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 HSF1⅐HSP90 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 heatinducible expression of HSP70, whereas the dominant negative form (RalA28N) suppresses HSP70 expression. We further find that ␣-tubulin and HSP90 are also present in the RalBP1⅐HSF1 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 RalBP1⅐HSF1⅐HSP90⅐␣-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.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 Ser 303 , Ser 307 , and Ser 363 is likely to be involved in repression of HSF1 transcriptional activity (1-7). 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 Ser 230 by calcium calmodulin protein kinase II (8). HSF1 is also found in multichaperone complexes under physiological conditions and during its repression (9 -11). Specifically, HSP90 has been coimmunoprecipitated with the monomeric form of HSF1, suggesting that an HSP90⅐HSF1 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 HSF1⅐HSP90 heterocomplexes during stress is the accumulation of denatured polypeptides and the ability of HSP90 to bind such denatured polypeptides (9 -11). During recovery from stress, HSF1 trimers h...

Self-regulatory role of 4-hydroxynonenal in signaling for stress-induced programmed cell death

“…This is indicated by our unpublished studies showing that in response to stress, their functions are mutually regulated through RalBP1-mediated sequestration of HSF1 into cytoskeleton and HSF1-mediated inhibition of the transport activity of RalBP1 toward glutathione conjugates such as GS-HNE. Thus HNE, being a determinant of GS-HNE concentrations, RalBP1-mediated transport activity can regulate the transcription of HSF1-associated genes [108,109].…”

Association and Regulation of Heat Shock Transcription Factor 4b with both Extracellular Signal-Regulated Kinase Mitogen-Activated Protein Kinase and Dual-Specificity Tyrosine Phosphatase DUSP26

“…Transfected DNA mixes included 4 to 8 g of expression plasmid DNA and, when required, empty plasmid DNA added to a total of 8 g. The DNA mix was added to 5 ϫ10 5 to 6 ϫ10 5 cells. The transfection efficiency varied between 60 to 70% in all experiments, as determined by immunofluorescence analysis (22).…”

“…For yeast two-hybrid screening, we used a CytoTrap two-hybrid system (Stratagene) as described previously (3,22). The fulllength human Hsf4b was subcloned into the pSos vector and was used to screen a human heart tissue cDNA library that had been subcloned into the pMyr vector.…”

“…The signaling pathways regulating heat shock factors are complex and only partially understood. Hsf1 is phosphorylated by multiple protein kinases (extracellular signal-related kinase [ERK], c-Jun NH 2 -terminal kinase [JNK], p38, calcium calmodulin protein kinase, and perhaps others) (11-13, 19, 20, 26, 27, 46, 48) and interacts with many partner proteins (Hsp90, Hsp70, 14-3-3, RalBP1, and HSBP1) (1,5,22,38). Hsf2 is not phosphorylated and interacts with different sets of proteins such as protein phosphatase 2A (21).…”

REPS2 (RALBP1 associated Eps domain containing 2)

“…When prostate cancer cell lines are transfected with a deletion mutant of REPS2/POB1, lacking the RalBP1 binding domain, only 30-40% of the transfected cells became apoptotic after 72-96 hours (Oosterhoff et al, 2003). REPS2/POB1 (514-660) binds RalBP1 C-terminal amino acids, 499-655, while an almost overlapping region of RalBP1 (440-655) binds the heat shock factor 1 Hsf-1 (Hu and Mivechi, 2003). Shingal et al show a ternary complex formation between RalBP1, Hsf-1, and REPS2/POB1.…”