera et al, 1993;Iyer et al, 2003Iyer et al, , 2006. The physiological importance of these modifications remains unsettled.…”

Four isoforms of serum response factor that increase or inhibit smooth-muscle-specific promoter activity

“…The activity of SRF is modified by phosphorylation at multiple sites throughout the protein. Phosphorylation at the N-terminus of SRF, in the DNA-binding domain of the proteins, occurs at serines 75, 79, 83, 85 and 103, probably via casein kinase II, which is activated by growth-factor stimulation [28][29][30]. Phosphorylation of these residues increases the binding of SRF to DNA and increases SRF-dependent transcription from target promoters.…”