title: Modulation of in vivo HSP70 chaperone activity by Hip and Bag-1.
abstract: The chaperone activity of Hsp70 is influenced by the activities of both positive and negative regulatory proteins. In this study, we provide first time evidence for the stimulating effect of the Hsp70-interacting protein Hip on the chaperone activity in the mammalian cytosol. Overexpressing Hip enhances the refolding of the heat-inactivated reporter enzyme luciferase expressed in hamster lung fibroblasts. Also, it protects luciferase from irreversible denaturation under conditions of ATP depletion. We demonstrate that these stimulating actions depend on both the presence of the central Hsp70-binding site and the amino-terminal homo-oligomerization domain of Hip. The carboxyl terminus (amino acids 257-368) comprising the 7 GGMP repeats (Hsc70-like domain) and the Sti1p-like domain are dispensable for the Hip-mediated stimulation of the cellular chaperone activity. Bag-1, which inhibits the Hsp70 chaperone activity both in vitro and in vivo, was found to compete with the stimulatory action of Hip. In cells overexpressing both Hip and Bag-1, the inhibitory effects of Bag-1 were found to be dominant. Our results reveal that in vivo a complex level of regulation of the cellular chaperone activity exists that not only depends on the concentration of Hsp70 but also on the concentration, affinity, and intracellular localization of positive and negative coregulators. As the Hsp70 chaperone machine is also protective in the absence of ATP, our data also demonstrate that cycling between an ATP/ADP-bound state is not absolutely required for the Hsp70 chaperone machine to be active in vivo.
authors.affiliation: Department of Radiation and Stress Cell Biology, Faculty of Medical Sciences, University of Groningen, A. Deusinglaan 1, 9713 AV Groningen, The Netherlands.
authors.forename: E A
authors.initials: EA
authors.lastname: Nollen
authors.forename: A E
authors.initials: AE
authors.lastname: Kabakov
authors.forename: J F
authors.initials: JF
authors.lastname: Brunsting
authors.forename: B
authors.initials: B
authors.lastname: Kanon
authors.forename: J
authors.initials: J
authors.lastname: Höhfeld
authors.forename: H H
authors.initials: HH
authors.lastname: Kampinga
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D016466:CHO Cells; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D006224:Cricetinae; D004268:DNA-Binding Proteins; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism*; D008156:Luciferases / Q000378:metabolism; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D014157:Transcription Factors; D014162:Transfection; D025521:Tumor Suppressor Proteins; D018832:Molecular Chaperones
chemical_list: D000255:Adenosine Triphosphate; D002352:Carrier Proteins; D004268:DNA-Binding Proteins; D018840:HSP70 Heat-Shock Proteins; D008156:Luciferases; D014157:Transcription Factors; D025521:Tumor Suppressor Proteins; C091732:BCL2-associated athanogene 1 protein; C101119:ST13 protein, human; D018832:Molecular Chaperones