Identification of multiple SRF N-terminal phosphorylation sites affecting DNA binding properties.
Human serum response factor (SRF) bearing a histidine tag was expressed using vaccinia virus. The recombinant protein was purified and shown to be phosphorylated mainly in its N-terminal part. The corresponding phosphorylation sites were mapped by microsequencing and also appear to be phosphorylated in endogenous serum response factor. Four phosphorylation sites are located on serines within amino acids 77-85, while another phosphorylation site has been identified at Ser103. Mutations that considerably reduced or abolished phosphorylation at amino acids 77-85 caused a decrease in binding to the c-fos serum response element accompanied by markedly reduced association and dissociation rates. In contrast, replacing Ser103 by alanine decreased DNA binding activity without drastically affecting the on/off rates. The combination of abolishing phosphorylation at amino acids 77-85 and 103 displayed greatly reduced on/off rates of DNA binding, but the reduction of DNA binding activity was partially alleviated. None of these mutations affect either the ability to interact with p62TCF or stimulation of transcription in vitro. These findings imply possible roles for SRF phosphorylation in the regulation of c-fos transcription.
Janknecht
R
R
Hannover Medical School, Institute for Molecular Biology, Germany.
Hipskind
R A
RA
Houthaeve
T
T
Nordheim
A
A
Stunnenberg
H G
HG
D000595:Amino Acid Sequence; D015153:Blotting, Western; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D006367:HeLa Cells / Q000382:microbiology; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D009687:Nuclear Proteins / Q000235:genetics* / Q000378:metabolism; D010766:Phosphorylation; D026362:Serum Response Factor; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic; D014162:Transfection; D014616:Vaccinia virus
D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D026362:Serum Response Factor; D014157:Transcription Factors; D004247:DNA