The 70-kDa Heat Shock Proteins Associate with Glandular Intermediate Filaments in an ATP-dependent Manner (*)
- From the Palo Alto Veterans Affairs Medical Center, Palo Alto, California 94304 and the Digestive Disease Center, Stanford University School of Medicine, Stanford, California 94305-5487
- § Please address reprint requests to Dr. Liao and other correspondence to Dr. Omary at the following address: Stanford University School of Medicine, Lab Surge Bldg., Rm. P304, Stanford, CA 94305-5487.
Abstract
Keratin polypeptides 8 and 18 (K8/18) are intermediate filament proteins expressed preferentially in glandular epithelia. We describe the identification, by co-immunoprecipitation from normal human colonic tissues and cultured cell lines, of the 70-kDa heat shock protein (hsp) and its related heat shock cognate protein as K8/18-associated proteins (hsp/c). The association is significant but sub-stoichiometric and occurs preferentially with the soluble rather than the cytoskeletal K8/18 fractions. Heat stress increases the level of soluble K8/18 in association with an increase in hsp70 levels and an increase in the stoichiometry of K8/18-hsp70 association. Identity of the associated proteins was confirmed by microsequencing of a tryptic digest of the purified associated protein and by using anti-hsp/c70-specific antibodies. The K8/18-hsp/c70 complex can be dissociated in a Mg-ATP-dependent manner that requires ATP hydrolysis. Binding of hsp to K8/18 can be reconstituted using purified bovine hsp70 and human K8/18 immunoprecipitates that have been depleted of bound hsp/c70 and increases slightly in the presence of ATP. The reconstituted K8/18-hsp70 complex can be again released in the presence of Mg-ATP. In addition, hsp70 binds to K8/18 without having a significant effect on in vitro filament assembly when added during or after assembly. Using an overlay assay, hsp70 binds exclusively to K8 in the presence of ATP. Our results show direct association of the hsp/c70 proteins with K8/18. This interaction may serve, at least in part, to regulate the function of these two abundant protein groups.
Footnotes
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↵* This work was supported by a Veterans Affairs Merit Award, National Institute on Alcohol Abuse and Alcoholism Grant AA0947A-01, the PEW Scholars Program, and Digestive Disease Center Grant DK38707. 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.
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↵1 The abbreviations used are:
- IF
-
intermediate filaments
- hsc
-
heat shock cognate protein
- hsp
-
heat shock protein
- HSP
-
heat shock protein family
- MAb
-
monoclonal antibody
- PBS
-
phosphate-buffered saline
- PAGE
-
polyacrylamide gel electrophoresis
- S and P fractions
-
soluble and pellet fractions
- IFAP
-
intermediate filament-associated proteins
- BSA
-
bovine serum albumin
- ATP
S -
adenosine 5′-O-(thiotriphosphate).
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- Received July 25, 1994.
- Revision received September 29, 1994.
- © 1995 by The American Society for Biochemistry and Molecular Biology, Inc.
