The 70-kDa Heat Shock Proteins Associate with Glandular Intermediate Filaments in an ATP-dependent Manner

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.

Rescuing of deficient killing and phagocytic activities of macrophages derived from non-obese diabetic mice by treatment with geldanamycin or heat shock: potential clinical implications

“…The mechanisms by which hsp expression modulates the inflammatory response of professional phagocytes may also be related to the control of vesicle trafficking and/or cytoskeleton rearrangements. Previous reports have shown F-actin formation after HS (Vega and De Maio 2005;Collier and Schlesinger 1986;Han et al 2000) as well as a close association of Hsp70 with actin-filaments, microtubules, tubulin, and intermediate filaments (Kirby et al 1994;Liao et al 1995;Dou et al 2003). Small hsp, in particular Hsp27, have also been implicated in the regulation of the cytoskeleton by participating in F-actin formation around plasma membrane rufflings and pinocytosis (Lavoie et al 1993) and by inducing stabilization of actin filaments (Piotrowicz and Levin 1997).…”

“…This function is accomplished due to the proteins' intracellular chaperone activity and is responsible for the refolding of stressdenatured proteins and stabilization of transcription and translation (Lindquist and Craig 1988;Morimoto 1991;De Maio 1999). More recently, it has been shown that hsp also regulate other cellular activities such as cytoskeleton rearrangement (Han et al 2000;Kirby et al 1994;Liao et al 1995;Dou et al 2003;Lavoie et al 1993;Piotrowicz and Levin 1997), endocytosis and phagocytosis (Vega et al 2010;Vega and De Maio 2005), and activation of immune cells (Vega et al 2008;Asea et al 2000Asea et al , 2002. Especially interesting is the capacity of both extra-and intracellular hsp to regulate the immune system by activation of key cells such as macrophages (Mfs) and dendritic cells, thereby controlling inflammatory and immune responses (Vega et al 2010;De Maio 2010;Asea et al 2000Asea et al , 2002Basu et al 2001).…”

BAG-1 accelerates cell motility of human gastric cancer cells

“…Though immunomicroscopic colocalization of BAG-1 with cytokeratin and apparent concentration of BAG-1 protein at membrane ru es support the idea of physical association, this remains to be demonstrated directly. In this context, Hsp70/Hsc70 can bind cytokeratin K8/K18 (Liao et al, 1995). Since Hsc70/ BAG-1 complexes can bind to various other proteins, it is tempting to speculate that BAG-1 may be able to indirectly bind to cytoskeletal proteins via Hsp70/ Hsc70.…”

Cytoplasmic retention of mutant tsp53 is dependent on an intermediate filament protein (Vimentin) scaffold

“…The constitutively expressed heat shock protein hsc70 which preferentially binds to tsp53 in its mutant conformation (Gannon and Lane, 1991;Hinds et al, 1987;Pinhasi-Kimhi et al, 1986) in theory could represent such an anchor protein. Chaperones themselves have an a nity for intermediate ®laments (Cheng and Lai, 1994;Liao et al, 1995) and therefore could link p53 to the cytoskeleton. However, tsp53 can be retained in the cytoplasm of IF-positive cells even when present in its properly folded wild-type conformation, to which hsc70 does not bind (Knippschild et al, 1996).…”