The carboxyl-terminal transactivation domain of human serum response factor contains DNA-activated protein kinase phosphorylation sites.

The serum response factor (SRF) is a 67-kDa phosphoprotein that, together with auxiliary factors, modulates transcription of immediate early genes containing serum response elements in their promoters. Here we show that the carboxyl-terminal domain of human SRF is phosphorylated in vivo and is recognized in vitro by the double-stranded DNA-activated serine/threonine-specific protein kinase, DNA-PK. SRF phosphorylation by DNA-PK was stimulated by its cognate binding site. Protein microsequence analysis of a 22-amino acid synthetic SRF peptide and phosphopeptide analysis of genetically altered glutathione S-transferase-SRF fusion proteins identified Ser-435 and Ser-446 of human SRF as sites phosphorylated by DNA-PK. Both serines are followed by glutamine. Changing Gln-436 and Gln-447 to other residues reduced or eliminated phosphorylation by DNA-PK, confirming that these glutamines are important determinants for kinase recognition. The carboxyl-terminal transcription activation domain was mapped within a 71-amino acid region that contains both DNA-PK phosphorylation sites. Amino acid substitutions that interfered with phosphorylation by DNA-PK at Ser-435/446 in GAL4-SRF fusion proteins were reduced in transactivation potency. From these data we suggest that DNA-PK phosphorylation may modulate SRF activity in vivo.