Title

Phosphorylation of human heat shock factor 1(hHSF1)

Date of Award

2002

Availability

Article

Degree Name

Doctor of Philosophy (Ph.D.)

Department

Biochemistry and Molecular Biology

First Committee Member

Richard W. Voellmy, Committee Chair

Abstract

Upon heat shock, human heat shock factor 1 (HSF1) homotrimerizes, accumulates in the nucleus, undergoes inducible phosphorylation and activates transcription of heat shock genes. HSF1 has been shown to be phosphorylated at multiple sites. Several phosphorylation sites have been identified previously that appear to repress the transcriptional activity of the factor. The present study was aimed at finding out whether there might exist other sites whose phosphorylation enhances the transcriptional competence of HSF1. An alanine scan of all serine, threonine and tyrosine residues of HSF1 was carried out. Activity of mutants was tested using a dual luciferase reporter assay. In addition, mass spectrometric analysis was employed to directly identify HSF1 residues that are phosphorylated after heat shock. Here the identification of several previously unknown phosphorylated residues is reported. Surprisingly, alanine substitution of only one of these residues, S326, substantially reduced HSF1 activity. The S326A mutant was indistinguishable from HSF1 regarding stability, DNA-binding ability and nuclear localization. Mutant S326A exhibited clearly increased SDS-PAGE mobility. It is therefore concluded that phosphorylation of S326 occurs with a high stoichiometry and substantially enhances the transactivation function of HSF1.

Keywords

Biology, Molecular

Link to Full Text

http://access.library.miami.edu/login?url=http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqdiss&rft_dat=xri:pqdiss:3050735