Ternary complex factors (TCFs), one of which is Elk‐1, have been implicated in mediation of c‐fos induction. They have been shown to be phosphorylated by mitogen‐activated protein kinases (MAPKs) in vitro. We demonstrate that recombinant Elk‐1 is hyperphosphorylated in vivo upon joint overexpression of MAPKs and constitutively activated Raf‐1 kinase, the latter serving as an indirect in vivo activator of MAPKs. This phosphorylation is accompanied by a conformational change and results in an elevated transactivation potential of Elk‐1. Mutation of mapped in vivo phosphorylation sites, which are potential targets for MAPKs, reduced Elk‐1‐mediated transcription. Thus, MAPKs are very probably controlling Elk‐1 activity by direct phosphorylation in vivo. Furthermore, Elk‐1 was shown to stimulate transcription from both the c‐fos serum response element and also from an Ets binding site. While binding of TCFs to the c‐fos promoter is dependent on the serum response factor, TCFs can autonomously interact with Ets binding sites. This indicates that TCFs may participate in the transcriptional regulation of two different sets of genes.

An important function of transcription factors may be to sequester coactivators or corepressors of transcription. In this manner transcription factors could regulate in trans the activity of promoters to which they do not bind. This may be of widespread significance as a mechanism to control cell cycle-dependent and differentiation-specific transcriptional activity within eukaryotic cells. Therefore squelching in vivo may be more important than hitherto appreciated.

The serum‐response factor (SRF) is essential for the induction and repression of the protooncogene c‐fos. Phosphorylation of SRF has been implicated to be involved in these processes and five phosphorylation sites have already been mapped within the N‐terminal region. Here we show that in vivo additional phosphorylation of SRF does occur. This modification is located primarily within amino acids 206–289, which probably contain more than one phosphorylation site. Microsequencing allowed the identification of one phosphorylation site at Ser253, which is a potential target of casein kinase II. Mutational analysis revealed that, in contrast to N‐terminal phosphorylation, Ser253 phosphorylation does not affect DNA‐binding properties. In addition, phosphorylation at Ser253 does not seem to change transactivation activity of SRF but rather influences its contribution to transcriptional repression. Thus, C‐terminal phosphorylation of SRF may modulate c‐fos basal repression.

The serum response element (SRE) contributes to transcriptional repression of the c-fos proto-oncogene. We show that the transcription factor SRF is able to repress SRE-dependent transcription, apparently by sequestering a co-activator. Only the DNA-binding core region is required for this SRE-dependent repression. Furthermore the phosphorylation status at potential casein kinase II sites within an N-terminal repression domain affects SRE-independent transcription. SRF may thus pleiotropically influence cellular transcription, representing a novel aspect of SRF function.