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.
Mesh Terms:
3T3 Cells, Amino Acid Sequence, Animals, Base Sequence, Binding Sites, Cells, Cultured, DNA, DNA, Complementary, DNA-Activated Protein Kinase, DNA-Binding Proteins, Fungal Proteins, Hela Cells, Humans, Mice, Molecular Sequence Data, Nuclear Proteins, Phosphorylation, Protein-Serine-Threonine Kinases, Recombinant Fusion Proteins, Saccharomyces cerevisiae Proteins, Serum Response Factor, Transcription Factors, Transcription, Genetic, Transcriptional Activation
3T3 Cells, Amino Acid Sequence, Animals, Base Sequence, Binding Sites, Cells, Cultured, DNA, DNA, Complementary, DNA-Activated Protein Kinase, DNA-Binding Proteins, Fungal Proteins, Hela Cells, Humans, Mice, Molecular Sequence Data, Nuclear Proteins, Phosphorylation, Protein-Serine-Threonine Kinases, Recombinant Fusion Proteins, Saccharomyces cerevisiae Proteins, Serum Response Factor, Transcription Factors, Transcription, Genetic, Transcriptional Activation
J. Biol. Chem.
Date: Oct. 05, 1993
PubMed ID: 8407951
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