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, toget her 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-activate d serine/threonine-specific protein kinase, DNA-PK. SRF phosphorylatio n by DNA-PK was stimulated by its cognate binding site. Protein micros equence analysis of a 22-amino acid synthetic SRF peptide and phosphop eptide analysis of genetically altered glutathione S-transferase-SRF f usion proteins identified Ser-435 and Ser-446 of human SRF as sites ph osphorylated by DNA-PK. Both serines are followed by glutamine. Changi ng Gln-436 and Gln-447 to other residues reduced or eliminated phospho rylation by DNA-PK, confirming that these glutamines are important det erminants for kinase recognition. The carboxyl-terminal transcription activation domain was mapped within a 71-amino acid region that contai ns both DNA-PK phosphorylation sites. Amino acid substitutions that in terfered with phosphorylation by DNA-PK at Ser-435/446 in GAL4-SRF fus ion proteins were reduced in transactivation potency. From these data we suggest that DNA-PK phosphorylation may modulate SRF activity in vi vo.