A two-step mechanism for recruitment of Pip by PU.1.
Transcription of the Ig kappa light chain gene is controlled in part by the 3' kappa enhancer. Two of the proteins that bind to the 3' enhancer, PU.1 and Pip, show tissue-restricted expression and may be responsible for the tissue specificity of 3' enhancer activity. PU.1 alone can bind to DNA; however, Pip cannot bind to its 3' enhancer site in electrophoretic mobility shift assays, unless recruited by PU.1. Previously, we showed that the PU.1 PEST domain (rich in the amino acids proline, 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 recruitment 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 sequences between residues 88-118 and 154-168 had no effect. Interestingly, we found that the PU.1 ETS domain (residues 170 to 255) is both necessary and sufficient for Pip interaction in solution and that other ETS domain proteins can physically interact with Pip as well. Our results suggest that Pip recruitment to DNA by PU.1 occurs via a two-step mechanism. First, a physical interaction that is not sufficient to recruit Pip occurs via the PU.1 ETS domain. Second, a conformational change in the PU.1 PEST domain, apparently mediated by serine phosphorylation, induces a conformational change in Pip enabling it to bind to DNA. We also show that the PU.1 PEST domain does not target PU.1 for rapid turnover.
Perkel
J M
JM
Graduate Group of Molecular Biology, Department of Animal Biology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia 19104, USA.
Atchison
M L
ML
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000378:metabolism*; D001665:Binding Sites; D004268:DNA-Binding Proteins / Q000378:metabolism; D004742:Enhancer Elements, Genetic; D005786:Gene Expression Regulation; D007145:Immunoglobulin kappa-Chains / Q000235:genetics*; D008565:Membrane Proteins; D051379:Mice; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism; D011485:Protein Binding; D011518:Proto-Oncogene Proteins / Q000737:chemistry / Q000378:metabolism*; D050783:Proto-Oncogene Proteins c-ets; D015534:Trans-Activators / Q000378:metabolism*; D014157:Transcription Factors / Q000737:chemistry; D000099232:Proto-Oncogene Protein Spi-1
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D007145:Immunoglobulin kappa-Chains; D008565:Membrane Proteins; D009687:Nuclear Proteins; D011518:Proto-Oncogene Proteins; D050783:Proto-Oncogene Proteins c-ets; D015534:Trans-Activators; D014157:Transcription Factors; D000099232:Proto-Oncogene Protein Spi-1; C082934:phage infection protein, Lactococcus lactis