Mammalian protein RAP46: an interaction partner and modulator of 70kDa heat shock proteins

Abstract

which was identified by screening an expression library 1 Corresponding author using as probe the glucocorticoid receptor overexpressed in the baculovirus system. It also interacts with other A ubiquitously expressed nuclear receptor-associating members of the nuclear receptor superfamily (Zeiner and protein of~46 kDa (RAP46) was identified recently.Gehring, 1995). Murine BAG-1 was obtained by use of a Interaction experiments with in vitro-translated prosimilar screening procedure in which baculovirusteins and proteins contained in cell extracts revealed expressed Bcl-2 or the hepatocyte growth factor receptor that a great variety of cellular regulators associate were employed to identify interacting proteins (Takayama with RAP46. However, in direct interaction tests Bardelli et al., 1996). Subsequent studies the far-Western technique, only 70 kDa proteins showed showed (see below) that the interaction of RAP46 is not up and were identified as members of the 70 kDa heat restricted to nuclear receptors but that various unrelated shock protein (hsp70) family. Interaction is specific regulatory proteins, including Bcl-2, also react with RAP46 since not all members of the hsp70 family bind to in pull-down experiments. This then raised the question RAP46; interaction occurs through their ATP-binding of whether such interactions might be mediated by some domain. RAP46 forms complexes with hsp70 in mamcommon interacting protein(s). In the present study, we malian cells and interacts with hsp70 in the yeast twoidentified members of the hsp70 family as direct and hybrid system. Consistent with the fact that hsp70 can specific interaction partners. Through binding to members bind a multitude of proteins, we identified heteromeric of the hsp70 family, a multitude of proteins might associate complexes of RAP46-hsp70 with some selected prowith RAP46. Indeed, we detected heteromeric complexes teins, most notably c-Jun. Complex formation is in which hsp70 serves as adaptor. The interaction of increased significantly by pre-treatment with alkaline RAP46 with hsp70s occurs via their ATP-binding domain. phosphatase, thus suggesting modulation of interactions by protein phosphorylation. We observed that RAP46 interferes with efficient refolding of thermally Results denatured luciferase. Moreover, ATP-dependent bind- Proteins interacting with RAP46 ing of misfolded proteins to hsp70 was greatly inhibitedOriginally, RAP46 was identified by binding the activated by RAP46. These data suggest that RAP46 functions glucocorticoid receptor (Zeiner and Gehring, 1995). In as a regulator of hsp70 in higher eukaryotes.pull-down assays with RAP46 fused to glutathione-SKeywords: c-Jun/dephosphorylation/hsp70 heat shock transferase (GST) and bound to glutathione (GSH)-proteins/nuclear receptors/protein refolding Sepharose, we obtained similar associations with other mammalian steroid hormone receptors (Zeiner and Gehring, 1995) and with ecdysteroid, thyroid and retinoic acid receptors (data not shown). Using this technique, we

BAG-1 accelerates cell motility of human gastric cancer cells

“…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. Though the nature of the functional relations between Hsp70/Hsc70 and K8/K18 remains uncertain, the evidence that BAG-1 is a regular of Hsp70/Hsc70 chaperone activity HoÈ hfeld and Jentsch, 1997;Zeiner et al, 1997;HoÈ hfeld, 1998) raises the possibilities that BAG-1 may function as a modulator of cytokeratin ®lament assembly. BAG-1 gene disruption experiments will clarify whether loss of BAG-1 function leads to disruption of cell migration.…”

“…BAG-1 binds tightly to Hsp70/Hsc70-family proteins and modulates their molecular chaperone activity HoÈ hfeld and Jentsch, 1997;Zeiner et al, 1997). Considering the many Hsp70/Hsc70 functions, which include protein folding, translocation and degradation (Craig et al, 1994), BAG-1 may participate in broad biological processes linked to Hsp70/Hsc70.…”

“…The carboxyl-domain of BAG-1 is required for Hsp70/Hsc70 binding and shows weak amino acid sequence homology with bacterial GrpE, an ADP/ATP exchange factor for the prokaryotic chaperone DNaJ (Stuart et al, 1998;Hartl, 1996). In this regard, BAG-1 has been shown to bind the ATPase domain of Hsc70, promote ADP/ ATP exchange and thereby modulating the chaperone activity in vitro HoÈ hfeld and Jentsch, 1997;Zeiner et al, 1997). However, in addition to its C-terminal Hsp70/Hsc70 binding site, BAG-1 appears to contain separate regions that mediate interactions with speci®c target proteins.…”

High level expression of differentially localized BAG-1 isoforms in some oestrogen receptor-positive human breast cancers

“…BAG-1 (also known as HAP; Zeiner et al, 1997) is a multifunctional protein which interacts with and modulates the activity of functionally diverse targets. BAG-1 was first identified as a BCL-2-binding protein that cooperated with BCL-2 to suppress apoptosis induced by staurosporine, Fas or cytotoxic T-lymphocytes in Jurkat cells (Takayama et al, 1995) and BAG-1 overexpression alone or in conjunction with BCL-2 has since been shown to contribute to suppression of apoptosis induced by diverse stimuli in a variety of cell types (Clevenger et al, 1997;Schulz et al, 1997;Takayama et al, 1997;Kullmann et al, 1998).…”

“…BAG-1 interacts with the human homologue of Drosophila seven in absentia (Siah) and inhibits p53-mediated growth arrest (Matsuzawa et al, 1998). Finally, BAG-1 binds directly and tightly to HSP/HSC70 heat shock proteins and modulates their chaperone activity (Hohfeld and Jentsch, 1997;Takayama et al, 1997;Zeiner et al, 1997). It is possible that binding to some BAG-1 partners, for example, the oestrogen receptor (ER), may be mediated by heat shock proteins, since HSP70 has been identified in ER complexes (Landel et al, 1994).…”