Phosphorylation represses Ets-1 DNA binding by reinforcing autoinhibition

Phosphorylation of transcription factors is a key link between cell signali ng and the control of gene expression. Here we report that phosphorylation regulates DNA binding of the Ets-l transcription factor by reinforcing an a utoinhibitory mechanism. Quantitative DNA-binding assays show that calcium- dependent phosphorylation inhibits Ets-1 DNA binding 50-fold. The four seri nes that mediate this inhibitory effect are distant from the DNA-binding do main but near structural elements required for autoinhibition. Mutational a nalyses demonstrate that an intact inhibitory module is required for phosph orylation-dependent regulation. Partial proteolysis studies indicate that p hosphorylation stabilizes an inhibitory conformation. These findings provid e a structural mechanism for phosphorylation-dependent inhibition of Ets-1 DNA binding and demonstrate a new function for inhibitory modules as struct ural mediators of negative signaling events.