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

J Biol Chem. 1993 Oct 5;268(28):21147-54.


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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • 3T3 Cells
  • Amino Acid Sequence
  • Animals
  • Base Sequence
  • Binding Sites
  • Cells, Cultured
  • DNA / metabolism*
  • DNA, Complementary
  • DNA-Activated Protein Kinase
  • DNA-Binding Proteins / metabolism*
  • Fungal Proteins / metabolism
  • HeLa Cells
  • Humans
  • Mice
  • Molecular Sequence Data
  • Nuclear Proteins / metabolism*
  • Phosphorylation
  • Protein-Serine-Threonine Kinases / metabolism*
  • Recombinant Fusion Proteins / metabolism
  • Saccharomyces cerevisiae Proteins*
  • Serum Response Factor
  • Transcription Factors / metabolism*
  • Transcription, Genetic
  • Transcriptional Activation*


  • DNA, Complementary
  • DNA-Binding Proteins
  • Fungal Proteins
  • GAL4 protein, S cerevisiae
  • Nuclear Proteins
  • Recombinant Fusion Proteins
  • Saccharomyces cerevisiae Proteins
  • Serum Response Factor
  • Transcription Factors
  • DNA
  • DNA-Activated Protein Kinase
  • PRKDC protein, human
  • Protein-Serine-Threonine Kinases