HSF-1 interacts with Ral-binding protein 1 in a stress-responsive, multiprotein complex with HSP90 in vivo

Abstract

Heat shock factor 1 (HSF1) regulates the rapid and transient expression of heat shock genes in response to stress. The transcriptional activity of HSF1 is tightly controlled, and under physiological growth conditions, the HSF1 monomer is in a heterocomplex with the molecular chaperone HSP90. Through unknown mechanisms, transcriptionally repressed HSF1·HSP90 heterocomplexes dissociate following stress, which triggers HSF1 activation and heat shock gene transcription. Using a yeast two-hybrid screening system, we have identified Ral-binding protein 1 (RalBP1) as an additional HSF1-interacting protein. We show that RalBP1 and HSF1 interact in vivo, and transient cotransfection of HSF1 and RalBP1 into hsf1−/− mouse embryo fibroblasts represses HSP70 expression. Furthermore, transient cotransfection of HSF1 and the constitutively active form of RalA (RalA23V), an upstream activator of the RalBP1 signaling pathway, increases the heat-inducible expression of HSP70, whereas the dominant negative form (RalA28N) suppresses HSP70 expression. We further find that α-tubulin and HSP90 are also present in the RalBP1·HSF1 heterocomplexes in unstressed cells. Upon heat shock, the Ral signaling pathway is activated, and the resulting RalGTP binds RalBP1. Concurrently, HSF1 is activated, leaves the RalBP1·HSF1·HSP90·α-tubulin heterocomplexes, and translocates into the nucleus, where it then activates transcription. In conclusion, these observations reveal that the RalGTP signal transduction pathway is critical for activation of the stress-responsive HSF1 and perhaps HSP90 molecular chaperone system. Heat shock factor 1 (HSF1) regulates the rapid and transient expression of heat shock genes in response to stress. The transcriptional activity of HSF1 is tightly controlled, and under physiological growth conditions, the HSF1 monomer is in a heterocomplex with the molecular chaperone HSP90. Through unknown mechanisms, transcriptionally repressed HSF1·HSP90 heterocomplexes dissociate following stress, which triggers HSF1 activation and heat shock gene transcription. Using a yeast two-hybrid screening system, we have identified Ral-binding protein 1 (RalBP1) as an additional HSF1-interacting protein. We show that RalBP1 and HSF1 interact in vivo, and transient cotransfection of HSF1 and RalBP1 into hsf1−/− mouse embryo fibroblasts represses HSP70 expression. Furthermore, transient cotransfection of HSF1 and the constitutively active form of RalA (RalA23V), an upstream activator of the RalBP1 signaling pathway, increases the heat-inducible expression of HSP70, whereas the dominant negative form (RalA28N) suppresses HSP70 expression. We further find that α-tubulin and HSP90 are also present in the RalBP1·HSF1 heterocomplexes in unstressed cells. Upon heat shock, the Ral signaling pathway is activated, and the resulting RalGTP binds RalBP1. Concurrently, HSF1 is activated, leaves the RalBP1·HSF1·HSP90·α-tubulin heterocomplexes, and translocates into the nucleus, where it then activates transcription. In conclusion, these observations reveal that the RalGTP signal transduction pathway is critical for activation of the stress-responsive HSF1 and perhaps HSP90 molecular chaperone system. heat shock factor 1 Ral-binding protein 1 Ral binding domain mouse embryo fibroblast phosphate-buffered saline heat shock protein Mammalian heat shock factor 1 (HSF1),1 a phosphorylated protein, regulates the stress inducibility of heat shock genes. Phosphorylation of HSF1 is indicative of its complex mode of regulation by various signaling pathways. Studies using phosphopeptide analysis of HSF1 protein as well as studies analyzing the transactivation properties of HSF1 using chimeric constructs containing GAL4-HSF1 or LexA-HSF1 have suggested that phosphorylation of serine residues Ser303, Ser307, and Ser363 is likely to be involved in repression of HSF1 transcriptional activity (1Dai R. He B. Freitag W. Zhang Y. Mivechi N.F. J. Biol. Chem. 2000; 275: 18210-18218Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 2Knauf U. Newton E.M. Kyriakins J. Kingston R.E. Genes Dev. 1996; 10: 2782-2793Crossref PubMed Scopus (184) Google Scholar, 3Soncin F. Asea A. Zhang X. Stevenson M.A. Calderwood S.K. Int. J. 2000; Google Scholar, Biol. PubMed Scopus Google Scholar, B. Mivechi N.F. Biol. PubMed Scopus Google Scholar, B. F. Stevenson M.A. Calderwood S.K. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, W. R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google protein and are for these HSF1 be phosphorylated its activation as perhaps by protein A. A. J. J. PubMed Scopus Google HSF1 is also in under physiological and its repression A. Biol. PubMed Scopus Google Scholar, J. Y. R. Full Text Full Text PDF PubMed Scopus Google Scholar, Y. F. R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google HSP90 with the form of that an heterocomplex HSF1 in a repressed of heterocomplex by stress HSF1 to form and binding