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HDAC6-ubiquitin interaction controls the duration of HSF1 activation after heat shock.

Pernet L, Faure V, Gilquin B, Dufour-Guérin S, Khochbin S, Vourc'h C - Mol. Biol. Cell (2014)

Bottom Line: Here we show that a full response to heat shock (activation of both HSP70 and HSP25) depends on the duration of HSF1 activation, which is itself controlled by HDAC6, a unique deacetylase known to bind monoubiquitin and polyubiquitin with high affinity.In cells expressing HDAC6 mutated in the ubiquitin-binding domain, the AAA ATPase factor p97/VCP mediates rapid inactivation of HSF1, precluding late activation of the HSP25 gene.In these cells, knockdown of p97/VCP rescues HSF1 from this rapid inactivation and restores HSP25 expression.

View Article: PubMed Central - PubMed

Affiliation: University Grenoble-Alpes, CRI INSERM, U823, Institut Albert Bonniot, La Tronche 38042, Grenoble Cedex 9, France.

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Absence of heat-induced HSP25 expression in cells expressing HDAC6 mutated in its ubiquitin-binding domain. (A) Schematic representation of HDAC6 and HDAC6 mutants. (B) Comparative analysis of HSF1 phosphorylation under HS and MG132 treatments. Proteins were extracted from WT cells and KO HDAC6 cells heat shocked or not for 1 h or incubated with MG132 inhibitor for 8 h. Phosphorylation of HSF1 was analyzed by Western blot using an anti-HSF1 antibody. Ponceau staining was used as a loading control. (C) Schematic representation of the heat shock kinetics. Cells were maintained at 37°C (NHS) or submitted to different conditions of recovery period (Rec) after 1 h of heat shock at 43°C. (D) HSP70 and HSP25 expression in HDAC6 mutants. Western blot analysis of HSP70 and HSP25 was performed in WT cells, HDAC6 KO cells, and cells expressing HDAC6 mutated in the ubiquitin-binding (Ubm) or HDAC domain (HDm). Tubulin (Tub) was used as a loading control. (E) HSP25 expression in HDAC6 mutants. Immunodetection of HSP25 was performed in the different cell lines submitted or not (NHS) to a recovery period of 8 h after heat shock. Bar, 10 μm.
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Figure 1: Absence of heat-induced HSP25 expression in cells expressing HDAC6 mutated in its ubiquitin-binding domain. (A) Schematic representation of HDAC6 and HDAC6 mutants. (B) Comparative analysis of HSF1 phosphorylation under HS and MG132 treatments. Proteins were extracted from WT cells and KO HDAC6 cells heat shocked or not for 1 h or incubated with MG132 inhibitor for 8 h. Phosphorylation of HSF1 was analyzed by Western blot using an anti-HSF1 antibody. Ponceau staining was used as a loading control. (C) Schematic representation of the heat shock kinetics. Cells were maintained at 37°C (NHS) or submitted to different conditions of recovery period (Rec) after 1 h of heat shock at 43°C. (D) HSP70 and HSP25 expression in HDAC6 mutants. Western blot analysis of HSP70 and HSP25 was performed in WT cells, HDAC6 KO cells, and cells expressing HDAC6 mutated in the ubiquitin-binding (Ubm) or HDAC domain (HDm). Tubulin (Tub) was used as a loading control. (E) HSP25 expression in HDAC6 mutants. Immunodetection of HSP25 was performed in the different cell lines submitted or not (NHS) to a recovery period of 8 h after heat shock. Bar, 10 μm.

Mentions: To assess the role of HDAC6 in the heat-induced accumulation of HSP proteins, we examined the profile of HSP25 and HSP70 accumulation in wild-type (WT) or in HDAC6-knockout (KO) mouse 3T3 cells reexpressing or not HDAC6 bearing inactivating mutations in the two catalytic deacetylase domains (HDm) or in the ubiquitin-binding domain (Ubm; Figure 1A). Similar amounts of HDAC6 were detected by Western blot analysis in these cells, whereas acetylated tubulin (a well-known substrate of HDAC6) was only detected in HDAC6 HDm and HDAC6 KO cells (Supplemental Figure S1).


HDAC6-ubiquitin interaction controls the duration of HSF1 activation after heat shock.

Pernet L, Faure V, Gilquin B, Dufour-Guérin S, Khochbin S, Vourc'h C - Mol. Biol. Cell (2014)

Absence of heat-induced HSP25 expression in cells expressing HDAC6 mutated in its ubiquitin-binding domain. (A) Schematic representation of HDAC6 and HDAC6 mutants. (B) Comparative analysis of HSF1 phosphorylation under HS and MG132 treatments. Proteins were extracted from WT cells and KO HDAC6 cells heat shocked or not for 1 h or incubated with MG132 inhibitor for 8 h. Phosphorylation of HSF1 was analyzed by Western blot using an anti-HSF1 antibody. Ponceau staining was used as a loading control. (C) Schematic representation of the heat shock kinetics. Cells were maintained at 37°C (NHS) or submitted to different conditions of recovery period (Rec) after 1 h of heat shock at 43°C. (D) HSP70 and HSP25 expression in HDAC6 mutants. Western blot analysis of HSP70 and HSP25 was performed in WT cells, HDAC6 KO cells, and cells expressing HDAC6 mutated in the ubiquitin-binding (Ubm) or HDAC domain (HDm). Tubulin (Tub) was used as a loading control. (E) HSP25 expression in HDAC6 mutants. Immunodetection of HSP25 was performed in the different cell lines submitted or not (NHS) to a recovery period of 8 h after heat shock. Bar, 10 μm.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4263459&req=5

Figure 1: Absence of heat-induced HSP25 expression in cells expressing HDAC6 mutated in its ubiquitin-binding domain. (A) Schematic representation of HDAC6 and HDAC6 mutants. (B) Comparative analysis of HSF1 phosphorylation under HS and MG132 treatments. Proteins were extracted from WT cells and KO HDAC6 cells heat shocked or not for 1 h or incubated with MG132 inhibitor for 8 h. Phosphorylation of HSF1 was analyzed by Western blot using an anti-HSF1 antibody. Ponceau staining was used as a loading control. (C) Schematic representation of the heat shock kinetics. Cells were maintained at 37°C (NHS) or submitted to different conditions of recovery period (Rec) after 1 h of heat shock at 43°C. (D) HSP70 and HSP25 expression in HDAC6 mutants. Western blot analysis of HSP70 and HSP25 was performed in WT cells, HDAC6 KO cells, and cells expressing HDAC6 mutated in the ubiquitin-binding (Ubm) or HDAC domain (HDm). Tubulin (Tub) was used as a loading control. (E) HSP25 expression in HDAC6 mutants. Immunodetection of HSP25 was performed in the different cell lines submitted or not (NHS) to a recovery period of 8 h after heat shock. Bar, 10 μm.
Mentions: To assess the role of HDAC6 in the heat-induced accumulation of HSP proteins, we examined the profile of HSP25 and HSP70 accumulation in wild-type (WT) or in HDAC6-knockout (KO) mouse 3T3 cells reexpressing or not HDAC6 bearing inactivating mutations in the two catalytic deacetylase domains (HDm) or in the ubiquitin-binding domain (Ubm; Figure 1A). Similar amounts of HDAC6 were detected by Western blot analysis in these cells, whereas acetylated tubulin (a well-known substrate of HDAC6) was only detected in HDAC6 HDm and HDAC6 KO cells (Supplemental Figure S1).

Bottom Line: Here we show that a full response to heat shock (activation of both HSP70 and HSP25) depends on the duration of HSF1 activation, which is itself controlled by HDAC6, a unique deacetylase known to bind monoubiquitin and polyubiquitin with high affinity.In cells expressing HDAC6 mutated in the ubiquitin-binding domain, the AAA ATPase factor p97/VCP mediates rapid inactivation of HSF1, precluding late activation of the HSP25 gene.In these cells, knockdown of p97/VCP rescues HSF1 from this rapid inactivation and restores HSP25 expression.

View Article: PubMed Central - PubMed

Affiliation: University Grenoble-Alpes, CRI INSERM, U823, Institut Albert Bonniot, La Tronche 38042, Grenoble Cedex 9, France.

Show MeSH
Related in: MedlinePlus