Figure 2
Figure 2. EZH2 Y244 site-specific phosphorylation mediates the effects of activated JAK3. (A) Coimmunoprecipitation assay showing that EZH2 is subject to tyrosine phosphorylation. NKYS cells were used to immunoprecipitate EZH2 and to probe antiphosphotyrosine antibody. (B) Coimmunoprecipitation assay showing that tyrosine phosphorylation of EZH2 in NKYS cells in the presence or absence of JAK3 inhibitor PF. EZH2 was immunoprecipitated from cells and were immunoblotted with antiphosphotyrosine antibodies to reveal the tyrosine phosphorylation levels of EZH2. (C) The top 3 phosphorylated tyr residues predicted by multiple phosphorylation site prediction programs. The 3 phosphorylation defective mutants were generated by mutating Y to A. (D) MTS cell viability assay showing different abilities of ectopic expression of EZH2 WT/mutants to affect cell growth of NKYS cells. Cells were transfected with the control empty vector pcDNA4.1 or EZH2 expression plasmids. The mean values of triplicate samples are shown, and error bars indicate standard deviation (SD). n = 4 independent experiments. *P < .05 (Student t test). (E) Luciferase reporter assay showing the effect of different EZH2 mutants on CCND1 promoter activity. NKYS cells were transfected with the luciferase reporter construct pGL4 containing the CCND1 promoter and various amounts of EZH2 WT/mutant plasmids or a control vector. Luciferase activities were measured after 24 hours. Luciferase readings were further normalized to the internal control pRL null. Results are presented as averages of triplicate experiments. Error bars indicate SD. *P < .05 (Student t test). (F) Coimmunoprecipitation assay of EZH2 tyrosine phosphorylation in 293T cells cotransfected with EZH2 WT/Y244A and JAK3 A572A for 48 hours. Transfected EZH2 was immunoprecipitated from cells using antihemagglutinin (HA) antibody and was immunoblotted with pEZH2-Y244 antibody or antiphosphotyrosine antibody to reveal the tyrosine phosphorylation level of EZH2. (G) Immunoblot analysis of pEZH2-Y244 levels in NKTL cells exposed to JAK3 inhibitor PF for 2 hours. (H) Immunofluorescent staining of H3 K27 trimethylation in NKYS cells transfected with EZH2 WT or Y244A expression plasmids for 2 days. Scale bars, 10 μm. The HA-EZH2-positive staining cells from EZH2 Y244A in general exhibit a stronger increase in the staining (relative to those nontransfected cells in the same field) for H3 K27 trimethylation compared with those positive staining cells from EZH2 WT. (I) Representative of immunoblot (left) and densitometric quantification (right; n = 3 independent experiments) of H3K27 trimethylation in 293T cells transfected with EZH2 WT/Y244A expression plasmids or control vector for 48 hours. Results are means ± SD. *P < .05 (Student t test). Expression of EZH2 WT and the Y244A mutant was detected by the HA-tag antibody. H3 and actin were used as loading controls. (J) Immunoblot analysis of H3 K27 trimethylation in 293T cells cotransfected with JAK3 A572V and EZH2 WT/Y244A expression plasmid or control vectors for 48 hours. Expression of EZH2 WT and the Y244A mutant was detected by the HA-tag antibody.

EZH2 Y244 site-specific phosphorylation mediates the effects of activated JAK3. (A) Coimmunoprecipitation assay showing that EZH2 is subject to tyrosine phosphorylation. NKYS cells were used to immunoprecipitate EZH2 and to probe antiphosphotyrosine antibody. (B) Coimmunoprecipitation assay showing that tyrosine phosphorylation of EZH2 in NKYS cells in the presence or absence of JAK3 inhibitor PF. EZH2 was immunoprecipitated from cells and were immunoblotted with antiphosphotyrosine antibodies to reveal the tyrosine phosphorylation levels of EZH2. (C) The top 3 phosphorylated tyr residues predicted by multiple phosphorylation site prediction programs. The 3 phosphorylation defective mutants were generated by mutating Y to A. (D) MTS cell viability assay showing different abilities of ectopic expression of EZH2 WT/mutants to affect cell growth of NKYS cells. Cells were transfected with the control empty vector pcDNA4.1 or EZH2 expression plasmids. The mean values of triplicate samples are shown, and error bars indicate standard deviation (SD). n = 4 independent experiments. *P < .05 (Student t test). (E) Luciferase reporter assay showing the effect of different EZH2 mutants on CCND1 promoter activity. NKYS cells were transfected with the luciferase reporter construct pGL4 containing the CCND1 promoter and various amounts of EZH2 WT/mutant plasmids or a control vector. Luciferase activities were measured after 24 hours. Luciferase readings were further normalized to the internal control pRL null. Results are presented as averages of triplicate experiments. Error bars indicate SD. *P < .05 (Student t test). (F) Coimmunoprecipitation assay of EZH2 tyrosine phosphorylation in 293T cells cotransfected with EZH2 WT/Y244A and JAK3 A572A for 48 hours. Transfected EZH2 was immunoprecipitated from cells using antihemagglutinin (HA) antibody and was immunoblotted with pEZH2-Y244 antibody or antiphosphotyrosine antibody to reveal the tyrosine phosphorylation level of EZH2. (G) Immunoblot analysis of pEZH2-Y244 levels in NKTL cells exposed to JAK3 inhibitor PF for 2 hours. (H) Immunofluorescent staining of H3 K27 trimethylation in NKYS cells transfected with EZH2 WT or Y244A expression plasmids for 2 days. Scale bars, 10 μm. The HA-EZH2-positive staining cells from EZH2 Y244A in general exhibit a stronger increase in the staining (relative to those nontransfected cells in the same field) for H3 K27 trimethylation compared with those positive staining cells from EZH2 WT. (I) Representative of immunoblot (left) and densitometric quantification (right; n = 3 independent experiments) of H3K27 trimethylation in 293T cells transfected with EZH2 WT/Y244A expression plasmids or control vector for 48 hours. Results are means ± SD. *P < .05 (Student t test). Expression of EZH2 WT and the Y244A mutant was detected by the HA-tag antibody. H3 and actin were used as loading controls. (J) Immunoblot analysis of H3 K27 trimethylation in 293T cells cotransfected with JAK3 A572V and EZH2 WT/Y244A expression plasmid or control vectors for 48 hours. Expression of EZH2 WT and the Y244A mutant was detected by the HA-tag antibody.

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