A Constitutive Heat Shock Element-binding Factor Is Immunologically Identical to the Ku Autoantigen

doi:10.1074/jbc.270.25.15277

A Constitutive Heat Shock Element-binding Factor Is Immunologically Identical to the Ku Autoantigen (*)

From the (1)Departments of Medical Physics and Radiation Oncology, Memorial Sloan-Kettering Cancer Center, New York, New York 10021

^§ To whom correspondence should be addressed:
Depts. of Medical Physics and Radiation Oncology, Memorial Sloan-Kettering Cancer Center, 1275 York Ave., New York, NY 10021.
Tel.: 212-639-6028; Fax: 212-639-2611.

Abstract

Analysis of the heat shock element (HSE)-binding proteins in extracts of rodent cells, during heat shock and their post-heat shock recovery, indicates that the regulation of heat shock response involves a constitutive HSE-binding factor (CHBF), in addition to the heat-inducible heat shock factor HSF1. We purified the CHBF to apparent homogeneity from HeLa cells using column chromatographic techniques including an HSE oligonucleotide affinity column. The purified CHBF consists of two polypeptides with apparent molecular masses of 70 and 86 kDa. Immunoblot and gel mobility shift analysis verify that CHBF is identical or closely related to the Ku autoantigen. The DNA binding characteristics of CHBF to double-stranded or single-stranded DNA are similar to that of Ku autoantigen. In gel mobility shift analysis using purified CHBF and recombinant human HSF1, CHBF competes with HSF1 for the binding of DNA sequences containing HSEs in vitro. Furthermore, when Rat-1 cells were co-transfected with human Ku expression vectors and the hsp70-promoter-driven luciferase reporter gene, thermal induction of luciferase is significantly suppressed relative to cells transfected with only the hsp70-luciferase construct. These data suggest a role of CHBF (or Ku protein) in the regulation of heat response in vivo.

Footnotes

↵* This work was supported in part by Grants CA 31397 and CA56909 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

HSE

heat shock element

hsp

heat shock protein

HSF

heat shock factor

HSF1

heat shock factor 1

CHBF

constitutive heat shock element-binding factor

ss

single-stranded

ds

double-stranded

DTT

dithiothreitol

PAGE

polyacrylamide gel electrophoresis

PMSF

phenylmethylsulfonyl fluoride.
↵²G. C. Li, unpublished results.