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.
Highlights
National Institute on Alcohol Abuse and Alcoholism Grant AA0947A01, the PEW Scholars Program, and Digestive Disease Center Grant DK38707
Association ofK8 / 18 with HSP70-In carrying out immunoprecipitations of K8/18 from the colonic epithelial cell line HT29, we consistently noted co-immunoprecipitation of a 70kDa band with KS/IS
This is exemplified in Fig. lA, where a 70-kDa species is noted to co-immunoprecipitate with KS/IS using our high capacity anti-KS/IS Monoclonal antibodies (MAb) L2Al
Summary
Other IFAP include ankyrin and lamin B (Georgatos et al, 1987; Djabali et al, 1991), desmoplakin (Stappenbeck et al, 1993), a 140-kDa desmosomal antigen (Cartaud et al, 1990), IFAP 300 (Skalli et al, 1994), plectin (Foisner et al, 1991), gyronemin ( called filamin) (Brown and Binder, 1992), and KAP85 (Chou et al, 1994) Another highly abundant protein family is the heat shock protein family (HSP) with major classes including HSP25, -60, -70, -80, -90, and -110 (numbers correspond to approximate M; of class members) (Gething and Sambrook, 1992; Georgopoulos and Welch, 1993; Hendrick and Hartl, 1993; Becker and Craig, 1994). 1) a ubiquitous 70-kDa protein termed ,B-internexin was initially identified as a microtubule-associated protein and shown indirectly to associate with IF (Napolitano et al, 1985) and was subsequently shown to be identical to hsp (Green and Liem, 1989); 2) hsp was shown to co-purify with microtubules in murine mastocytoma cells (Ohtsuka et al, 1986) and with retinal microtubule and IF fractions (Clark and Brown, 1986); 3) in vitro interaction of
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