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. 2017 Oct;31(10):2132-2142.
doi: 10.1038/leu.2017.4. Epub 2017 Jan 11.

O-GlcNAcylation of STAT5 Controls Tyrosine Phosphorylation and Oncogenic Transcription in STAT5-dependent Malignancies

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Free PMC article

O-GlcNAcylation of STAT5 Controls Tyrosine Phosphorylation and Oncogenic Transcription in STAT5-dependent Malignancies

P Freund et al. Leukemia. .
Free PMC article

Abstract

The signal transducer and activator of transcription 5 (STAT5) regulates differentiation, survival, proliferation and transformation of hematopoietic cells. Upon cytokine stimulation, STAT5 tyrosine phosphorylation (pYSTAT5) is transient, while in diverse neoplastic cells persistent overexpression and enhanced pYSTAT5 are frequently found. Post-translational modifications might contribute to enhanced STAT5 activation in the context of transformation, but the strength and duration of pYSTAT5 are incompletely understood. We found that O-GlcNAcylation and tyrosine phosphorylation act together to trigger pYSTAT5 levels and oncogenic transcription in neoplastic cells. The expression of a mutated hyperactive gain-of-function (GOF) STAT5 without O-GlcNAcylation resulted in decreased tyrosine phosphorylation, oligomerization and transactivation potential and complete loss of oncogenic transformation capacity. The lack of O-GlcNAcylation diminished phospho-ERK and phospho-AKT levels. Our data show that O-GlcNAcylation of STAT5 is an important process that contributes to oncogenic transcription through enhanced STAT5 tyrosine phosphorylation and oligomerization driving myeloid transformation. O-GlcNAcylation of STAT5 could be required for nutrient sensing and metabolism of cancer cells.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
O-GlcNAcylation in the STAT5 N-terminus at position threonine 92. (a) Schematic representation of murine STAT5A and the mutant STAT5 proteins. (b) Sequence of the human STAT5A N-terminus from amino-acid 1 to 136 with the prediction for the position of the eight alpha helices. (c) The crystal structure of the STAT4 N-terminus in two different orientations. The numbers indicate the eight helices as shown in b.
Figure 2
Figure 2
O-GlcNAcylation of STAT5 and tetramer formation. (a) Immunoblot of pull down and flow through from WGA assay for mouse fibroblasts gpE+86 and human cell lines (n=2). (b) STAT5-tetramer electrophoretic mobility shift assays (EMSAs) on a 2 × β-casein site with whole-cell extracts of parental and cS5 or cS5-T92A transfected Ba/F3 cells with and without 10 ng/ml IL-3 stimulation (n=2).
Figure 3
Figure 3
The lack of O-GlcNAcylation in STAT5A led to decreased pYSTAT5 and reduced STAT5 target gene expression in cS5 context. (a) pYSTAT5 and phospho-serine (pSSTAT5) immunoblots of parental and transfected (cS5 and cS5-T92A) Ba/F3 cells with and without IL-3 (20 ng/ml) stimulation (n=2). (b) Analysis of transcriptional expression of STAT5 target genes in Ba/F3 cells after IL-3 removal by semi quantitative real-time PCR with Rpl13a as housekeeping gene, performed in triplicates. Parental vs cS5 and parental vs cS5-T92A (ns=not significant; *P<0.05; **P<0.01; ***P<0.001 two-way analysis of variance (ANOVA)) cS5 vs cS5-T92A (#P<0.01; ##P<0.001 two-way ANOVA). (c) pYSTAT5 western blot of parental and transfected Ba/F3 cells, treated with vehicle (aqua) or incubated with 10 mM, 20 mM, 30 mM Alloxan for 1 h and stimulated with IL-3 (10 ng/ml) for 15 min (n=2).
Figure 4
Figure 4
Hyperactive, O-GlcNAcylated STAT5 maintains tyrosine phosphorylation and phospho-AKT and phospho-ERK levels. (a) Phospho-tyrosine immunoblot of transfected Ba/F3 cells after IL-3 removal for indicated time points (n=2). (b) Phospho- and total-protein immunoblot for AKT, ERK and STAT5 in Ba/F3 cells, electroporated with cS5 or cS5-T92A (n=2).
Figure 5
Figure 5
Lack of STAT5 O-GlcNAcylation in context of cS5 re-establishes cytokine dependence for its proliferative activity. (a) Growth curve of transfected Ba/F3 cells upon IL-3 starvation. Cells were grown and counted in triplicates (ns=not significant; ***P<0.001 two-way analysis of variance (ANOVA)) (n=2). (b) FACS analysis of transduced T-cells for Thy1.2/GFP double-positive cells (n=2). (c) Viable cells were examined for proliferation upon IL-2 and IL-4 stimulation by 3H-thymidine incorporation assay. Mean and s.d. were calculated from four single wells (ns=not significant; *P<0.05; ***P<0.001 one-way ANOVA) (n=2).
Figure 6
Figure 6
The transforming potential of cS5 requires O-GlcNAcylation. (a) white blood cell count of transplanted mice at 4 weeks (left) and 12 months (right) (n⩾4, each) (***P<0.001 one-way analysis of variance (ANOVA)) post transplant. (b) Kaplan–Meier blot of transplanted mice (n=12, each) (P<0.0001 log-rank test). (c) Histo-pathology analysis of blood, liver, spleen and BM of 4-weeks-old mice transplanted with MSCV-GFP, cS5 or cS5-T92A (Magnifications: 40 × blood; 20 × liver; 10 × spleen; 20 × BM). Blood smears were stained with Benzidine, all other organ slides were hematoxylin and eosin stained.
Figure 7
Figure 7
Pre-leukemic cell populations of cS5 transplanted mice showed myeloproliferative neoplasia, but cS5-T92A supports normal hematopoiesis. (a) Flow cytometry analysis for myeloid markers (Gr-1, Mac-1) of transplanted mice from blood, BM and spleen, 4 weeks after transplantation (n=4). (b) FACS analysis of BM from MSCV-GFP, cS5 and cS5-T92A transplanted mice for lineage-negative (Lin-) stem-cell antigen 1 (SCA1+) KIT+ (LSK) cells at 4 weeks post transplantation (n=4). (c) Flow cytometry analysis for Ter119 and GFP of peripheral blood from cS5-T92A transplanted mice drawn at indicated time points (n=4).

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