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An ID2-dependent mechanism for VHL inactivation in cancer

View Article: PubMed Central - PubMed

ABSTRACT

Mechanisms that maintain cancer stem cells are crucial to tumor progression. The ID2 protein underpins cancer hallmarks including the cancer stem cell state. HIFα transcription factors, most notably HIF2α, are expressed in and required for maintenance of cancer stem cells (CSCs). However, the pathways that are engaged by ID2 or drive HIF2α accumulation in CSCs have remained unclear. We report that DYRK1A and DYRK1B kinases phosphorylate ID2 on Threonine-27 (T27). Hypoxia down regulates this phosphorylation via inactivation of DYRK1, whose activity is stimulated in normoxia by the oxygen sensing prolyl hydroxylase PHD1. ID2 binds to the VHL ubiquitin ligase complex, displaces VHL-associated Cullin-2, and impairs HIF2α ubiquitylation and degradation. Phosphorylation of ID2-T27 by DYRK1 blocks ID2-VHL interaction and preserves HIF2α ubiquitylation. In glioblastoma ID2 positively modulates HIF2α activity. Conversely, elevated expression of DYRK1 phosphorylates ID2- T27, leading to HIF2α destabilization, loss of glioma stemness, inhibition of tumor growth, and a more favorable outcome for patients with glioblastoma.

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The DYRK1-ID2-T27 pathway modulates HIFα stability by regulating the interaction between ID2 and VHLa, In vivo ubiquitylation of HIF1α protein. U87 cells transfected with the expression plasmids HIF1α and MYC-ubiquitin were co-transfected with FLAG-ID2, FLAG-ID2-T27A, or the empty vector in the presence or in the absence of GFP-DYRK1B. After treatment with MG132 (20 μM) for 6 h, lysates were prepared in denaturing buffer and identical aliquots were immunoprecipitated with antibodies directed against MYC. An anti-HA antibody was used to detect HIF1α ubiquitin conjugates (left panels); Cellular lysates, WCL, were analyzed by western blot using the indicated antibodies (right panels). b, U87 cells were co-transfected with plasmids expressing HA-HIF2α and GFP-DYRK1B or GFP-vector. Cells were treated with 50 μg/ml of C HX for the indicated times and analyzed by western blot. c, Quantification of HIF2α protein from the experiment in panel b as the Log2 of the percentage of HIF2α relative to untreated cells. d, IMR32 cells were co-transfected with ID2 and FLAG-VHL or FLAG-HIF1α expressing vectors. Immunoprecipitation was performed using FLAG antibody and immunocomplexes and whole cell lysates were analyzed by western blot using the indicated antibodies. e, IMR32 cells transfected with FLAG-VHL expression vector were used for IgG or ID2 antibody immunoprecipitation. Immunocomplexes and WCL were analyzed by western blot. Arrow points to the specific FLAG-VHL band; asterisk indicates IgG light chain. f, FLAG immunoprecipitation of binding reactions of in vitro translated FLAG-ID and HA-Elongin C proteins. Immunocomplexes were analyzed by western blot for HA and FLAG. g, FLAG-ID proteins and HA-VHL were translated and incubated in vitro. FLAG immunocomplexes were analyzed by western blot for HA and FLAG. h, In vitro streptavidin pull down assay of biotinylated ID2 peptides (amino acid 14-34 WT, pT27, and T27W) and in vitro translated HA-VHL. Bound polypeptides were detected by western blot.
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Figure 5: The DYRK1-ID2-T27 pathway modulates HIFα stability by regulating the interaction between ID2 and VHLa, In vivo ubiquitylation of HIF1α protein. U87 cells transfected with the expression plasmids HIF1α and MYC-ubiquitin were co-transfected with FLAG-ID2, FLAG-ID2-T27A, or the empty vector in the presence or in the absence of GFP-DYRK1B. After treatment with MG132 (20 μM) for 6 h, lysates were prepared in denaturing buffer and identical aliquots were immunoprecipitated with antibodies directed against MYC. An anti-HA antibody was used to detect HIF1α ubiquitin conjugates (left panels); Cellular lysates, WCL, were analyzed by western blot using the indicated antibodies (right panels). b, U87 cells were co-transfected with plasmids expressing HA-HIF2α and GFP-DYRK1B or GFP-vector. Cells were treated with 50 μg/ml of C HX for the indicated times and analyzed by western blot. c, Quantification of HIF2α protein from the experiment in panel b as the Log2 of the percentage of HIF2α relative to untreated cells. d, IMR32 cells were co-transfected with ID2 and FLAG-VHL or FLAG-HIF1α expressing vectors. Immunoprecipitation was performed using FLAG antibody and immunocomplexes and whole cell lysates were analyzed by western blot using the indicated antibodies. e, IMR32 cells transfected with FLAG-VHL expression vector were used for IgG or ID2 antibody immunoprecipitation. Immunocomplexes and WCL were analyzed by western blot. Arrow points to the specific FLAG-VHL band; asterisk indicates IgG light chain. f, FLAG immunoprecipitation of binding reactions of in vitro translated FLAG-ID and HA-Elongin C proteins. Immunocomplexes were analyzed by western blot for HA and FLAG. g, FLAG-ID proteins and HA-VHL were translated and incubated in vitro. FLAG immunocomplexes were analyzed by western blot for HA and FLAG. h, In vitro streptavidin pull down assay of biotinylated ID2 peptides (amino acid 14-34 WT, pT27, and T27W) and in vitro translated HA-VHL. Bound polypeptides were detected by western blot.

Mentions: Expression of DYRK1 in GSC#34 and GSC#31 reduced HIF2α, the HIF2α target TGFα and the glioma stem cell marker SOX2 (Fig. 3a, Extended Data Fig. 4g, h). Also in this case HIF2α mRNA was unchanged (Extended Data Fig. 4h, j). LDA and serial clonal experiments showed that DYRK1-induced decrease of HIF2α attenuated glioma stemness (Fig. 3b-d, Extended Data Fig. 4k). However, accumulation of HIF2α, expression of SOX2 and frequency of GSCs were restored by co-expression of DYRK1 and ID2-T27A but not ID2 WT (Fig. 3a-d, Extended Data Fig. 4k). DYRK1-mediated inhibition of gliomasphere formation was overridden by co-expression of non-degradable HIF2α (HIF2α-TM, Extended Fig. 4l)20. Furthermore, silencing of DYRK1A or DYRK1B up-regulated HIF2α and reduced pT27-ID2, with maximal effects after co-silencing of both kinases (Fig. 3e). Next, we investigated the effects of DYRK1 and ID2-T27A on ubiquitylation and stability of HIFα. DYRK1-mediated phosphorylation of T27 triggered HIFα ubiquitylation and expression of ID2-T27A reverted DYRK1 effect (Fig. 3f, Extended Data Figure 5a). Similarly, expression of DYRK1B prevented accumulation of HIF2α under hypoxia and co-expression of ID2-T27A abrogated this response (Fig. 3g). DYRK1 accelerated the decay of HIF2α during recovery from exposure to CoCl2 and reduced HIF2α half-life and ID2-T27A countered these effects (Fig. 3h, i, Extended Data Figure 5b, c).


An ID2-dependent mechanism for VHL inactivation in cancer
The DYRK1-ID2-T27 pathway modulates HIFα stability by regulating the interaction between ID2 and VHLa, In vivo ubiquitylation of HIF1α protein. U87 cells transfected with the expression plasmids HIF1α and MYC-ubiquitin were co-transfected with FLAG-ID2, FLAG-ID2-T27A, or the empty vector in the presence or in the absence of GFP-DYRK1B. After treatment with MG132 (20 μM) for 6 h, lysates were prepared in denaturing buffer and identical aliquots were immunoprecipitated with antibodies directed against MYC. An anti-HA antibody was used to detect HIF1α ubiquitin conjugates (left panels); Cellular lysates, WCL, were analyzed by western blot using the indicated antibodies (right panels). b, U87 cells were co-transfected with plasmids expressing HA-HIF2α and GFP-DYRK1B or GFP-vector. Cells were treated with 50 μg/ml of C HX for the indicated times and analyzed by western blot. c, Quantification of HIF2α protein from the experiment in panel b as the Log2 of the percentage of HIF2α relative to untreated cells. d, IMR32 cells were co-transfected with ID2 and FLAG-VHL or FLAG-HIF1α expressing vectors. Immunoprecipitation was performed using FLAG antibody and immunocomplexes and whole cell lysates were analyzed by western blot using the indicated antibodies. e, IMR32 cells transfected with FLAG-VHL expression vector were used for IgG or ID2 antibody immunoprecipitation. Immunocomplexes and WCL were analyzed by western blot. Arrow points to the specific FLAG-VHL band; asterisk indicates IgG light chain. f, FLAG immunoprecipitation of binding reactions of in vitro translated FLAG-ID and HA-Elongin C proteins. Immunocomplexes were analyzed by western blot for HA and FLAG. g, FLAG-ID proteins and HA-VHL were translated and incubated in vitro. FLAG immunocomplexes were analyzed by western blot for HA and FLAG. h, In vitro streptavidin pull down assay of biotinylated ID2 peptides (amino acid 14-34 WT, pT27, and T27W) and in vitro translated HA-VHL. Bound polypeptides were detected by western blot.
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Figure 5: The DYRK1-ID2-T27 pathway modulates HIFα stability by regulating the interaction between ID2 and VHLa, In vivo ubiquitylation of HIF1α protein. U87 cells transfected with the expression plasmids HIF1α and MYC-ubiquitin were co-transfected with FLAG-ID2, FLAG-ID2-T27A, or the empty vector in the presence or in the absence of GFP-DYRK1B. After treatment with MG132 (20 μM) for 6 h, lysates were prepared in denaturing buffer and identical aliquots were immunoprecipitated with antibodies directed against MYC. An anti-HA antibody was used to detect HIF1α ubiquitin conjugates (left panels); Cellular lysates, WCL, were analyzed by western blot using the indicated antibodies (right panels). b, U87 cells were co-transfected with plasmids expressing HA-HIF2α and GFP-DYRK1B or GFP-vector. Cells were treated with 50 μg/ml of C HX for the indicated times and analyzed by western blot. c, Quantification of HIF2α protein from the experiment in panel b as the Log2 of the percentage of HIF2α relative to untreated cells. d, IMR32 cells were co-transfected with ID2 and FLAG-VHL or FLAG-HIF1α expressing vectors. Immunoprecipitation was performed using FLAG antibody and immunocomplexes and whole cell lysates were analyzed by western blot using the indicated antibodies. e, IMR32 cells transfected with FLAG-VHL expression vector were used for IgG or ID2 antibody immunoprecipitation. Immunocomplexes and WCL were analyzed by western blot. Arrow points to the specific FLAG-VHL band; asterisk indicates IgG light chain. f, FLAG immunoprecipitation of binding reactions of in vitro translated FLAG-ID and HA-Elongin C proteins. Immunocomplexes were analyzed by western blot for HA and FLAG. g, FLAG-ID proteins and HA-VHL were translated and incubated in vitro. FLAG immunocomplexes were analyzed by western blot for HA and FLAG. h, In vitro streptavidin pull down assay of biotinylated ID2 peptides (amino acid 14-34 WT, pT27, and T27W) and in vitro translated HA-VHL. Bound polypeptides were detected by western blot.
Mentions: Expression of DYRK1 in GSC#34 and GSC#31 reduced HIF2α, the HIF2α target TGFα and the glioma stem cell marker SOX2 (Fig. 3a, Extended Data Fig. 4g, h). Also in this case HIF2α mRNA was unchanged (Extended Data Fig. 4h, j). LDA and serial clonal experiments showed that DYRK1-induced decrease of HIF2α attenuated glioma stemness (Fig. 3b-d, Extended Data Fig. 4k). However, accumulation of HIF2α, expression of SOX2 and frequency of GSCs were restored by co-expression of DYRK1 and ID2-T27A but not ID2 WT (Fig. 3a-d, Extended Data Fig. 4k). DYRK1-mediated inhibition of gliomasphere formation was overridden by co-expression of non-degradable HIF2α (HIF2α-TM, Extended Fig. 4l)20. Furthermore, silencing of DYRK1A or DYRK1B up-regulated HIF2α and reduced pT27-ID2, with maximal effects after co-silencing of both kinases (Fig. 3e). Next, we investigated the effects of DYRK1 and ID2-T27A on ubiquitylation and stability of HIFα. DYRK1-mediated phosphorylation of T27 triggered HIFα ubiquitylation and expression of ID2-T27A reverted DYRK1 effect (Fig. 3f, Extended Data Figure 5a). Similarly, expression of DYRK1B prevented accumulation of HIF2α under hypoxia and co-expression of ID2-T27A abrogated this response (Fig. 3g). DYRK1 accelerated the decay of HIF2α during recovery from exposure to CoCl2 and reduced HIF2α half-life and ID2-T27A countered these effects (Fig. 3h, i, Extended Data Figure 5b, c).

View Article: PubMed Central - PubMed

ABSTRACT

Mechanisms that maintain cancer stem cells are crucial to tumor progression. The ID2 protein underpins cancer hallmarks including the cancer stem cell state. HIFα transcription factors, most notably HIF2α, are expressed in and required for maintenance of cancer stem cells (CSCs). However, the pathways that are engaged by ID2 or drive HIF2α accumulation in CSCs have remained unclear. We report that DYRK1A and DYRK1B kinases phosphorylate ID2 on Threonine-27 (T27). Hypoxia down regulates this phosphorylation via inactivation of DYRK1, whose activity is stimulated in normoxia by the oxygen sensing prolyl hydroxylase PHD1. ID2 binds to the VHL ubiquitin ligase complex, displaces VHL-associated Cullin-2, and impairs HIF2α ubiquitylation and degradation. Phosphorylation of ID2-T27 by DYRK1 blocks ID2-VHL interaction and preserves HIF2α ubiquitylation. In glioblastoma ID2 positively modulates HIF2α activity. Conversely, elevated expression of DYRK1 phosphorylates ID2- T27, leading to HIF2α destabilization, loss of glioma stemness, inhibition of tumor growth, and a more favorable outcome for patients with glioblastoma.

No MeSH data available.


Related in: MedlinePlus