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FBXW7 modulates cellular stress response and metastatic potential through ​HSF1 post-translational modification.

Kourtis N, Moubarak RS, Aranda-Orgilles B, Lui K, Aydin IT, Trimarchi T, Darvishian F, Salvaggio C, Zhong J, Bhatt K, Chen EI, Celebi JT, Lazaris C, Tsirigos A, Osman I, Hernando E, Aifantis I - Nat. Cell Biol. (2015)

Bottom Line: ​Heat-shock factor 1 (​HSF1) orchestrates the heat-shock response in eukaryotes.Although this pathway has evolved to help cells adapt in the presence of challenging conditions, it is co-opted in cancer to support malignancy.Here we show that the ubiquitin ligase ​FBXW7α interacts with ​HSF1 through a conserved motif phosphorylated by ​GSK3β and ​ERK1. ​FBXW7α ubiquitylates ​HSF1 and loss of ​FBXW7α results in impaired degradation of nuclear ​HSF1 and defective heat-shock response attenuation. ​FBXW7α is either mutated or transcriptionally downregulated in melanoma and ​HSF1 nuclear stabilization correlates with increased metastatic potential and disease progression. ​FBXW7α deficiency and subsequent ​HSF1 accumulation activates an invasion-supportive transcriptional program and enhances the metastatic potential of human melanoma cells.

View Article: PubMed Central - PubMed

ABSTRACT
​Heat-shock factor 1 (​HSF1) orchestrates the heat-shock response in eukaryotes. Although this pathway has evolved to help cells adapt in the presence of challenging conditions, it is co-opted in cancer to support malignancy. However, the mechanisms that regulate ​HSF1 and thus cellular stress response are poorly understood. Here we show that the ubiquitin ligase ​FBXW7α interacts with ​HSF1 through a conserved motif phosphorylated by ​GSK3β and ​ERK1. ​FBXW7α ubiquitylates ​HSF1 and loss of ​FBXW7α results in impaired degradation of nuclear ​HSF1 and defective heat-shock response attenuation. ​FBXW7α is either mutated or transcriptionally downregulated in melanoma and ​HSF1 nuclear stabilization correlates with increased metastatic potential and disease progression. ​FBXW7α deficiency and subsequent ​HSF1 accumulation activates an invasion-supportive transcriptional program and enhances the metastatic potential of human melanoma cells. These findings identify a post-translational mechanism of regulation of the ​HSF1 transcriptional program both in the presence of exogenous stress and in cancer.

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HSF1 interacts with FBXW7α through a conserved degron sequence phosphorylated by GSK3β and ERK1(a) HSF1 binds FBXW7α through a conserved degron. HEK293T cells were transfected with FLAG-HA tagged FBXW7α and constructs encoding FLAG tagged HSF1 or HSF1(Ser303/307Ala) or HSF1(Ser363/367Ala). HA-tagged FBXW7α was immunoprecipitated (IP) from cell extracts with anti-HA resin, followed by immunoblotting as indicated. The left panel shows inputs. (b) Both Ser303 and Ser307 in HSF1 are required for the interaction with FBXW7α. HEK293T cells were transfected with FLAG-HA tagged FBXW7α and constructs encoding FLAG tagged HSF1 or HSF1(Ser303/307Ala) or HSF1(Ser303Ala) or HSF1(Ser307Ala). HA-tagged FBXW7α was immunoprecipitated (IP) from cell extracts with anti-HA resin, followed by immunoblotting as indicated. (c) Interaction between HSF1 and FBXW7α depends on GSK3β activity. HEK293T cells were transfected with constructs encoding FLAG tagged HSF1 and FLAG-HA tagged FBXW7α. Cells were treated with GSK3i IX (10 μM for 10 h) or DMSO. HA-tagged FBXW7α was immunoprecipitated (IP) from cell extracts with anti-HA resin, followed by immunoblotting as indicated. (d) Interaction between HSF1 and FBXW7α depends on ERK1 activity. HEK293T cells were transfected with constructs encoding FLAG tagged HSF1 and FLAG-HA tagged FBXW7α. Cells were treated with MEK1 inhibitor U0126 (10 μM for 2 h) or DMSO. HA-tagged FBXW7α was immunoprecipitated (IP) from cell extracts with anti-HA resin, followed by immunoblotting as indicated. (e) FBXW7α controls the half-life of nuclear HSF1. HEK293T cells were infected with the indicated shRNA-encoding lentiviruses. Cells were treated with 2 μg/ml cycloheximide for the indicated length of time. Nuclear fractions were analyzed by immunoblotting as indicated. TATA-binding protein (TBP) was used as loading control. (f) FBXW7α depletion abolishes HSF1 ubiquitylation in vivo. HEK293T cells were transfected with constructs encoding FLAG tagged HSF1 and Histidine-Myc tagged ubiquitin and infected with the indicated shRNA-encoding lentiviruses. Cells were heat shocked at 42°C for 1 h to induce ubiquitylation. Histidine tagged proteins were immunoprecipitated from whole cell extracts with nickel (Ni)-NTA beads, followed by immunoblotting for HSF1. Uncropped blots are shown in Supplementary Fig. 8.
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Figure 2: HSF1 interacts with FBXW7α through a conserved degron sequence phosphorylated by GSK3β and ERK1(a) HSF1 binds FBXW7α through a conserved degron. HEK293T cells were transfected with FLAG-HA tagged FBXW7α and constructs encoding FLAG tagged HSF1 or HSF1(Ser303/307Ala) or HSF1(Ser363/367Ala). HA-tagged FBXW7α was immunoprecipitated (IP) from cell extracts with anti-HA resin, followed by immunoblotting as indicated. The left panel shows inputs. (b) Both Ser303 and Ser307 in HSF1 are required for the interaction with FBXW7α. HEK293T cells were transfected with FLAG-HA tagged FBXW7α and constructs encoding FLAG tagged HSF1 or HSF1(Ser303/307Ala) or HSF1(Ser303Ala) or HSF1(Ser307Ala). HA-tagged FBXW7α was immunoprecipitated (IP) from cell extracts with anti-HA resin, followed by immunoblotting as indicated. (c) Interaction between HSF1 and FBXW7α depends on GSK3β activity. HEK293T cells were transfected with constructs encoding FLAG tagged HSF1 and FLAG-HA tagged FBXW7α. Cells were treated with GSK3i IX (10 μM for 10 h) or DMSO. HA-tagged FBXW7α was immunoprecipitated (IP) from cell extracts with anti-HA resin, followed by immunoblotting as indicated. (d) Interaction between HSF1 and FBXW7α depends on ERK1 activity. HEK293T cells were transfected with constructs encoding FLAG tagged HSF1 and FLAG-HA tagged FBXW7α. Cells were treated with MEK1 inhibitor U0126 (10 μM for 2 h) or DMSO. HA-tagged FBXW7α was immunoprecipitated (IP) from cell extracts with anti-HA resin, followed by immunoblotting as indicated. (e) FBXW7α controls the half-life of nuclear HSF1. HEK293T cells were infected with the indicated shRNA-encoding lentiviruses. Cells were treated with 2 μg/ml cycloheximide for the indicated length of time. Nuclear fractions were analyzed by immunoblotting as indicated. TATA-binding protein (TBP) was used as loading control. (f) FBXW7α depletion abolishes HSF1 ubiquitylation in vivo. HEK293T cells were transfected with constructs encoding FLAG tagged HSF1 and Histidine-Myc tagged ubiquitin and infected with the indicated shRNA-encoding lentiviruses. Cells were heat shocked at 42°C for 1 h to induce ubiquitylation. Histidine tagged proteins were immunoprecipitated from whole cell extracts with nickel (Ni)-NTA beads, followed by immunoblotting for HSF1. Uncropped blots are shown in Supplementary Fig. 8.

Mentions: We next mapped the FBXW7α -binding motif of human HSF1. To investigate which amino acids participate in the interaction of HSF1 with FBXW7α, we mutated the two putative FBXW7α degrons (amino acid positions 303-307 and 363-367). We found that an HSF1 mutant containing alanine substitutions at both Ser303 and Ser307 [HSF1(Ser303/307Ala)] failed to bind FBXW7α (Fig. 2a). Further mutational analysis revealed both Ser303 and Ser307 as necessary residues contributing to its interaction with FBXW7α (Fig. 2b). Notably, phosphorylation of HSF1 on Ser303/307 by GSK3β and ERK1 respectively, has been suggested to play a role in down-modulating HSF1's activity during recovery from stress31-36. Treatment of cells with a GSK3 inhibitor (GSK3i IX and XVI) or a MEK inhibitor (U0126) markedly decreased the affinity of FBXW7α for HSF1 (Fig. 2c, d; Supplementary Fig. 1b). This is consistent with the notion of a priming role for the Ser307 phosphorylation for subsequent phosphorylation on Ser30337. Moreover, depletion of FBXW7 increased the half-life of nuclear HSF1 (Fig. 2e).


FBXW7 modulates cellular stress response and metastatic potential through ​HSF1 post-translational modification.

Kourtis N, Moubarak RS, Aranda-Orgilles B, Lui K, Aydin IT, Trimarchi T, Darvishian F, Salvaggio C, Zhong J, Bhatt K, Chen EI, Celebi JT, Lazaris C, Tsirigos A, Osman I, Hernando E, Aifantis I - Nat. Cell Biol. (2015)

HSF1 interacts with FBXW7α through a conserved degron sequence phosphorylated by GSK3β and ERK1(a) HSF1 binds FBXW7α through a conserved degron. HEK293T cells were transfected with FLAG-HA tagged FBXW7α and constructs encoding FLAG tagged HSF1 or HSF1(Ser303/307Ala) or HSF1(Ser363/367Ala). HA-tagged FBXW7α was immunoprecipitated (IP) from cell extracts with anti-HA resin, followed by immunoblotting as indicated. The left panel shows inputs. (b) Both Ser303 and Ser307 in HSF1 are required for the interaction with FBXW7α. HEK293T cells were transfected with FLAG-HA tagged FBXW7α and constructs encoding FLAG tagged HSF1 or HSF1(Ser303/307Ala) or HSF1(Ser303Ala) or HSF1(Ser307Ala). HA-tagged FBXW7α was immunoprecipitated (IP) from cell extracts with anti-HA resin, followed by immunoblotting as indicated. (c) Interaction between HSF1 and FBXW7α depends on GSK3β activity. HEK293T cells were transfected with constructs encoding FLAG tagged HSF1 and FLAG-HA tagged FBXW7α. Cells were treated with GSK3i IX (10 μM for 10 h) or DMSO. HA-tagged FBXW7α was immunoprecipitated (IP) from cell extracts with anti-HA resin, followed by immunoblotting as indicated. (d) Interaction between HSF1 and FBXW7α depends on ERK1 activity. HEK293T cells were transfected with constructs encoding FLAG tagged HSF1 and FLAG-HA tagged FBXW7α. Cells were treated with MEK1 inhibitor U0126 (10 μM for 2 h) or DMSO. HA-tagged FBXW7α was immunoprecipitated (IP) from cell extracts with anti-HA resin, followed by immunoblotting as indicated. (e) FBXW7α controls the half-life of nuclear HSF1. HEK293T cells were infected with the indicated shRNA-encoding lentiviruses. Cells were treated with 2 μg/ml cycloheximide for the indicated length of time. Nuclear fractions were analyzed by immunoblotting as indicated. TATA-binding protein (TBP) was used as loading control. (f) FBXW7α depletion abolishes HSF1 ubiquitylation in vivo. HEK293T cells were transfected with constructs encoding FLAG tagged HSF1 and Histidine-Myc tagged ubiquitin and infected with the indicated shRNA-encoding lentiviruses. Cells were heat shocked at 42°C for 1 h to induce ubiquitylation. Histidine tagged proteins were immunoprecipitated from whole cell extracts with nickel (Ni)-NTA beads, followed by immunoblotting for HSF1. Uncropped blots are shown in Supplementary Fig. 8.
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Figure 2: HSF1 interacts with FBXW7α through a conserved degron sequence phosphorylated by GSK3β and ERK1(a) HSF1 binds FBXW7α through a conserved degron. HEK293T cells were transfected with FLAG-HA tagged FBXW7α and constructs encoding FLAG tagged HSF1 or HSF1(Ser303/307Ala) or HSF1(Ser363/367Ala). HA-tagged FBXW7α was immunoprecipitated (IP) from cell extracts with anti-HA resin, followed by immunoblotting as indicated. The left panel shows inputs. (b) Both Ser303 and Ser307 in HSF1 are required for the interaction with FBXW7α. HEK293T cells were transfected with FLAG-HA tagged FBXW7α and constructs encoding FLAG tagged HSF1 or HSF1(Ser303/307Ala) or HSF1(Ser303Ala) or HSF1(Ser307Ala). HA-tagged FBXW7α was immunoprecipitated (IP) from cell extracts with anti-HA resin, followed by immunoblotting as indicated. (c) Interaction between HSF1 and FBXW7α depends on GSK3β activity. HEK293T cells were transfected with constructs encoding FLAG tagged HSF1 and FLAG-HA tagged FBXW7α. Cells were treated with GSK3i IX (10 μM for 10 h) or DMSO. HA-tagged FBXW7α was immunoprecipitated (IP) from cell extracts with anti-HA resin, followed by immunoblotting as indicated. (d) Interaction between HSF1 and FBXW7α depends on ERK1 activity. HEK293T cells were transfected with constructs encoding FLAG tagged HSF1 and FLAG-HA tagged FBXW7α. Cells were treated with MEK1 inhibitor U0126 (10 μM for 2 h) or DMSO. HA-tagged FBXW7α was immunoprecipitated (IP) from cell extracts with anti-HA resin, followed by immunoblotting as indicated. (e) FBXW7α controls the half-life of nuclear HSF1. HEK293T cells were infected with the indicated shRNA-encoding lentiviruses. Cells were treated with 2 μg/ml cycloheximide for the indicated length of time. Nuclear fractions were analyzed by immunoblotting as indicated. TATA-binding protein (TBP) was used as loading control. (f) FBXW7α depletion abolishes HSF1 ubiquitylation in vivo. HEK293T cells were transfected with constructs encoding FLAG tagged HSF1 and Histidine-Myc tagged ubiquitin and infected with the indicated shRNA-encoding lentiviruses. Cells were heat shocked at 42°C for 1 h to induce ubiquitylation. Histidine tagged proteins were immunoprecipitated from whole cell extracts with nickel (Ni)-NTA beads, followed by immunoblotting for HSF1. Uncropped blots are shown in Supplementary Fig. 8.
Mentions: We next mapped the FBXW7α -binding motif of human HSF1. To investigate which amino acids participate in the interaction of HSF1 with FBXW7α, we mutated the two putative FBXW7α degrons (amino acid positions 303-307 and 363-367). We found that an HSF1 mutant containing alanine substitutions at both Ser303 and Ser307 [HSF1(Ser303/307Ala)] failed to bind FBXW7α (Fig. 2a). Further mutational analysis revealed both Ser303 and Ser307 as necessary residues contributing to its interaction with FBXW7α (Fig. 2b). Notably, phosphorylation of HSF1 on Ser303/307 by GSK3β and ERK1 respectively, has been suggested to play a role in down-modulating HSF1's activity during recovery from stress31-36. Treatment of cells with a GSK3 inhibitor (GSK3i IX and XVI) or a MEK inhibitor (U0126) markedly decreased the affinity of FBXW7α for HSF1 (Fig. 2c, d; Supplementary Fig. 1b). This is consistent with the notion of a priming role for the Ser307 phosphorylation for subsequent phosphorylation on Ser30337. Moreover, depletion of FBXW7 increased the half-life of nuclear HSF1 (Fig. 2e).

Bottom Line: ​Heat-shock factor 1 (​HSF1) orchestrates the heat-shock response in eukaryotes.Although this pathway has evolved to help cells adapt in the presence of challenging conditions, it is co-opted in cancer to support malignancy.Here we show that the ubiquitin ligase ​FBXW7α interacts with ​HSF1 through a conserved motif phosphorylated by ​GSK3β and ​ERK1. ​FBXW7α ubiquitylates ​HSF1 and loss of ​FBXW7α results in impaired degradation of nuclear ​HSF1 and defective heat-shock response attenuation. ​FBXW7α is either mutated or transcriptionally downregulated in melanoma and ​HSF1 nuclear stabilization correlates with increased metastatic potential and disease progression. ​FBXW7α deficiency and subsequent ​HSF1 accumulation activates an invasion-supportive transcriptional program and enhances the metastatic potential of human melanoma cells.

View Article: PubMed Central - PubMed

ABSTRACT
​Heat-shock factor 1 (​HSF1) orchestrates the heat-shock response in eukaryotes. Although this pathway has evolved to help cells adapt in the presence of challenging conditions, it is co-opted in cancer to support malignancy. However, the mechanisms that regulate ​HSF1 and thus cellular stress response are poorly understood. Here we show that the ubiquitin ligase ​FBXW7α interacts with ​HSF1 through a conserved motif phosphorylated by ​GSK3β and ​ERK1. ​FBXW7α ubiquitylates ​HSF1 and loss of ​FBXW7α results in impaired degradation of nuclear ​HSF1 and defective heat-shock response attenuation. ​FBXW7α is either mutated or transcriptionally downregulated in melanoma and ​HSF1 nuclear stabilization correlates with increased metastatic potential and disease progression. ​FBXW7α deficiency and subsequent ​HSF1 accumulation activates an invasion-supportive transcriptional program and enhances the metastatic potential of human melanoma cells. These findings identify a post-translational mechanism of regulation of the ​HSF1 transcriptional program both in the presence of exogenous stress and in cancer.

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Related in: MedlinePlus