Regulation of molecular chaperone gene transcription involves the serine phosphorylation, 14-3-3ε binding, and cytoplasmic sequestration of heat shock factor 1

Heat shock transcription factor 1 (HSF1) is the mammalian regulator of the heat shock response and activates the transcription of heat shock protein (Hsp) molecular chaperone genes (43, 47, 49). Inactivation of the murine hsf1gene has been shown to confer a complex phenotype, indicating an essential function for hsf1 in growth, in development, and in acute response to stress (35). Disruption of hsf1 (i.e., hsf1−/−) in mouse embryonic fibroblasts leads to a profound loss of thermotolerance and markedly increased susceptibility to heat-induced apoptosis (11, 35). hsf1 is required as a maternal factor during the early cleavage stage of development in the −/− mouse embryo (11). hsf1-deficient mice can survive to adulthood but display severe defects in the chorioallantoic placenta that result in increased prenatal lethality (56). In addition, the aging process is associated with degeneration of the heat shock response, and transcriptional activity of HSF1 protein was significantly reduced with age in a cell-free system, as well as in isolated hepatocytes (26). Understanding the processes involved in HSF1 regulation may therefore aid in delineating its role in resistance to stress, development, and aging. Under normal conditions, cellular HSF1 exists in a predominantly transcriptionally repressed state (44, 59). Such HSF1 is monomeric, is constitutively phosphorylated, and lacks the ability to bind the cis-acting heat shock elements (HSEs) located in the promoters of Hsp genes (50, 55). Induction of transcriptional activity by heat shock then results in the conversion of HSF1 from inactive monomer to a DNA-binding trimer (44, 46, 53). Activation of HSF1 is a multistep process, involving trimerization, acquisition of HSE-binding activity, novel phosphorylation, and transactivation of Hsp genes (38, 50, 55). Trimerization of HSF1 is governed by leucine zipper domains in the N terminus and is subject to intramolecular negative regulation by a fourth leucine zipper domain in the C terminus (44). The molecular chaperone Hsp90 functions as the principal cellular repressor of HSF1 in unstressed cells and plays a major role in retaining HSF1 in an inactive state; HSF1 trimerization is accompanied by the sequestration of Hsp90 in protein aggregates and escape from Hsp90-containing HSF1 complexes in response to stress (59). Hsp70, is also important, but not sufficient, to negatively regulate HSF1 activity in the absence of stress (1, 5). HSF1 also appears to have additional layers of regulation in the cell (25, 42). Our studies, as well as the work of others, have demonstrated the hierarchical phosphorylation of human HSF1 within its transcriptional regulatory domain by extracellular signal-regulated kinase 1 (ERK1; on serine 307) and by glycogen synthase kinase 3 (GSK3; on serine 303) (12, 13, 23, 29, 30). The regulatory domain of HSF1 functions as a molecular switch coupling hsp gene transcription to cellular conditions, repressing C-terminal transactivation domains under growth conditions, and under stress conditions causing powerful stimulation of the same activation domains (22, 30, 40). In the present study, we have examined how the regulatory domain responds to extracellular conditions. Since phosphorylation can directly regulate distinct aspects of transcription factor function, including subcellular localization, protein stability, protein-protein interactions, DNA binding, and transactivation, we have examined the role of phosphorylation in the activity of the regulatory domain (14, 54). Our previous studies indicate that the protein kinases ERK1 and GSK3 phosphorylate HSF1 on serine residues within a proline-rich region (RVKEEPPS303PPQS307PRV) of the regulatory domain (12, 13). Many recent studies show that phosphorylation on serine and threonine can be converted into an intracellular signal by association of the phosphorylated domain with regulatory proteins that recognize serine/threonine phosphorylated domains (14). The first such proteins to be identified were the highly conserved 14-3-3 family, which bind to a wide array of cellular proteins largely through recognition of phosphoserine-containing domains (2, 57). At least seven 14-3-3 genes exist in vertebrates, and these give rise to nine protein isoforms (α, β, δ, ɛ, γ, η, σ, τ, and ζ) (3). 14-3-3 proteins are found largely as dimers within the cell and are able to bind either to multiple sites within proteins such as c-Raf1 or to act as a bridge, with one 14-3-3 dimer binding to two different proteins (20). 14-3-3 dimers can thus act as molecular scaffold proteins, bringing together proteins that interact functionally and effecting phosphorylation-dependent cell regulation (20). These highly abundant proteins are thus involved in key cellular processes, such as signal transduction, cell cycle control, and apoptosis (20, 34, 57, 58). In the present study, we investigated the potential role of 14-3-3 in the function of the transcriptional regulatory domain of HSF1. We show for the first time that HSF1 can bind to 14-3-3. In addition, we have demonstrated that extracellular ERK activation by mitogenic stimulation, a process that controls phosphorylation of serines 303 and 307 of HSF1, leads to HSF1 association with 14-4-3 species, including 14-3-3ɛ and 14-3-3ζ. In addition, inhibiting the ERK cascade with the specific MEK1 inhibitor PD98059 in vivo strongly suppresses the binding of HSF1 to 14-3-3ɛ. We next demonstrated the direct in vitro binding of 14-3-3ɛ to synthetic HSF1-derived peptides phosphorylated at serines 303 and 307. Our studies further showed that 14-3-3ɛ binding inhibits both the transcriptional activity and the nuclear accumulation of HSF1 in HeLa cells at 37°C. These effects of 14-3-3ɛ on nuclear exclusion and transactivation of HSF1 required serines 307 and 303 within the regulatory domain. Leptomycin B (LMB), specific inhibitor of nuclear export receptor CRM1, blocked the cytoplasmic localization of HSF1 in 14-3-3ɛ-overexpressing cells. These experiments suggest that 14-3-3ɛ mediates interaction between phospho-S307-S303-HSF1 and CRM1 in either a direct or an indirect manner and thus contributes to cytoplasmic sequestration and transcriptional repression in response to ERK stimulation. Our experiments therefore indicate a pathway whereby HSF1 activity and molecular chaperone expression are regulated at the intracellular level by protein kinases and within larger biological systems through the mediation of extracellular factors.