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• W2005431141 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. Mol. Med. 2000; 6: 705-710PubMed Google Scholar, 4Kline M.P. Morimoto R.I. Mol. Cell. Biol. 1997; 17: 2107-2115Crossref PubMed Scopus (243) Google Scholar, 5He B. Meng Y.-H. Mivechi N.F. Mol. Cell. Biol. 1998; 18: 6624-6633Crossref PubMed Scopus (133) Google Scholar, 6Chu B. Soncin F. Price B.D. Stevenson M.A. Calderwood S.K. J. Biol. Chem. 1996; 271: 30847-30857Abstract Full Text Full Text PDF PubMed Scopus (308) Google Scholar, 7Xia W. Voellmy R. J. Biol. Chem. 1997; 272: 4094-4102Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). Mitogen-activated protein kinases and glycogen synthase kinase 3 are candidates for phosphorylating these residues. HSF1 could potentially be phosphorylated during its activation process as well, perhaps at Ser230 by calcium calmodulin protein kinase II (8Holmberg C.I. Hietakangas V. Mikhailov A. Rantanen J.O. Kallio M. Meinander A. Hellman J. Morrice N. Mackintosh C. Morimoto R.I. Eriksson J.E. Sistonen L. EMBO J. 2001; 20: 3800-3810Crossref PubMed Scopus (248) Google Scholar). HSF1 is also found in multichaperone complexes under physiological conditions and during its repression (9Bharadwaj S. Ali A. Ovsenek N. Mol. Cell. Biol. 1999; 19: 8033-8041Crossref PubMed Scopus (145) Google Scholar, 10Zou J. Guo Y. Guettouche T. Smith D.F. Voellmy R. Cell. 1998; 94: 471-480Abstract Full Text Full Text PDF PubMed Scopus (925) Google Scholar, 11Guo Y. Guettouche T. Fenna M. Boellmann F. Pratt W.B. Toft D.O. Smith D.F. Voellmy R. J. Biol. Chem. 2001; 276: 45791-45799Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar). Specifically, HSP90 has been co-immunoprecipitated with the monomeric form of HSF1, suggesting that an HSP90·HSF1 heterocomplex may keep HSF1 in a repressed state. Disruption of this heterocomplex by stress would allow HSF1 to form trimers and acquire DNA binding capability. One likely outcome of the disruption of HSF1·HSP90 heterocomplexes during stress is the accumulation of denatured polypeptides and the ability of HSP90 to bind such denatured polypeptides (9Bharadwaj S. Ali A. Ovsenek N. Mol. Cell. Biol. 1999; 19: 8033-8041Crossref PubMed Scopus (145) Google Scholar, 10Zou J. Guo Y. Guettouche T. Smith D.F. Voellmy R. Cell. 1998; 94: 471-480Abstract Full Text Full Text PDF PubMed Scopus (925) Google Scholar, 11Guo Y. Guettouche T. Fenna M. Boellmann F. Pratt W.B. Toft D.O. Smith D.F. Voellmy R. J. Biol. Chem. 2001; 276: 45791-45799Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar). During recovery from stress, HSF1 trimers have been found in separate complexes with HSP70 and in HSP90-immunophilin (FKBP52)-p23 chaperone complexes (10Zou J. Guo Y. Guettouche T. Smith D.F. Voellmy R. Cell. 1998; 94: 471-480Abstract Full Text Full Text PDF PubMed Scopus (925) Google Scholar). Heat shock factor-binding protein 1, which interacts with HSF1 trimers together with HSP70, has also been isolated (12Satyal S.H. Chen D. Fox S.G. Kramer J.M. Morimoto R.I. Genes Dev. 1998; 12: 1962-1974Crossref PubMed Scopus (184) Google Scholar), and this interaction appears to also be a negative regulator of HSF1 transcriptional activity.Ral proteins are GTPases present in the plasma membrane and cytoplasmic vesicles and become biologically active through exchange of GDP for GTP (13Bielinske D.F. Pyun N.Y. Linko-Stentz K. Macara I. Fine R.E. Biochim. Biophys. Acta. 1993; 1151: 246-256Crossref PubMed Scopus (60) Google Scholar, 14Volknandt W. Pevsner J. Elferink L.A. Scheller R.H. FEBS Lett. 1993; 317: 53-56Crossref PubMed Scopus (39) Google Scholar, 15Urano T. Emkey R. Feig L.A. EMBO J. 1996; 16: 810-816Crossref Scopus (299) Google Scholar, 16Reuther G.W. Der C.J. Curr. Opin. Cell Biol. 2000; 12: 157-165Crossref PubMed Scopus (345) Google Scholar, 17Feig L.A. Urano T. Cantor S. Trends Biochem. Sci. 1996; 21: 438-441Abstract Full Text PDF PubMed Scopus (179) Google Scholar). Epidermal growth factor, other receptor tyrosine kinases, and G protein-coupled receptor-induced Ral activation is dependent on Ras activation, suggesting that Ral guanine nucleotide exchange factors can also function as Ras effector molecules (17Feig L.A. Urano T. Cantor S. Trends Biochem. Sci. 1996; 21: 438-441Abstract Full Text PDF PubMed Scopus (179) Google Scholar, 18White M.A. Vale T. Camonis J.H. Schaefer E. Wigler M.H. J. Biol. Chem. 1996; 271: 16439-16442Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar, 19Wolthuis R.M. Franke B. van Triest M. Bauer B. Cool R.H. Camonis J.H. Mol. Cell. Biol. 1998; 18: 2486-2491Crossref PubMed Scopus (130) Google Scholar, 20Wolthuis R.M. Zwartkruis F. Moen T.C. Bos J.L. Curr. Biol. 1998; 8: 471-474Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 21Wolthuis R.M. Bos J.L. Curr. Opin. Genet. Dev. 1999; 9: 112-117Crossref PubMed Scopus (128) Google Scholar, 22Goi T. Shipitsin M. Lu Z. Foster D.A. Klinz S.G. Feig L.A. EMBO J. 2000; 19: 623-630Crossref PubMed Scopus (116) Google Scholar). Ras-dependent activation of Ral functions in parallel to the Ras-Raf-Mek-extracellular signal-regulated kinase pathway in a number of cell types (15Urano T. Emkey R. Feig L.A. EMBO J. 1996; 16: 810-816Crossref Scopus (299) Google Scholar, 17Feig L.A. Urano T. Cantor S. Trends Biochem. Sci. 1996; 21: 438-441Abstract Full Text PDF PubMed Scopus (179) Google Scholar, 18White M.A. Vale T. Camonis J.H. Schaefer E. Wigler M.H. J. Biol. Chem. 1996; 271: 16439-16442Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar, 20Wolthuis R.M. Zwartkruis F. Moen T.C. Bos J.L. Curr. Biol. 1998; 8: 471-474Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). In addition, Ral can be activated by a Ras-independent pathway that involves a phospholipase C-mediated increase in intracellular Ca2+ (19Wolthuis R.M. Franke B. van Triest M. Bauer B. Cool R.H. Camonis J.H. Mol. Cell. Biol. 1998; 18: 2486-2491Crossref PubMed Scopus (130) Google Scholar, 20Wolthuis R.M. Zwartkruis F. Moen T.C. Bos J.L. Curr. Biol. 1998; 8: 471-474Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 23Hofer F. Berdeaux R. Martin G.S. Curr. Biol. 1998; 8: 839-842Abstract Full Text Full Text PDF PubMed Google Scholar). Ras-independent activation of Ral signaling via stimulation of formyl-Met-Leu-Phe receptor and dissociation of Ral-GDS from औ-arrestin has been shown to cause cytoskeletal rearrangement (24Bhattacharya M. Anborgh P.H. Babwah A.V. Dale L.B. Bobransky T. Benovic J.L. Feldman R.D. Verdi J.M. Rylett R.J. Ferguson S.S. Nat. Cell Biol. 2002; 8: 547-555Crossref Scopus (117) Google Scholar). The physiological consequence of RalGTP signaling is unknown. However, control of transcription could be one end point of the RalGTP signaling (22Goi T. Shipitsin M. Lu Z. Foster D.A. Klinz S.G. Feig L.A. EMBO J. 2000; 19: 623-630Crossref PubMed Scopus (116) Google Scholar, 25de Ruiter N.D. Burgering B.M. Bos J.L. Mol. Cell. Biol. 2001; 21: 8225-8235Crossref PubMed Scopus (81) Google Scholar, 26McCormick F. Mol. Reprod. Dev. 1995; 42: 500-506Crossref PubMed Scopus (98) Google Scholar, 27Franke B. Akkerman J.W. Bos J.L. EMBO J. 1997; 16: 252-259Crossref PubMed Scopus (364) Google Scholar, 28de Ruiter N.D. Wolthuis R.M. van Dam H. Burgering B.M. Bos J.L. Mol. Cell. Biol. 2000; 20: 8480-8488Crossref PubMed Scopus (61) Google Scholar, 29Wolthuis R.M. de Ruiter N.D. Cool R.H. Bos J.L. EMBO J. 1997; 16: 6748-6761Crossref PubMed Scopus (144) Google Scholar, 30Ouwens D.M. de Ruiter N.D. van der Zon G.C. Carter A.P. Schouten J. van der Burgt C. Kooistra K. Bos J.L. Maassen J.A. van Dam H. EMBO J. 2002; 21: 3782-3793Crossref PubMed Scopus (185) Google Scholar, 31Henry D.O. Moskalenko S.A. Kaur K.J. Fu M. Pestell R.G. Camonis J.H. White M.A. Mol. Cell. Biol. 2000; 20: 8084-8092Crossref PubMed Scopus (87) Google Scholar, 32Kops G.J. de Ruiter N.D. DeVeries-Smits A.M. Powell D.R. Bos J.L. Burgering B.M. Nature. 1999; 398: 630-634Crossref PubMed Scopus (947) Google Scholar). Ral activation in response to insulin induces phosphorylation of c-Jun transcription factor through activation of Jun N-terminal kinase and cellular Src (28de Ruiter N.D. Wolthuis R.M. van Dam H. Burgering B.M. Bos J.L. Mol. Cell. Biol. 2000; 20: 8480-8488Crossref PubMed Scopus (61) Google Scholar). Similarly, activated Ral-GTPase leads to phosphorylation of Stat3 (22Goi T. Shipitsin M. Lu Z. Foster D.A. Klinz S.G. Feig L.A. EMBO J. 2000; 19: 623-630Crossref PubMed Scopus (116) Google Scholar). Other reports suggest that expression of constitutive active Ral leads to activation of NF-κB and its downstream gene cyclin D1 (31Henry D.O. Moskalenko S.A. Kaur K.J. Fu M. Pestell R.G. Camonis J.H. White M.A. Mol. Cell. Biol. 2000; 20: 8084-8092Crossref PubMed Scopus (87) Google Scholar). Ras-dependent Ral signaling pathway leads to phosphorylation of Forkhead transcription factor on threonine residues 447 and 451, leading to its activation. The protein kinase involved in such phosphorylation is unknown (25de Ruiter N.D. Burgering B.M. Bos J.L. Mol. Cell. Biol. 2001; 21: 8225-8235Crossref PubMed Scopus (81) Google Scholar). Ras-dependent activation of RalGTP signaling has been implicated in cellular transformation; in addition, cells expressing constitutively active RalGTP show an enhanced growth rate and can form colonies in soft agar (15Urano T. Emkey R. Feig L.A. EMBO J. 1996; 16: 810-816Crossref Scopus (299) Google Scholar, 18White M.A. Vale T. Camonis J.H. Schaefer E. Wigler M.H. J. Biol. Chem. 1996; 271: 16439-16442Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar, 33Ward Y. Wang W. Woodhouse E. Linnoila I. Liotta L. Kelly K. Mol. Cell. Biol. 2001; 21: 5958-5969Crossref PubMed Scopus (134) Google Scholar, 34Rodriguez-Viciana P. Warne P. Khwaja A. Marte B. Pappin D. Ridley A. Downward J. Cell. 1997; 89: 457-467Abstract Full Text Full Text PDF PubMed Scopus (954) Google Scholar). RalGTP has been shown to bind mammalian Sec 5, a subunit of the exocyst complex. Inhibition of RalA binding to Sec 5 prevents filopod production by tumor necrosis factor α and interleukin-1 (35Sugihara K. Asano S. Tanaka K. Iwamatsu A. Okawa K. Ohta Y. Nat. Cell Biol. 2002; 4: 73-78Crossref PubMed Scopus (202) Google Scholar, 36Moskalenko S. Henry D.O. Rosse C. Mirey G. Camonis J.H. White M.A. Nat. Cell Biol. 2001; 4: 66-72Crossref Scopus (349) Google Scholar). Several Ral targets that provide clues in Ral guanine nucleotide exchange factor-induced signaling pathways have been identified. Ral has been shown to interact with phospholipase D and Arf, which suggests a role for Ral in phospholipase D-mediated vesicle transport and membrane trafficking (37Luo J.Q. Liu X. Frankel P. Rotunda T. Ramos M. Flom J. Jiang L.A. Feig L.A. Morris A.J. Kahn R.A. Foster D.A. Proc. Natl. Acad. Sci. 1998; 95: 3632-3637Crossref PubMed Scopus (119) Google Scholar, 38Jiang H. Luo J.Q. Urano T. Frankel P. Lu Z. Foster D.A. Fieg L.A. Nature. 1995; 378: 409-412Crossref PubMed Scopus (246) Google Scholar). Ral could also function in regulating the cytoskeleton through interaction with Ral-binding protein 1 (RalBP1) (22Goi T. Shipitsin M. Lu Z. Foster D.A. Klinz S.G. Feig L.A. EMBO J. 2000; 19: 623-630Crossref PubMed Scopus (116) Google Scholar, 23Hofer F. Berdeaux R. Martin G.S. Curr. Biol. 1998; 8: 839-842Abstract Full Text Full Text PDF PubMed Google Scholar, 24Bhattacharya M. Anborgh P.H. Babwah A.V. Dale L.B. Bobransky T. Benovic J.L. Feldman R.D. Verdi J.M. Rylett R.J. Ferguson S.S. Nat. Cell Biol. 2002; 8: 547-555Crossref Scopus (117) Google Scholar, 27Franke B. Akkerman J.W. Bos J.L. EMBO J. 1997; 16: 252-259Crossref PubMed Scopus (364) Google Scholar, 39Cantor S.B. Urano T. Feig L.A. Mol. Cell. Biol. 1995; 15: 4578-4584Crossref PubMed Scopus (260) Google Scholar, 40Park S.H. Weinberg R.A. Oncogene. 1995; 11: 2349-2355PubMed Google Scholar). RalBP1 (also known as RLIP76 or RIP1) is a Ral effector molecule and associates in epidermal growth factor receptor complexes with the tyrosine-phosphorylated proteins POB1 and Reps1 (41Ikeda M. Ishida O. Hinoi T. Kishida S. Kikuchi A. J. Biol. Chem. 1998; 273: 814-821Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar, 42Nakashima S. Morinaka K. Koyama S. Ikeda M. Kishida M. Okawa K. Iwamatsu A. Kishida S. Kikuchi A. EMBO J. 1999; 18: 3629-3642Crossref PubMed Scopus (185) Google Scholar, 43Yamaguchi A. Urano T. Goi T. Feig L.A. J. Biol. Chem. 1997; 272: 31230-31234Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). Reps1 can also bind to the adaptor proteins Crk and Grb2 (43Yamaguchi A. Urano T. Goi T. Feig L.A. J. Biol. Chem. 1997; 272: 31230-31234Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). The significance of or the signaling pathway leading from the RalGTP-RalBP1 interaction is not known, but RalBP1 contains a weak GTPase (GAP) activity for Cdc42 and Rac GTPases in vitro (39Cantor S.B. Urano T. Feig L.A. Mol. Cell. Biol. 1995; 15: 4578-4584Crossref PubMed Scopus (260) Google Scholar, 44Jullien-Flores V. Dorseuil O. Romero F. Letourneur F. Saragosti S. Berger R. Tavitian A. Gacon G. Camonis J.H. J. Biol. Chem. 1995; 270: 22473-22477Abstract Full Text Full Text PDF PubMed Scopus (286) Google Scholar). Ral has therefore been shown to affect cellular proliferation, receptor-mediated endocytosis, Src kinase activation, and phospholipase D activation (15Urano T. Emkey R. Feig L.A. EMBO J. 1996; 16: 810-816Crossref Scopus (299) Google Scholar, 17Feig L.A. Urano T. Cantor S. Trends Biochem. Sci. 1996; 21: 438-441Abstract Full Text PDF PubMed Scopus (179) Google Scholar, 37Luo J.Q. Liu X. Frankel P. Rotunda T. Ramos M. Flom J. Jiang L.A. Feig L.A. Morris A.J. Kahn R.A. Foster D.A. Proc. Natl. Acad. Sci. 1998; 95: 3632-3637Crossref PubMed Scopus (119) Google Scholar, 38Jiang H. Luo J.Q. Urano T. Frankel P. Lu Z. Foster D.A. Fieg L.A. Nature. 1995; 378: 409-412Crossref PubMed Scopus (246) Google Scholar, 39Cantor S.B. Urano T. Feig L.A. Mol. Cell. Biol. 1995; 15: 4578-4584Crossref PubMed Scopus (260) Google Scholar, 45Miller M.J. Prigent S. Kupperman E. Rioux L. Park S.H. Framisco J.R. White M.A. Rutkowski J.L. Meinkoth J.L. J. Biol. Chem. 1997; 272: 5600-5605Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 46Ohta Y. Suzuki N. Nakamura S. Hartwig J.H. Stossel T.P. Proc. Natl. Acad. Sci. 1999; 96: 2122-2128Crossref PubMed Scopus (368) Google Scholar).In these studies, we show that one pathway leading to HSF1 activation is mediated through the activation of the RalGTP signal transduction pathway. HSF1 interacts with a Ral effector molecule, RalBP1, bothin vivo and in vitro. The HSF1·RalBP1 complex dissociates upon Ral activation by heat shock, since RalGTP has a high affinity for the Ral binding domain of RalBP1, thus leading to HSF1 activation. We also show that the HSF1·RalBP1 heterocomplexes contain HSP90 and α-tubulin.DISCUSSIONIn this study, we show evidence that the Ral-binding protein RalBP1 interacts with HSF1 in vivo. The RalPB1-HSF1 interaction occurs at 37 °C, and RalBP1·HSF1 heterocomplexes dissociate after heat shock. We also find significantly high levels of HSP90 and α-tubulin in the RalBP1·HSF1 heterocomplexes. This extends the previous observations indicating that the monomeric, repressed form of HSF1 is in heterocomplexes with HSP90 (11Guo Y. Guettouche T. Fenna M. Boellmann F. Pratt W.B. Toft D.O. Smith D.F. Voellmy R. J. Biol. Chem. 2001; 276: 45791-45799Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar) and extends a previous observation that HSF1·HSP90 heterocomplexes are located on α-tubulin that is a component of the cytoskeleton. Under stress conditions, HSP90·HSF1 heterocomplexes dissociate, perhaps because of the affinity of HSP90 for denatured polypeptides (9Bharadwaj S. Ali A. Ovsenek N. Mol. Cell. Biol. 1999; 19: 8033-8041Crossref PubMed Scopus (145) Google Scholar, 11Guo Y. Guettouche T. Fenna M. Boellmann F. Pratt W.B. Toft D.O. Smith D.F. Voellmy R. J. Biol. Chem. 2001; 276: 45791-45799Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar). These results indicate that RalBP1·HSF1·HSP90·α-tubulin heterocomplexes receive signals from stresses that activate the Ral signaling pathway, which also leads to HSF1 activation. HSF1·HSP90 heterocomplexes are reported to be dynamic, and immunodepletion of HSP90 leads to activation of HSF1 (11Guo Y. Guettouche T. Fenna M. Boellmann F. Pratt W.B. Toft D.O. Smith D.F. Voellmy R. J. Biol. Chem. 2001; 276: 45791-45799Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar). We have not detected HSP70/HSC70 proteins in RalBP1· HSF1·HSP90·α-tubulin complexes, a result that is also consistent with previous studies indicating that the repressed form of HSF1 interacts only with HSP90 and not with HSP70 (11Guo Y. Guettouche T. Fenna M. Boellmann F. Pratt W.B. Toft D.O. Smith D.F. Voellmy R. J. Biol. Chem. 2001; 276: 45791-45799Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar). However, HSP70 interacts with the trimeric form of HSF1 together with HSP90 and multiple other co-chaperones during the HSF1 inactivation process; this interaction occurs in the nucleus (9Bharadwaj S. Ali A. Ovsenek N. Mol. Cell. Biol. 1999; 19: 8033-8041Crossref PubMed Scopus (145) Google Scholar, 10Zou J. Guo Y. Guettouche T. Smith D.F. Voellmy R. Cell. 1998; 94: 471-480Abstract Full Text Full Text PDF PubMed Scopus (925) Google Scholar, 12Satyal S.H. Chen D. Fox S.G. Kramer J.M. Morimoto R.I. Genes Dev. 1998; 12: 1962-1974Crossref PubMed Scopus (184) Google Scholar).We also show that the Ral signal transduction pathway is highly activated by heat shock. Activation of the Ral signaling pathway is associated with conversion of RalGDP to RalGTP and, because of the high affinity of RalGTP for RalBP1, the RalBP1·HSF1·HSP90·α-tubulin heterocomplexes can potentially dissociate, leading to release of HSF1 and allowing it to translocate into the nucleus. It is conceivable that RalBP1·HSF1·HSP90·α-tubulin heterocomplexes also contain protein kinases, and such a kinase could be activated upon activation of the Ral signaling pathway. This protein kinase could then phosphorylate and activate HSF1. Similarly to the Ras-Raf-Mek-extracellular signal-regulating kinase signaling pathway, activation of the Ral signaling pathway could also lead to activation of an as yet unknown protein kinase cascade. Since Ral is found not only in the plasma membrane but also in membrane vesicles, and Ras-independent activation of Ral is calcium-dependent (13Bielinske D.F. Pyun N.Y. Linko-Stentz K. Macara I. Fine R.E. Biochim. Biophys. Acta. 1993; 1151: 246-256Crossref PubMed Scopus (60) Google Scholar, 14Volknandt W. Pevsner J. Elferink L.A. Scheller R.H. FEBS Lett. 1993; 317: 53-56Crossref PubMed Scopus (39) Google Scholar, 15Urano T. Emkey R. Feig L.A. EMBO J. 1996; 16: 810-816Crossref Scopus (299) Google Scholar, 16Reuther G.W. Der C.J. Curr. Opin. Cell Biol. 2000; 12: 157-165Crossref PubMed Scopus (345) Google Scholar, 17Feig L.A. Urano T. Cantor S. Trends Biochem. Sci. 1996; 21: 438-441Abstract Full Text PDF PubMed Scopus (179) Google Scholar, 23Hofer F. Berdeaux R. Martin G.S. Curr. Biol. 1998; 8: 839-842Abstract Full Text Full Text PDF PubMed Google Scholar), we hypothesize that activation of calcium-dependent protein kinases such as, for example, protein kinase C, protein kinase G, or calcium/calmodulin-dependent protein kinase, could phosphorylate and activate HSF1. Calcium/calmodulin-dependent protein kinase has been implicated in phosphorylation of HSF1 on serine 230, leading to its activation (8Holmberg C.I. Hietakangas V. Mikhailov A. Rantanen J.O. Kallio M. Meinander A. Hellman J. Morrice N. Mackintosh C. Morimoto R.I. Eriksson J.E. Sistonen L. EMBO J. 2001; 20: 3800-3810Crossref PubMed Scopus (248) Google Scholar); however, more evidence is required to implicate this or any other enzyme in the pathway.Similar to the other small GTP binding proteins Ras and Rac, no protein kinase has been discovered to be downstream of the Ral signal transduction pathway. Ral, through its interacting partners, has been implicated in multiple cellular processes such as endocytosis, actin/cytoskeletal organization, and vesicle function (17Feig L.A. Urano T. Cantor S. Trends Biochem. Sci. 1996; 21: 438-441Abstract Full Text PDF PubMed Scopus (179) Google Scholar, 41Ikeda M. Ishida O. Hinoi T. Kishida S. Kikuchi A. J. Biol. Chem. 1998; 273: 814-821Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar, 43Yamaguchi A. Urano T. Goi T. Feig L.A. J. Biol. Chem. 1997; 272: 31230-31234Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 53Quaroni A. Paul E.C.A. J. Cell Sci. 1999; 112: 707-718Crossref PubMed Google Scholar). The significance of the RalBP1 binding to the active form of RalGTP is not understood. RalBP1 contains a weak GTPase activity toward Cdc42 and Rac, which influences the actin cytoskeleton and can modulate the Jun N-terminal kinase signaling pathway (33Ward Y. Wang W. Woodhouse E. Linnoila I. Liotta L. Kelly K. Mol. Cell. Biol. 2001; 21: 5958-5969Crossref PubMed Scopus (134) Google Scholar, 39Cantor S.B. Urano T. Feig L.A. Mol. Cell. Biol. 1995; 15: 4578-4584Crossref PubMed Scopus (260) Google Scholar, 40Park S.H. Weinberg R.A. Oncogene. 1995; 11: 2349-2355PubMed Google Scholar, 44Jullien-Flores V. Dorseuil O. Romero F. Letourneur F. Saragosti S. Berger R. Tavitian A. Gacon G. Camonis J.H. J. Biol. Chem. 1995; 270: 22473-22477Abstract Full Text Full Text PDF PubMed Scopus (286) Google Scholar, 54Scita G. Tenca P. Frittoli E. Tocchetti A. Innocenti M. Giardina G. Di Fiore P.P. EMBO J. 2000; 19: 2393-2398Crossref PubMed Google Scholar). The RalGTP-RalBP1 interaction represses the activity of Rac. Since Jun N-terminal kinase has been shown to be activated by heat shock (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) and it has been suggested that it also phosphorylates HSF1 and represses its transcriptional activity during its inactivation cycle (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), slowing down Rac activation and similarly slowing down activation of Jun N-terminal kinase after heat shock could perhaps provide a sufficient amount of time for a rapid activation of HSF1 and transcription of downstream genes followed by its subsequent inactivation by phosphorylation by Jun N-terminal kinase and perhaps other protein kinases. RalBP1 has also been shown to interact with the ॖ2 subunit of AP2, which is involved in endocytosis (49Jullien-Flores V. Mahe Y. Mirey G. Leprince C. Meunier-Bisceuil B. Sorkin A. Camonis J.H. J. Cell Sci. 2000; 113: 2837-2844Crossref PubMed Google Scholar,55Pishvaee B. Payne G.S. Cell. 1998; 95: 443-446Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). Therefore, active RalGTP has been suggested to stabilize association of ॖ2-RalBP1 with the membrane, leading to inhibition of clathrin-dependent endocytosis of certain receptors such as epidermal growth factor (49Jullien-Flores V. Mahe Y. Mirey G. Leprince C. Meunier-Bisceuil B. Sorkin A. Camonis J.H. J. Cell Sci. 2000; 113: 2837-2844Crossref PubMed Google Scholar).Interestingly, RalBP1 has a Ral binding region, and this region of RalBP1 partially overlaps with the region that binds HSF1. This suggests that Ral and HSF1 could compete for a binding site on RalBP1. However, the HSF1 binding region in RalBP1 requires a number of amino acids that extend to the POB1 binding region. The exact boundaries of the Ral binding region and POB1 binding region in RalBP1, however, has not been fully defined. POB1 (partner of RalBP1) is a protein with unknown function. It contains an Eps15 homology domain, and proteins with such domains appear to be involved in clathrin-dependent endocytosis (56Chen H. Fre S. Slepnev V.I. Capua M.R. Takei K. Butler M.H. Di Fiore P.P. De Camilli P. Nature. 1998; 394: 793-797Crossref PubMed Scopus (270) Google Scholar). RalBP1 and POB1 may link the tyrosine kinase and Src homology 3-containing proteins to Ral. We also have further found that RalBP1 colocalizes and is in the same complex with α-tubulin. Furthermore, HSF1 and HSP90 also colocalize to this same complex. This is significant, since these results locate the repressed HSF1 on the cytoskeletal proteins that have been shown to undergo major morphological changes upon heat stress (47Liang P. MacRae T.H. J. Cell Sci. 1997; 110: 1431-1440Crossref PubMed Google Scholar). We further show that the Ral signaling pathway is activated by heat shock, and transient transfection of the constitutively active form of Ral (RalA23V) increases HSP70 expression in both H1299 cells, which contain endogenous HSF1, andhsf1−/− MEF cells in which HSF1 was transiently expressed. These results strongly suggest that activation of the Ral signaling pathway increases RalGTP binding to RalBP1, and therefore, there is less RalBP1 that is present in cells to bind and repress HSF1. Similarly, transient transfection of RalBP1 inhsf1−/− MEF reduced HSF1-mediated HSP70 expression.In conclusion, we show that HSF1 interacts with RalBP1 in vivo at 37 °C. HSF1·RalBP1 heterocomplexes also contain HSP90 and α-tubulin. Upon heat shock, HSF1 dissociates from RalBP1 and translocates into the nucleus. This coincides with the activation of the Ral-GTP signaling pathway as shown by an increase in Ral-GTP binding to the Ral binding domain of RalBP1. Expression of constitutive active RalGTP leads to an increase in HSP70 e" @default.
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