Phosphorylation of serine 230 promotes inducible transcriptional activity of heat shock factor 1

Abstract

Heat shock factor 1 (HSF1) is a serine-rich constitutively phosphorylated mediator of the stress response. Upon stress, HSF1 forms DNA-binding trimers, relocalizes to nuclear granules, undergoes inducible phosphorylation and acquires the properties of a transactivator. HSF1 is phosphorylated on multiple sites, but the sites and their function have remained an enigma. Here, we have analyzed sites of endogenous phosphorylation on human HSF1 and developed a phosphopeptide antibody to identify Ser230 as a novel in vivo phosphorylation site. Ser230 is located in the regulatory domain of HSF1, and promotes the magnitude of the inducible transcriptional activity. Ser230 lies within a consensus site for calcium/calmodulin-dependent protein kinase II (CaMKII), and CaMKII overexpression enhances both the level of in vivo Ser230 phosphorylation and transactivation of HSF1. The importance of Ser230 was further established by the S230A HSF1 mutant showing markedly reduced activity relative to wild-type HSF1 when expressed in hsf1(-/-) cells. Our study provides the first evidence that phosphorylation is essential for the transcriptional activity of HSF1, and hence for induction of the heat shock response.

Fig. 1. Heterogeneous in vivo phosphorylation of HSF1. K562 cells were in vivo labeled with [³²P]orthophosphate for 3 h before they were subjected to heat shock (HS) or left untreated (C). HSF1 was immunoprecipitated with anti-hHSF1 antibodies and resolved on 8% SDS–PAGE. (A) Autoradiograph of the immunoprecipitated HSF1. The asterisk indicates unknown phosphoprotein. (B) Phosphoamino acid analysis of the immunoprecipitated HSF1. The relative positions of phosphoSer, phosphoThr and phosphoTyr are indicated. (C) Tryptic phosphopeptide mapping of HSF1. The black arrowhead indicates a new phosphopeptide detected upon heat shock, and the white arrowheads indicate phosphopeptides, the intensity of which is markedly enhanced upon heat stress. Phosphopeptide-1, -2 and -z are explained in Results.

Fig. 2. Identification of Ser230 phosphorylation of HSF1 in vivo. (A) Manual Edman degradation was performed on the phospho peptide-1 and -2 derived from in vivo ³²P-labeled endogenous HSF1 (Figure 1C). The cycle of ³²P release, the corresponding phospho peptide sequence and the phosphorylated residues are shown. (B) Schematic representation of human HSF1. The position of Ser230 in the regulatory domain is indicated. DBD, DNA-binding domain; HR-A/B and -C, hydrophobic repeat A/B and C; RD, regulatory domain; AD, activation domain.

Fig. 3. CaMKII phosphorylates rhHSF1 on Ser230. (A) Autoradiograph of rhHSF1 phosphorylated in vitro by CaMKII. (B) Tryptic phosphopeptide mapping of immunoprecipitated ³²P-labeled HSF1 from heat-shocked K562 cells (HS), or of rhHSF1 phosphorylated in vitro by CaMKII. The arrows mark phosphopeptides with similar migration patterns in vivo and in vitro. (C) Tryptic phosphopeptide mapping of mutant HSF1. K562 cells were transfected with wild-type or S230A HSF1-Myc-His, in vivo labeled with [³²P]orthophosphate, and subjected to heat shock. HSF1 was immuno precipitated with anti-Myc antibodies followed by tryptic phosphopeptide mapping. The arrowheads represent phosphopeptides that are absent in S230A.

Fig. 3. CaMKII phosphorylates rhHSF1 on Ser230. (A) Autoradiograph of rhHSF1 phosphorylated in vitro by CaMKII. (B) Tryptic phosphopeptide mapping of immunoprecipitated ³²P-labeled HSF1 from heat-shocked K562 cells (HS), or of rhHSF1 phosphorylated in vitro by CaMKII. The arrows mark phosphopeptides with similar migration patterns in vivo and in vitro. (C) Tryptic phosphopeptide mapping of mutant HSF1. K562 cells were transfected with wild-type or S230A HSF1-Myc-His, in vivo labeled with [³²P]orthophosphate, and subjected to heat shock. HSF1 was immuno precipitated with anti-Myc antibodies followed by tryptic phosphopeptide mapping. The arrowheads represent phosphopeptides that are absent in S230A.

Fig. 3. CaMKII phosphorylates rhHSF1 on Ser230. (A) Autoradiograph of rhHSF1 phosphorylated in vitro by CaMKII. (B) Tryptic phosphopeptide mapping of immunoprecipitated ³²P-labeled HSF1 from heat-shocked K562 cells (HS), or of rhHSF1 phosphorylated in vitro by CaMKII. The arrows mark phosphopeptides with similar migration patterns in vivo and in vitro. (C) Tryptic phosphopeptide mapping of mutant HSF1. K562 cells were transfected with wild-type or S230A HSF1-Myc-His, in vivo labeled with [³²P]orthophosphate, and subjected to heat shock. HSF1 was immuno precipitated with anti-Myc antibodies followed by tryptic phosphopeptide mapping. The arrowheads represent phosphopeptides that are absent in S230A.

Fig. 4. Characterization of in vivo phosphorylated Ser230 on hHSF1 with phosphopeptide-specific antibodies. (A) Whole-cell extracts (60 µg) of untreated (C) and heat-shocked (HS) K562 cells, as well as rhHSF1 (5 ng) and rhHSF1 phosphorylated in vitro by CaMKII (pS230-rhHSF1, 5 ng) were resolved on 8% SDS–PAGE and immunoblotted with the anti-pS230-hHSF1 antibody (–). The antibody was pre-incubated with free phosphopeptide for competitive inhibition of the immunoreaction (+). (B) HeLa cells were exposed to heat shock (HS) and CdSO₄ (100 µM) for the indicated time periods or were left untreated (C). Whole-cell extracts were analyzed by western blotting using antibodies to phosphorylated Ser230 (pS230-HSF1), HSF1, Hsp70 and Hsc70 (left panel). The HSF1 DNA-binding activity in the whole-cell extracts was examined by gel mobility shift assay (right panel). HSF, HSF1–HSE complex; NS, non-specific DNA-binding activity. (C) Whole-cell extracts from HeLa cells exposed to heat shock (HS) were incubated with anti-pS230-hHSF1 (α-pS230), anti-hHSF1 (α-hHSF1), anti-HSF2 (α-HSF2) antibodies or with pre-immune serum (PS) prior to the gel mobility shift assay. (D) HeLa cells were heat shocked (HS) or kept at 37°C (control) and the immunofluorescence analysis was performed using an anti-pS230-hHSF1 (pS230, green) and anti-hHSF1 (HSF1, red) antibody. The DNA was stained with DAPI. The arrows indicate HSF1 nuclear granules.

Fig. 4. Characterization of in vivo phosphorylated Ser230 on hHSF1 with phosphopeptide-specific antibodies. (A) Whole-cell extracts (60 µg) of untreated (C) and heat-shocked (HS) K562 cells, as well as rhHSF1 (5 ng) and rhHSF1 phosphorylated in vitro by CaMKII (pS230-rhHSF1, 5 ng) were resolved on 8% SDS–PAGE and immunoblotted with the anti-pS230-hHSF1 antibody (–). The antibody was pre-incubated with free phosphopeptide for competitive inhibition of the immunoreaction (+). (B) HeLa cells were exposed to heat shock (HS) and CdSO₄ (100 µM) for the indicated time periods or were left untreated (C). Whole-cell extracts were analyzed by western blotting using antibodies to phosphorylated Ser230 (pS230-HSF1), HSF1, Hsp70 and Hsc70 (left panel). The HSF1 DNA-binding activity in the whole-cell extracts was examined by gel mobility shift assay (right panel). HSF, HSF1–HSE complex; NS, non-specific DNA-binding activity. (C) Whole-cell extracts from HeLa cells exposed to heat shock (HS) were incubated with anti-pS230-hHSF1 (α-pS230), anti-hHSF1 (α-hHSF1), anti-HSF2 (α-HSF2) antibodies or with pre-immune serum (PS) prior to the gel mobility shift assay. (D) HeLa cells were heat shocked (HS) or kept at 37°C (control) and the immunofluorescence analysis was performed using an anti-pS230-hHSF1 (pS230, green) and anti-hHSF1 (HSF1, red) antibody. The DNA was stained with DAPI. The arrows indicate HSF1 nuclear granules.

Fig. 4. Characterization of in vivo phosphorylated Ser230 on hHSF1 with phosphopeptide-specific antibodies. (A) Whole-cell extracts (60 µg) of untreated (C) and heat-shocked (HS) K562 cells, as well as rhHSF1 (5 ng) and rhHSF1 phosphorylated in vitro by CaMKII (pS230-rhHSF1, 5 ng) were resolved on 8% SDS–PAGE and immunoblotted with the anti-pS230-hHSF1 antibody (–). The antibody was pre-incubated with free phosphopeptide for competitive inhibition of the immunoreaction (+). (B) HeLa cells were exposed to heat shock (HS) and CdSO₄ (100 µM) for the indicated time periods or were left untreated (C). Whole-cell extracts were analyzed by western blotting using antibodies to phosphorylated Ser230 (pS230-HSF1), HSF1, Hsp70 and Hsc70 (left panel). The HSF1 DNA-binding activity in the whole-cell extracts was examined by gel mobility shift assay (right panel). HSF, HSF1–HSE complex; NS, non-specific DNA-binding activity. (C) Whole-cell extracts from HeLa cells exposed to heat shock (HS) were incubated with anti-pS230-hHSF1 (α-pS230), anti-hHSF1 (α-hHSF1), anti-HSF2 (α-HSF2) antibodies or with pre-immune serum (PS) prior to the gel mobility shift assay. (D) HeLa cells were heat shocked (HS) or kept at 37°C (control) and the immunofluorescence analysis was performed using an anti-pS230-hHSF1 (pS230, green) and anti-hHSF1 (HSF1, red) antibody. The DNA was stained with DAPI. The arrows indicate HSF1 nuclear granules.

Fig. 4. Characterization of in vivo phosphorylated Ser230 on hHSF1 with phosphopeptide-specific antibodies. (A) Whole-cell extracts (60 µg) of untreated (C) and heat-shocked (HS) K562 cells, as well as rhHSF1 (5 ng) and rhHSF1 phosphorylated in vitro by CaMKII (pS230-rhHSF1, 5 ng) were resolved on 8% SDS–PAGE and immunoblotted with the anti-pS230-hHSF1 antibody (–). The antibody was pre-incubated with free phosphopeptide for competitive inhibition of the immunoreaction (+). (B) HeLa cells were exposed to heat shock (HS) and CdSO₄ (100 µM) for the indicated time periods or were left untreated (C). Whole-cell extracts were analyzed by western blotting using antibodies to phosphorylated Ser230 (pS230-HSF1), HSF1, Hsp70 and Hsc70 (left panel). The HSF1 DNA-binding activity in the whole-cell extracts was examined by gel mobility shift assay (right panel). HSF, HSF1–HSE complex; NS, non-specific DNA-binding activity. (C) Whole-cell extracts from HeLa cells exposed to heat shock (HS) were incubated with anti-pS230-hHSF1 (α-pS230), anti-hHSF1 (α-hHSF1), anti-HSF2 (α-HSF2) antibodies or with pre-immune serum (PS) prior to the gel mobility shift assay. (D) HeLa cells were heat shocked (HS) or kept at 37°C (control) and the immunofluorescence analysis was performed using an anti-pS230-hHSF1 (pS230, green) and anti-hHSF1 (HSF1, red) antibody. The DNA was stained with DAPI. The arrows indicate HSF1 nuclear granules.

Fig. 5. CaMKII increases heat-induced transactivation and phosphorylation of Ser230 on HSF1. K562 cells were cotransfected with a plasmid coding for constitutively active CaMKII and an LSN reporter construct. The transfectants were exposed to heat shock followed by 6 h recovery at 37°C (R) or were left untreated (C). Mock, cells cotransfected with an empty vector and the reporter plasmid. (A) The CAT assay was quantified and the CAT activity in untreated mock transfectants was assigned a fold induction of 1. The data represent mean ± SEM from two independent experiments performed in duplicate. (B) Whole-cell extracts were prepared from the cells described above and analyzed by western blotting using the anti-pS230-hHSF1 (upper panel) and anti-hHSF1 (lower panel) antibody. The asterisk indicates an unknown protein band. (C) The HSF1 DNA-binding activity in the transfectants exposed to heat shock (HS) was examined by gel mobility shift assay. (D) HeLa cells were transfected with the LSN plasmid and exposed to heat shock followed by 6 h recovery at 37°C in the absence (R) or presence of KN-62 (KN+R, 10 µM KN-62 was added 15 min prior to heat shock) or KN-62 alone (KN). The data represent mean ± SEM from three independent experiments performed in duplicate. *p <0.05 when compared with the heat-shocked cells (R).

Fig. 5. CaMKII increases heat-induced transactivation and phosphorylation of Ser230 on HSF1. K562 cells were cotransfected with a plasmid coding for constitutively active CaMKII and an LSN reporter construct. The transfectants were exposed to heat shock followed by 6 h recovery at 37°C (R) or were left untreated (C). Mock, cells cotransfected with an empty vector and the reporter plasmid. (A) The CAT assay was quantified and the CAT activity in untreated mock transfectants was assigned a fold induction of 1. The data represent mean ± SEM from two independent experiments performed in duplicate. (B) Whole-cell extracts were prepared from the cells described above and analyzed by western blotting using the anti-pS230-hHSF1 (upper panel) and anti-hHSF1 (lower panel) antibody. The asterisk indicates an unknown protein band. (C) The HSF1 DNA-binding activity in the transfectants exposed to heat shock (HS) was examined by gel mobility shift assay. (D) HeLa cells were transfected with the LSN plasmid and exposed to heat shock followed by 6 h recovery at 37°C in the absence (R) or presence of KN-62 (KN+R, 10 µM KN-62 was added 15 min prior to heat shock) or KN-62 alone (KN). The data represent mean ± SEM from three independent experiments performed in duplicate. *p <0.05 when compared with the heat-shocked cells (R).

Fig. 6. S230A has diminished transcriptional activity. (A) K562 cells were transfected with wild-type or mutant GAL4-HSF1, the G₅BCAT reporter plasmid and the internal control RSVβ-gal. The transfectants were exposed to heat shock followed by 3 h recovery (+). Untreated transfectants are denoted by (–). The CAT activity in untreated wild-type GAL4-HSF1 transfectants was assigned a fold induction of 1. The data represent mean ± SEM from two independent experiments performed in duplicate. (B–D) hsf1^–/– MEFs (–/–) were transfected with wild-type (hHSF1) or S230A (S230A) hHSF1-Myc-His plasmids. +/+, MEFs containing endogenous HSF1. The cells were exposed to heat shock (HS), heat shock followed by 3 h recovery (R) or were left untreated (C). (B) Western blot analysis using antibodies against HSF1, Hsp70 and Hsc70. Note that the HSF1 blot of +/+ was exposed for a longer time than the HSF1 blot of –/– cells. For quantification, the Hsp70 level in wild-type hHSF1 transfectants was assigned to 100%. The data represent mean ± SEM from three independent experiments. (C) Western blotting using the anti-pS230-hHSF1 (upper panel) and anti-hHSF1 (lower panel) antibody. (D) Analysis of the HSF1 DNA-binding activity by gel mobility shift assay.

Fig. 6. S230A has diminished transcriptional activity. (A) K562 cells were transfected with wild-type or mutant GAL4-HSF1, the G₅BCAT reporter plasmid and the internal control RSVβ-gal. The transfectants were exposed to heat shock followed by 3 h recovery (+). Untreated transfectants are denoted by (–). The CAT activity in untreated wild-type GAL4-HSF1 transfectants was assigned a fold induction of 1. The data represent mean ± SEM from two independent experiments performed in duplicate. (B–D) hsf1^–/– MEFs (–/–) were transfected with wild-type (hHSF1) or S230A (S230A) hHSF1-Myc-His plasmids. +/+, MEFs containing endogenous HSF1. The cells were exposed to heat shock (HS), heat shock followed by 3 h recovery (R) or were left untreated (C). (B) Western blot analysis using antibodies against HSF1, Hsp70 and Hsc70. Note that the HSF1 blot of +/+ was exposed for a longer time than the HSF1 blot of –/– cells. For quantification, the Hsp70 level in wild-type hHSF1 transfectants was assigned to 100%. The data represent mean ± SEM from three independent experiments. (C) Western blotting using the anti-pS230-hHSF1 (upper panel) and anti-hHSF1 (lower panel) antibody. (D) Analysis of the HSF1 DNA-binding activity by gel mobility shift assay.

Fig. 7. Model of phosphorylation-mediated modulation of the magnitude of hHSF1 transcriptional activity. Based on the phosphorylation sites identified to date, HSF1 contains an activating site S230 (green) and three repressing sites, S303, S307 and S363 (red). Among these sites, the serines 230, 303 and 307 are localized in the regulatory domain of the factor. Upon stress, the Ser230 phosphorylation is enhanced, and the molar ratio between the activating and the repressing sites determines the magnitude of the transcriptional activity. Thickness of the arrow indicates degree of transcriptional activity. The transactivating potency is conferred by a heterogeneous population of phosphorylated HSF1. A modest transcriptional activity is acquired upon stress in the absence of Ser230 phosphorylation, which is likely to be mediated by the as yet uncharacterized inducible and constitutive phosphorylation sites. Ser230 phosphorylation is not needed for the DNA-binding activity of HSF1.