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 bi...

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Bibliographic Details
Published in:The Journal of immunology (1950) 1998-01, Vol.160 (1), p.241-252
Main Authors: Perkel, Jeffrey M, Atchison, Michael L
Format: Article
Language:English
Subjects:
3T3 Cells
Amino Acid Sequence
Animals
Bacterial Proteins - metabolism
Binding Sites
DNA-Binding Proteins - metabolism
Enhancer Elements, Genetic
Gene Expression Regulation
Immunoglobulin kappa-Chains - genetics
Membrane Proteins
Mice
Molecular Sequence Data
Nuclear Proteins - metabolism
Protein Binding
Proto-Oncogene Proteins - chemistry
Proto-Oncogene Proteins - metabolism
Proto-Oncogene Proteins c-ets
Trans-Activators - metabolism
Transcription Factors - chemistry
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Summary: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.
ISSN:0022-1767
1550-6606
DOI:10.4049/jimmunol.160.1.241