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An ID2-dependent mechanism for VHL inactivation in cancer

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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.


DYRK1-mediated phosphorylation of ID2 at T27 promotes NSC propertiesa, Reconstitution of NSCs ID2-/- with ID2, ID2-T27A, or the empty vector. b, Microphotographs of representative cultures from neurosphere forming assay. c, Percent neurospheres generated in serial clonal assays. (means of 3 biological replicates ± s.d.; ***: p=0.00883-0.000229 for ID2−/−-ID2-T27A compared with ID2−/−-ID2-WT). d, Cumulative cell number of cultures as in c; (means of 3 biological replicates ± s.d. ***: p=<0.0001 for ID2−/−-ID2-T27A compared with ID2−/−-ID2-WT). e, In vitro kinase assay shows phosphorylation of GST-ID2 proteins by recombinant DYRK1B. f, Phosphorylation of ID2 but not ID2-T27A by DYRK1B in IMR32 cells. g, Phosphorylation of endogenous ID2 by DYRK1A in U87 cells. h, Phosphorylation of endogenous ID2 by DYRK1B but not the kinase inactive GFP-DYRK1B-K140R in U87 cells. i, Binding between endogenous DYRK1A or DYRK1B and ID2. WCL: whole cellular lysate.
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Figure 11: DYRK1-mediated phosphorylation of ID2 at T27 promotes NSC propertiesa, Reconstitution of NSCs ID2-/- with ID2, ID2-T27A, or the empty vector. b, Microphotographs of representative cultures from neurosphere forming assay. c, Percent neurospheres generated in serial clonal assays. (means of 3 biological replicates ± s.d.; ***: p=0.00883-0.000229 for ID2−/−-ID2-T27A compared with ID2−/−-ID2-WT). d, Cumulative cell number of cultures as in c; (means of 3 biological replicates ± s.d. ***: p=<0.0001 for ID2−/−-ID2-T27A compared with ID2−/−-ID2-WT). e, In vitro kinase assay shows phosphorylation of GST-ID2 proteins by recombinant DYRK1B. f, Phosphorylation of ID2 but not ID2-T27A by DYRK1B in IMR32 cells. g, Phosphorylation of endogenous ID2 by DYRK1A in U87 cells. h, Phosphorylation of endogenous ID2 by DYRK1B but not the kinase inactive GFP-DYRK1B-K140R in U87 cells. i, Binding between endogenous DYRK1A or DYRK1B and ID2. WCL: whole cellular lysate.

Mentions: We used mass spectrometry to identify the phosphorylation sites of human ID2. Beside Serine-511, we found that ID2 is phosphorylated on Serine-14 and Threonine-27 (Extended Data Fig. 1a-c). A sequencing analysis of the ID2 gene in cancer revealed that the colorectal cancer cell line HRT-18 harbors and expresses a mutant ID2-T27A protein (Extended Data Fig. 2a, b). Threonine-27 of ID2 is highly conserved throughout evolution (Extended Data Fig. 2c). The primary role of ID proteins is to preserve stem cell properties, a function widely documented in neural stem cells (NSCs)12,13. Therefore, to interrogate the significance of the ID2-T27A mutation, we tested the self-renewing capacity of ID2- NSCs reconstituted with wild type (WT) or ID2-T27A (Fig. 1a). Introduction of ID2-T27A in ID2- NSCs increased neurosphere formation in serial passages by more than 50% when compared with WT ID2 (p = 0.00883-0.000229; t ratio = 4.772-12.597) and caused a 2.4-fold increase in cell expansion rate (40.5±1.7 vs. 16.7±0.831; p < 0.0001, Fig. 1b-d). From the analysis of 18 candidate kinases, the dual-specificity tyrosine-phosphorylation-regulated protein kinases 1A and 1B (DYRK1A and DYRK1B) emerged as the only enzymes able to phosphorylate T27 of ID2 (Fig. 1e, Extended Data Fig. 2d). The sequence surrounding the T27 residue in ID2 resembles the DYRK1 phosphorylation consensus motif RX(X)(S/T)P and is highly conserved in different species (Extended Data Fig. 2c)14. Antibodies against a phospho-T27-ID2 peptide confirmed that ID2 is phosphorylated by WT but not the inactive DYRK1B-K140R kinase (Fig. 1a, f-h)15. Endogenous and exogenous ID2 and ID2-T27A co-precipitated endogenous DYRK1A and DYRK1B (Fig. 1i, Extended Data Fig. 2e). Treatment of glioma cells with harmine, a small molecule inhibitor of DYRK116, or combined shRNA-mediated silencing of DYRK1A and DYRK1B reduced ID2-T27 phosphorylation (Extended Data Fig. 2f, Fig. 3f).


An ID2-dependent mechanism for VHL inactivation in cancer
DYRK1-mediated phosphorylation of ID2 at T27 promotes NSC propertiesa, Reconstitution of NSCs ID2-/- with ID2, ID2-T27A, or the empty vector. b, Microphotographs of representative cultures from neurosphere forming assay. c, Percent neurospheres generated in serial clonal assays. (means of 3 biological replicates ± s.d.; ***: p=0.00883-0.000229 for ID2−/−-ID2-T27A compared with ID2−/−-ID2-WT). d, Cumulative cell number of cultures as in c; (means of 3 biological replicates ± s.d. ***: p=<0.0001 for ID2−/−-ID2-T27A compared with ID2−/−-ID2-WT). e, In vitro kinase assay shows phosphorylation of GST-ID2 proteins by recombinant DYRK1B. f, Phosphorylation of ID2 but not ID2-T27A by DYRK1B in IMR32 cells. g, Phosphorylation of endogenous ID2 by DYRK1A in U87 cells. h, Phosphorylation of endogenous ID2 by DYRK1B but not the kinase inactive GFP-DYRK1B-K140R in U87 cells. i, Binding between endogenous DYRK1A or DYRK1B and ID2. WCL: whole cellular lysate.
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Figure 11: DYRK1-mediated phosphorylation of ID2 at T27 promotes NSC propertiesa, Reconstitution of NSCs ID2-/- with ID2, ID2-T27A, or the empty vector. b, Microphotographs of representative cultures from neurosphere forming assay. c, Percent neurospheres generated in serial clonal assays. (means of 3 biological replicates ± s.d.; ***: p=0.00883-0.000229 for ID2−/−-ID2-T27A compared with ID2−/−-ID2-WT). d, Cumulative cell number of cultures as in c; (means of 3 biological replicates ± s.d. ***: p=<0.0001 for ID2−/−-ID2-T27A compared with ID2−/−-ID2-WT). e, In vitro kinase assay shows phosphorylation of GST-ID2 proteins by recombinant DYRK1B. f, Phosphorylation of ID2 but not ID2-T27A by DYRK1B in IMR32 cells. g, Phosphorylation of endogenous ID2 by DYRK1A in U87 cells. h, Phosphorylation of endogenous ID2 by DYRK1B but not the kinase inactive GFP-DYRK1B-K140R in U87 cells. i, Binding between endogenous DYRK1A or DYRK1B and ID2. WCL: whole cellular lysate.
Mentions: We used mass spectrometry to identify the phosphorylation sites of human ID2. Beside Serine-511, we found that ID2 is phosphorylated on Serine-14 and Threonine-27 (Extended Data Fig. 1a-c). A sequencing analysis of the ID2 gene in cancer revealed that the colorectal cancer cell line HRT-18 harbors and expresses a mutant ID2-T27A protein (Extended Data Fig. 2a, b). Threonine-27 of ID2 is highly conserved throughout evolution (Extended Data Fig. 2c). The primary role of ID proteins is to preserve stem cell properties, a function widely documented in neural stem cells (NSCs)12,13. Therefore, to interrogate the significance of the ID2-T27A mutation, we tested the self-renewing capacity of ID2- NSCs reconstituted with wild type (WT) or ID2-T27A (Fig. 1a). Introduction of ID2-T27A in ID2- NSCs increased neurosphere formation in serial passages by more than 50% when compared with WT ID2 (p = 0.00883-0.000229; t ratio = 4.772-12.597) and caused a 2.4-fold increase in cell expansion rate (40.5±1.7 vs. 16.7±0.831; p < 0.0001, Fig. 1b-d). From the analysis of 18 candidate kinases, the dual-specificity tyrosine-phosphorylation-regulated protein kinases 1A and 1B (DYRK1A and DYRK1B) emerged as the only enzymes able to phosphorylate T27 of ID2 (Fig. 1e, Extended Data Fig. 2d). The sequence surrounding the T27 residue in ID2 resembles the DYRK1 phosphorylation consensus motif RX(X)(S/T)P and is highly conserved in different species (Extended Data Fig. 2c)14. Antibodies against a phospho-T27-ID2 peptide confirmed that ID2 is phosphorylated by WT but not the inactive DYRK1B-K140R kinase (Fig. 1a, f-h)15. Endogenous and exogenous ID2 and ID2-T27A co-precipitated endogenous DYRK1A and DYRK1B (Fig. 1i, Extended Data Fig. 2e). Treatment of glioma cells with harmine, a small molecule inhibitor of DYRK116, or combined shRNA-mediated silencing of DYRK1A and DYRK1B reduced ID2-T27 phosphorylation (Extended Data Fig. 2f, Fig. 3f).

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. HIF&alpha; transcription factors, most notably HIF2&alpha;, are expressed in and required for maintenance of cancer stem cells (CSCs). However, the pathways that are engaged by ID2 or drive HIF2&alpha; 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 HIF2&alpha; ubiquitylation and degradation. Phosphorylation of ID2-T27 by DYRK1 blocks ID2-VHL interaction and preserves HIF2&alpha; ubiquitylation. In glioblastoma ID2 positively modulates HIF2&alpha; activity. Conversely, elevated expression of DYRK1 phosphorylates ID2- T27, leading to HIF2&alpha; destabilization, loss of glioma stemness, inhibition of tumor growth, and a more favorable outcome for patients with glioblastoma.

No MeSH data available.