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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 is a substrate of the FBXW7α ubiquitin ligase(a) Network of FBXW7α-interacting partners. Serial immunoprecipitation experiments from HEK293 cells coupled to mass-spectrometry based analysis revealed a large number of known substrates (NFKB2, MYC, MED13L, MED13), already characterized members of the Cullin 1 complex (SKP1, CUL1) and putative interactors (MED1, HSF1). The FBXW7 degrons on various substrates are indicated. (b) FBXW7α binds to HSF1 through specific residues in the WD40 domain. HEK293T cells were transfected with constructs encoding FLAG tagged HSF1, and FLAG-HA tagged empty vector (EV), or FLAG-HA tagged FBXW7α or FLAG-HA tagged FBXW7α (WD40), a substrate binding mutant, in which three residues within one of the seven WD40 repeats of FBXW7α have been mutated57. HA-tagged FBXW7α was immunoprecipitated (IP) from cell extracts with anti-HA resin, followed by immunoblotting as indicated. The left panel shows inputs. (c) Alignment of the human HSF1 protein region containing the putative degron with HSF1 from various organisms. Conserved phospho-amino acids (amino acids 303 and 307 in human sequence) are highlighted. Uncropped blots are shown in Supplementary Fig. 8.
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Figure 1: HSF1 is a substrate of the FBXW7α ubiquitin ligase(a) Network of FBXW7α-interacting partners. Serial immunoprecipitation experiments from HEK293 cells coupled to mass-spectrometry based analysis revealed a large number of known substrates (NFKB2, MYC, MED13L, MED13), already characterized members of the Cullin 1 complex (SKP1, CUL1) and putative interactors (MED1, HSF1). The FBXW7 degrons on various substrates are indicated. (b) FBXW7α binds to HSF1 through specific residues in the WD40 domain. HEK293T cells were transfected with constructs encoding FLAG tagged HSF1, and FLAG-HA tagged empty vector (EV), or FLAG-HA tagged FBXW7α or FLAG-HA tagged FBXW7α (WD40), a substrate binding mutant, in which three residues within one of the seven WD40 repeats of FBXW7α have been mutated57. HA-tagged FBXW7α was immunoprecipitated (IP) from cell extracts with anti-HA resin, followed by immunoblotting as indicated. The left panel shows inputs. (c) Alignment of the human HSF1 protein region containing the putative degron with HSF1 from various organisms. Conserved phospho-amino acids (amino acids 303 and 307 in human sequence) are highlighted. Uncropped blots are shown in Supplementary Fig. 8.

Mentions: To identify substrates of the ubiquitin ligase FBXW7α, we performed tandem affinity purification of FBXW7α and identified its interacting proteins by 2D LC-MS/MS (Fig. 1a; Supplementary Table 1). Interestingly, HSF1, similar to MYC, was detected in FBXW7α immunoprecipitates (Fig. 1b). However, the HSF1 interaction with a WD40 domain mutant FBXW7α, that lacks the ability to bind protein substrates but binds the Cullin 1 complex, was significantly reduced (Fig. 1b). In addition, endogenous FBXW7 and HSF1 were found to interact (Supplementary Fig. 1a). Analysis of HSF1 protein sequence revealed the presence of two conserved amino-acid sequences resembling the canonical FBXW7 degradation motif (degron) S/TPPXS/T20, one of which (SPPQS), contains evolutionary conserved phosphoamino acids (Fig. 1c).


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 is a substrate of the FBXW7α ubiquitin ligase(a) Network of FBXW7α-interacting partners. Serial immunoprecipitation experiments from HEK293 cells coupled to mass-spectrometry based analysis revealed a large number of known substrates (NFKB2, MYC, MED13L, MED13), already characterized members of the Cullin 1 complex (SKP1, CUL1) and putative interactors (MED1, HSF1). The FBXW7 degrons on various substrates are indicated. (b) FBXW7α binds to HSF1 through specific residues in the WD40 domain. HEK293T cells were transfected with constructs encoding FLAG tagged HSF1, and FLAG-HA tagged empty vector (EV), or FLAG-HA tagged FBXW7α or FLAG-HA tagged FBXW7α (WD40), a substrate binding mutant, in which three residues within one of the seven WD40 repeats of FBXW7α have been mutated57. HA-tagged FBXW7α was immunoprecipitated (IP) from cell extracts with anti-HA resin, followed by immunoblotting as indicated. The left panel shows inputs. (c) Alignment of the human HSF1 protein region containing the putative degron with HSF1 from various organisms. Conserved phospho-amino acids (amino acids 303 and 307 in human sequence) are highlighted. Uncropped blots are shown in Supplementary Fig. 8.
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Figure 1: HSF1 is a substrate of the FBXW7α ubiquitin ligase(a) Network of FBXW7α-interacting partners. Serial immunoprecipitation experiments from HEK293 cells coupled to mass-spectrometry based analysis revealed a large number of known substrates (NFKB2, MYC, MED13L, MED13), already characterized members of the Cullin 1 complex (SKP1, CUL1) and putative interactors (MED1, HSF1). The FBXW7 degrons on various substrates are indicated. (b) FBXW7α binds to HSF1 through specific residues in the WD40 domain. HEK293T cells were transfected with constructs encoding FLAG tagged HSF1, and FLAG-HA tagged empty vector (EV), or FLAG-HA tagged FBXW7α or FLAG-HA tagged FBXW7α (WD40), a substrate binding mutant, in which three residues within one of the seven WD40 repeats of FBXW7α have been mutated57. HA-tagged FBXW7α was immunoprecipitated (IP) from cell extracts with anti-HA resin, followed by immunoblotting as indicated. The left panel shows inputs. (c) Alignment of the human HSF1 protein region containing the putative degron with HSF1 from various organisms. Conserved phospho-amino acids (amino acids 303 and 307 in human sequence) are highlighted. Uncropped blots are shown in Supplementary Fig. 8.
Mentions: To identify substrates of the ubiquitin ligase FBXW7α, we performed tandem affinity purification of FBXW7α and identified its interacting proteins by 2D LC-MS/MS (Fig. 1a; Supplementary Table 1). Interestingly, HSF1, similar to MYC, was detected in FBXW7α immunoprecipitates (Fig. 1b). However, the HSF1 interaction with a WD40 domain mutant FBXW7α, that lacks the ability to bind protein substrates but binds the Cullin 1 complex, was significantly reduced (Fig. 1b). In addition, endogenous FBXW7 and HSF1 were found to interact (Supplementary Fig. 1a). Analysis of HSF1 protein sequence revealed the presence of two conserved amino-acid sequences resembling the canonical FBXW7 degradation motif (degron) S/TPPXS/T20, one of which (SPPQS), contains evolutionary conserved phosphoamino acids (Fig. 1c).

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.

Show MeSH
Related in: MedlinePlus