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.