Analysis of the physical interactions between PU.1 and Pip, two immunoglobulin kappa 3' enhancer binding proteins.

Format:

Book

Thesis/Dissertation

Author/Creator:

Perkel, Jeffrey Michael.

Contributor:

Atchison, Michael L., advisor.

University of Pennsylvania.

Language:

English

Subjects (All):

Molecular biology.

0307.

Penn dissertations--Molecular biology.

Molecular biology--Penn dissertations.

Local Subjects:

Penn dissertations--Molecular biology.

Molecular biology--Penn dissertations.

0307.

Physical Description:

155 pages

Contained In:

Dissertation Abstracts International 58-07B.

System Details:

Mode of access: World Wide Web.

text file

Summary:

Transcription of the immunoglobulin kappa light chain gene is controlled in part by the activity of the immunoglobulin $kappa\ 3\sp\prime$ enhancer (E$\kappa3\sp\prime).$ Two proteins that bind to the 3$\sp\prime$ enhancer, PU.1 and Pip, show tissue restricted expression, making them candidates for providing the tissue specificity of 3$\sp\prime$ enhancer activity. PU.1 is able to bind to DNA in the absence of Pip; however, Pip binding to its 3$\sp\prime$ enhancer site, as measured by electrophoretic mobility shift assay (EMSA), cannot occur without recruitment by PU.1. Previously we showed that PU.1 residues 118-160 (the PEST domain) are necessary for Pip recruitment to DNA. Here we used a detailed mutagenic analysis of PU.1 to more precisely identify sequences required for Pip recruitment by EMSA. We found that three PU.1 sequences (118-125, 133-139, and 141-147) modulate the efficiency of Pip recruitment. Mutation of PU.1 sequences between residues 88 and 118 or between 154 and 168 had no effect on recruiting Pip to DNA. Kinetic and conformational studies carried out on mutants $\Delta$118-125 and $\Delta$133-139 suggest that these deletions cause a conformational change in the protein that results in altered Pip-recruitment kinetics. Interestingly, we found that the PU.1 ETS domain (residues 170 to 255) is both necessary and sufficient for Pip interaction in solution. Our results suggest that recruitment of Pip to DNA by PU.1 occurs via a two-step mechanism. First physical interaction occurs via the PU.1 ETS domain but this interaction is not sufficient to recruit Pip to DNA. Second, a conformational change in the PU.1 PEST domain apparently mediated by serine phosphorylation induces a conformational change in Pip enabling it to bind to DNA. Interestingly, using a GST chromatography DNA binding assay we demonstrate the Pip is able to specifically bind to the 3$\sp\prime$ enhancer PU.1:Pip sequence in the absence of PU.1, suggesting that Pip can weakly bind to this sequence by itself. We also show that other ETS domain proteins can physically interact with Pip and that the PU.1 PEST domain does not target PU.1 for rapid turnover. Finally, a functional analysis of the physical interaction between PU.1 and MKP-1 is presented.

Notes:

Thesis (Ph.D. in Molecular Biology) -- University of Pennsylvania, 1997.

Source: Dissertation Abstracts International, Volume: 58-07, Section: B, page: 3477.

Supervisor: Michael L. Atchison.

Local Notes:

School code: 0175.

ISBN:

9780591501582

Access Restriction:

Restricted for use by site license.