Phosphorylation represses Ets-1 DNA binding by reinforcing autoinhibition.
Phosphorylation of transcription factors is a key link between cell signaling and the control of gene expression. Here we report that phosphorylation regulates DNA binding of the Ets-1 transcription factor by reinforcing an autoinhibitory mechanism. Quantitative DNA-binding assays show that calcium-dependent phosphorylation inhibits Ets-1 DNA binding 50-fold. The four serines that mediate this inhibitory effect are distant from the DNA-binding domain but near structural elements required for autoinhibition. Mutational analyses demonstrate that an intact inhibitory module is required for phosphorylation-dependent regulation. Partial proteolysis studies indicate that phosphorylation stabilizes an inhibitory conformation. These findings provide a structural mechanism for phosphorylation-dependent inhibition of Ets-1 DNA binding and demonstrate a new function for inhibitory modules as structural mediators of negative signaling events.
Cowley
D O
DO
Huntsman Cancer Institute, University of Utah, Salt Lake City, Utah 84112-5550, USA.
Graves
B J
BJ
D000595:Amino Acid Sequence; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D002457:Cell Extracts; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008954:Models, Biological; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism; D017434:Protein Structure, Tertiary; D050784:Proto-Oncogene Protein c-ets-1; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism*; D050783:Proto-Oncogene Proteins c-ets; D013601:T-Lymphocytes / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
D002457:Cell Extracts; D004268:DNA-Binding Proteins; C494041:ETS1 protein, human; D050784:Proto-Oncogene Protein c-ets-1; D011518:Proto-Oncogene Proteins; D050783:Proto-Oncogene Proteins c-ets; D014157:Transcription Factors; D010768:Phosphoserine; D004247:DNA; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases