Overexpression of 14-3-3ɛ inhibits HSF1-HSE binding activity. HeLa cells were transfected with empty expression vector pcDNA3.1 (lane 4), with pcDNA3.1 HSF1 alone (lanes 5 and 6), or with pcDNA3.1 HSF1 and pHA-14-3-3ɛ (lanes 7, 8, and 9). Nuclear extracts were then prepared after 24 h of transfection, and EMSA was carried out by using a ³²P-labeled consensus HSE from the HSP70B promoter. Gel supershift assay with anti-HSF1 antibody was carried out as described earlier (52) (lanes 3, 6, and 8). No nuclear extract was added to the incubation in control lane 1. Nuclear extracts from heat-shocked (HS) HeLa cells (lanes 2 and 3) were used as positive controls to demonstrate the migration of HSF-HSE complexes. An arrow indicates the position of HSF1-HSE complex. ³²P-labeled OCT-1 was used as a control to confirm the loading of equal amounts of nuclear extracts (4 μg) in lanes 2 to 9 in the incubations. Quantitation of HSF1-HSE binding is shown below the panels. The experiment was carried out twice times with similar findings.

Mutations in the ERK1 and GSK3 phosphorylation sites (Ser-307 and Ser-303) on HSF1 block 14-3-3ɛ-mediated nuclear exclusion of HSF1. (A) HeLa cells expressing the HSF1-GFP serine mutant proteins as indicated without (rows 1 to 3) or with HA-14-3-3ɛ coexpression (rows 4 to 6) were stained with an anti-HA antibody to localize cotransfected HA-14-3-3ɛ and with DAPI to visualize the nuclei. Fixed cells were then analyzed for green autofluorescence (HSF1), red immunofluorescence (14-3-3ɛ), or blue fluorescence (nuclear stain) as described in Fig. 4A. The location of the cells was visualized by phase-contrast microscopy. Magnification, ×400. (B) Relative equilibration of HSF1wt-GFP and the serine mutants between nuclear (N), nuclear-cytoplasmic (N&C), and strictly cytoplasmic (C) distributions in HeLa cells was quantitated as described in Fig. 4D. Microscopic analysis was carried out as in panel A. The values shown in the histograms are the means ± the SDs from three separate experiments. (C) Effect of 14-3-3ɛ expression on relative localization of HSF1wt-GFP and the HSF1 serine-glycine mutants in HeLa cells. The experiment was performed as described in Fig. 4E. Values shown in the histograms are the means ± the SDs from three separate experiments. Columns: N, nuclear; N&C, nuclear-cytoplasmic; C, cytoplasmic.

Effects of 14-3-3ɛ overexpression on the transcriptional activation of the HSP70B promoter by wild-type HSF1 or serine mutants. (A) Schematic representation of the functional domain organization of HSF1. Graphic representations are as indicated: DBD, DNA-binding domain; OLIGO, leucine zipper motif oligomerization domain; RED, regulatory domain; CTA, C-terminal transcriptional activation domain. (B) HeLa cells were cotransfected in triplicate with the HSP70B promoter luciferase reporter plasmid pGLHSP70B and the expression plasmids pHSF1wt, pS303G, pS307G, pS303G-S307G, and pS363A, with (▪) or without (□) pHA-14-3-3ɛ as indicated. In addition, pCMV-lacZ was cotransfected into each culture as an internal control for the transfection efficiency. After the experiment, cells were quenched, and proteins were extracted and assayed for luciferase and β-galactosidase as described in Materials and Methods. Luciferase activity was then normalized to β-galactosidase activity. The relative luciferase activity was then expressed as a percentage of the activity in cells cotransfected with pGLHSP70B and HSF1wt (second column). The relative luciferase values shown in the histogram are the means ± the SDs from three independent experiments. Experiments were carried out four times with reproducible findings each time.

Overexpression of 14-3-3ɛ represses transcriptional activation of the HSP70B promoter by endogenous HSF1. HeLa cells were transfected with pGLHSP70B (HSP70B) alone or with increasing amounts of HA-14-3-3ɛ expression plasmid as indicated. In addition, pCMV-lacZ plasmid was cotransfected into each culture as an internal control for transfection efficiency. (A) After each experiment, cells were quenched and proteins were extracted and assayed for expression of transfected HA-14-3-3ɛ and endogenous HSF1. Transfected 14-3-3ɛ was detected by Western analysis with anti-HA antibodies and HSF1 with specific anti-HSF1 antibodies as described in Materials and Methods. (B) The relative luciferase activity was next examined in the extracts. Luciferase and β-galactosidase were assayed in triplicate samples as described in Materials and Methods. The luciferase activity in each extract was then normalized to the β-galactosidase transfection efficiency control activity. The relative luciferase activity was then expressed ± the standard deviation (SD) of the mean as a percentage of the activity in cells cotransfected with pGLHSP70B and HSF1wt (second column). Luciferase activity was not detected in control cells not transfected with reporter plasmid, and this control was not included in the figure. Experiments were carried out three times with similar results. (C) Plot of the total activity of the HSP70B-luciferase promoter and the pCMV-lacZ control promoter (determined as the β-galactosidase activity) calculated in the experiments above, illustrating the lack of effect of 14-3-3ɛ overexpression on the activity of the control promoter.

Intracellular expression of 14-3-3ɛ induces the cytoplasmic localization of HSF1. (A) HeLa cells were transfected with pHSF1wt-GFP without (rows 1, 2, and 3) or with pHA-14-3-3ɛ (row 4). After 48 h, cells were fixed and incubated with the primary antibodies anti-14-3-3ɛ or anti-HA antibody. Primary antibody staining was detected with a Texas red-conjugated secondary antibody to localize endogenous 14-3-3ɛ (row 3) or coexpressed HA-14-3-3ɛ (row 4) as indicated. HSF1wt-GFP and 14-3-3ɛ were visualized by green autofluorescence or red immunofluorescence as indicated. Cell nuclei were visualized with DAPI autofluorescence. “Merge (1)” indicates colocalization of green or red fluorescence and the DAPI-stained nuclei; “Merge (2)” indicates colocalization of HSF1wt-GFP and HA-14-3-3ɛ. Whole-cell morphology was visualized in the phase-contrast images as indicated. Magnification, ×400. The experiment was carried out three times with reproducible findings each time. (B) Quantitative analysis of the cytoplasmic localization of HSF1wt-GFP in HeLa cells coexpressing increasing amounts of HA-14-3-3ɛ as determined by fluorescence microscopy. The subcellular distribution of HSF1wt-GFP was scored according to whether it was predominantly expressed in the cytoplasm (C bars [▪]). The experiment was carried out three times with reproducible findings each time. (C) Coexpression of 14-3-3ɛ inhibits the nuclear accumulation of HSF1 at 37°C, as determined by biochemical extraction and Western blot analysis. Portions (50 μg) of nuclear extracts (NE) from HeLa cells transfected with pcDNA3.1 HSF1wt alone (lane 1) or with pHA-14-3-3ɛ (lane 2) at 37°C were immunoblotted by using anti-HSF1 antibody to detect the level of HSF1 (upper panel). Quantitation of HSF1 expression (lanes 1 and 2) is shown below the panels. The blot was reprobed with anti-HA antibody (middle panel) to confirm the expression of HA-14-3-3ɛ. Equal protein loading was confirmed by reprobing the same blot with anti-α-actin antibody (bottom panel). The experiment was carried out twice, with similar findings each time. (D) HeLa cells were transfected with pHA-14-3-3ɛ or pHA-P16. After 48 h, cells were fixed and incubated with the primary antibodies anti-HSF1, and anti-HA antibody. Anti-HSF1 was detected with a Texas red-conjugated secondary antibody to localize endogenous HSF1 (rows 1 and 2) and anti-HA was detected with an fluorescein isothiocyanate-conjugated secondary antibody to localize overexpressed HA-14-3-3ɛ (row 3) or HA-P16 (row 4). Endogenous HSF1 and HA-14-3-3ɛ and HA-P16 were visualized by red and green immunofluorescence as indicated. Cell nuclei were stained with DAPI. “Merge (1)” indicates the degree of colocalization of red and green fluorescence of endogenous HSF1 and HA-14-3-3ɛ or HA-P16; “Merge” and “Merge (2)” indicate relative colocalizations of red fluorescence of endogenous HSF1 and the DAPI-stained nuclei. Likewise, to study the effect of 14-3-3ɛ overexpression on intracellular localization of HSF2, cells were fixed and incubated with the primary antibodies anti-HSF2 and anti-HA 48 h after transfection with or without pHA-14-3-3ɛ. Detection with secondary antibodies was carried out as described above. Magnification, ×400. The experiment was carried out three times with reproducible findings. (E) LMB inhibits 14-3-3ɛ-mediated nuclear export of HSF1. HeLa cells cotransfected with pHSF1wt-GFP and pHA-14-3-3ɛ were treated without (Control) or with 10 ng of LMB/ml for 4, 6, and 16 h prior to fixation. Quantitative analysis of the distribution of HSF1wt-GFP in HeLa control cells or in cells coexpressing HA-14-3-3ɛ was performed by using fluorescence microscopy. The subcellular distribution of HSF1wt-GFP was scored according to whether it was predominantly expressed in the nucleus (N), in both the nucleus and the cytoplasm (N&C), or predominantly expressed in the cytoplasm (C). The results are the means ± the SDs from three separate experiments.

HSF1 binds to 14-3-3 after serum stimulation and requirement for HSF1 phosphorylation on serines 303 and 307. (A) Association of 14-3-3 with HSF1 is enhanced by serum. GST-tagged 14-3-3ɛ expression vector was transfected in equivalent amounts into four subconfluent cell cultures, and plates were serum starved as described in Materials and Methods. Subsequently, the cultures were harvested without stimulation (lanes 1 and 2) or after stimulation with 20% fetal calf serum. 14-3-3ɛ-GST fusion proteins were then purified by GSH affinity chromatography, fractionated by SDS-PAGE, and analyzed for their levels and associated proteins with anti-GST and anti-HSF1 antibodies as indicated. As a control, extracts from the two serum-stimulated duplicate cultures of lanes 3 and 4 were subjected to GSH affinity chromatography in the presence of 0.1% SDS-0.5 M NaCl (lanes 5 and 6). As additional control pull-downs, protein A-Sepharose was used in place of GSH-Sepharose for these two extracts. Finally, total extracts from each of the four transfections were analyzed by Western and enhanced chemiluminescence with antibodies against activated MEK or ERK or with control antibodies for ERK2 or HSF1 (bottom). For direct assessment of the relative levels of 14-3-3ɛ-GST expression, the same extracts were also blotted with anti-pan 14-3-3 antisera, which recognizes both the transfected 14-3-3ɛ-GST chimera and the endogenous 14-3-3 isoforms. To this end, please note the low levels of exogenous 14-3-3 expression in comparison with its endogenous counterpart. Experiments were carried out twice with reproducible results. (B) Association of 14-3-3 with HSF1 in vivo. 14-3-3ɛ was transiently expressed in cells as a GST fusion protein as described in Materials and Methods. As a control, in a parallel transfection, GST propeptide was expressed alone as indicated. Cycling cultures of each of the three transfections were harvested, solubilized in extraction buffer, and subjected to GSH affinity chromatography (lanes 1, 3, and 5). As controls, parallel pull-downs containing 0.1% SDS-0.5 M NaCl in the extraction and wash buffer were included (lanes 2, 4, and 6). After SDS-PAGE, anti-GST and anti-HSF1 blotting was performed to verify the levels of 14-3-3ɛ-GST expression and recovery and associated HSF1. For assessment of protein expression, control Western blots of total cellular extracts with anti-pan 14-3-3 antisera or antibodies directed against HSF1 are also shown (bottom panels). (C) HeLa cells were serum starved and pretreated with 50 μM PD98059 before stimulation with 10% FBS-Ham F-12 medium. Cells were then lysed in immunoprecipitation buffer and probed by immunoprecipitation with anti-HSF1 polyclonal antibody (A68-3), followed by immunoblotting with anti-14-3-3ɛ monoclonal antibody as indicated (upper panel). The blot was then reprobed to confirm efficient immunoprecipitation of HSF1 with anti-HSF1 polyclonal antibody (A68-3) (bottom panel). Experiments were carried out twice with reproducible findings. (D) Binding of ³⁵S-labeled 14-3-3ɛ to synthetic peptides. Microtiter wells were coated with synthetic phosphorylated and unphosphorylated peptides derived from the proline-rich domain of HSF1 (phospho-S303-S307, phospho-S307, phospho-S303, and unphospho-HSF1) as described in Materials and Methods. Different concentrations of ³⁵S-labeled 14-3-3ɛ protein (160 and 400 ng/ml) were then added to the coated microtiter wells and incubated at 22°C for 2 h. After an extensive washing, bound proteins were extracted and measured by liquid scintillation counting. The results are expressed as the mean of the ³⁵S-labeled 14-3-3ɛ activity extracted from triplicate wells ± the SD. Experiments were carried out three times with close agreement between experiments. (E) Competitive binding of phosphorylated HSF1 peptides to ³⁵S-labeled 14-3-3ɛ protein. ³⁵S-labeled 14-3-3ɛ (160 ng/ml) was incubated first with a 100 μM concentration of each of the phosphorylated or unphosphorylated peptides for 1 h at 22°C and then added to microtiter wells coated with each of the phosphorylated peptides (phospho-S303-S307, phospho-S307, and phospho-S303) (as described above), followed by incubation at 22°C for 2 h. After extensive washing of the plates, bound proteins were extracted and assayed by liquid scintillation counting. Competition experiments were also carried out with phospho-Cdc25c-S216, a well-characterized 14-3-3-binding peptide, as a positive control as indicated. Each competition assay was carried out in duplicate. The entire experiment was carried out three times with reproducible findings each time. (F) Effect of phospho-S303 and phospho-S307 peptides on the association of 14-3-3 with FLAG-HSF1. Purified 14-3-3-GST was mixed with serum-stimulated cell extracts prepared from cells expressing either FLAG-HSF1 (lane 1) or FLAG propeptide alone (lane 4). To test the potential effect of phosphorylation on the association of 14-3-3ɛ with FLAG-HSF1, parallel reactions included synthetic HSF1-based peptides phosphorylated either on Ser-303 (lanes 2 and 5) or Ser-307 (lanes 3 and 6). After washes, the 14-3-3ɛ-GST and associated FLAG-HSF1 were fractionated by SDS-PAGE and blotted with anti-FLAG and anti-14-3-3ɛ antibodies as indicated. Below each lane the densitometry value of bound FLAG-HSF1 is indicated as a percentage value, with a control (100%) in lane 1. As a control, 10-μl aliquots of the reactions taken before washes were also assessed for the levels of FLAG-HSF1 (bottom). Experiments were repeated reproducibly three times.