2-STEP MECHANISM FOR RECRUITMENT OF PIP BY PU.1

Transcription of the Ig kappa light chain gene is controlled in part b y the 3' kappa enhancer. Two of the proteins that bind to the 3' enhan cer, PU.1 and Pip, show tissue-restricted expression and may be respon sible for the tissue specificity of 3' enhancer activity, PU.1 alone c an bind to DNA; however, Pip cannot bind to its 3' enhancer site in el ectrophoretic mobility shift assays, unless recruited by PU.1. Previou sly, we showed that the PU.1 PEST domain (rich in the amino acids prol ine, glutamate, serine, and threonine; sequences 118-160) is necessary for Pip recruitment to DNA, Here we used detailed mutagenic analyzes of PU.1 to more precisely identify sequences required for Pip recruitm ent by electrophoretic mobility shift assay. We found that mutation of three segments within the PU.1 PEST domain (118-125, 133-139, and 141 -147) modulated the efficiency of Pip recruitment, while mutation of s equences between residues 88-118 and 154-168 had no effect. Interestin gly, we found that the PU.1 ETS domain (residues 170 to 255) is both n ecessary and sufficient for Pip interaction in solution and that other ETS domain proteins can physically interact with Pip as well. Our res ults suggest that Pip recruitment to DNA by PU.1 occurs via a two-step mechanism. First, a physical interaction that is not sufficient to re cruit Pip occurs via the PU.1 ETS domain. Second, a conformational cha nge in the PU.1 PEST domain, apparently mediated by serine phosphoryla tion, induces a conformational change in Pip enabling it to bind to DN A. We also show that the PU.1 PEST domain does not target PU.1 for rap id turnover.