Mutation of serum response factor phosphorylation sites and the mechanism by which its DNA-binding activity is increased by casein kinase II.
Casein kinase II (CKII) phosphorylates the mammalian transcription factor serum response factor (SRF) on a serine residue(s) located within a region of the protein spanning amino acids 70 to 92, thereby enhancing its DNA-binding activity in vitro. We report here that serine 83 appears to be the residue phosphorylated by CKII but that three other serines in this region can also be involved in phosphorylation and the enhancement of DNA-binding activity. A mutant that contained glutamate residues in place of these serines had only low-level binding activity; however, when the serines were replaced with glutamates and further mutations were made that increased the negative charge of the region, the resulting mutant showed a constitutively high level of binding equal to that achieved by phosphorylation of wild-type SRF. We have investigated the mechanism by which phosphorylation of SRF increases its DNA-binding activity. We have ruled out the possibilities that phosphorylation affects SRF dimerization or relieves inhibition due to masking of the DNA-binding domain by an amino-terminal region of the protein. Rather, using partial proteolysis to probe SRF's structure, we find that the conformation of SRF's DNA-binding domain is altered by phosphorylation.
Manak
J R
JR
Department of Biological Sciences, Columbia University, New York, New York 10027.
Prywes
R
R
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D047388:Casein Kinases; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009687:Nuclear Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D015345:Oligonucleotide Probes; D010766:Phosphorylation; D010957:Plasmids; D011487:Protein Conformation; D011494:Protein Kinases / Q000378:metabolism*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D026362:Serum Response Factor; D014157:Transcription Factors / Q000378:metabolism
D004268:DNA-Binding Proteins; D046911:Macromolecular Substances; D009687:Nuclear Proteins; D015345:Oligonucleotide Probes; D011994:Recombinant Proteins; D026362:Serum Response Factor; D014157:Transcription Factors; D011494:Protein Kinases; D047388:Casein Kinases