Figure 1
STAT3 mutations confer a GOF. (A) Schematic of human STAT3 protein showing the location of 9 different STAT3 missense mutations and the resulting amino acid changes. Mutations were located throughout the protein in the all alpha, DNA binding, SH2, and C-terminal (transactivation) protein domains. The numbers represent the amino acid location based on STAT3 transcript variant 1 (protein model based on the Pfam protein family database). The p.T716M variant was detected in 2 different families (patient 7 and family 2). (B) Western blot of STAT3 expression. WT, GOF, LOF, and the 9 different mutant STAT3 transcripts were expressed in the STAT3-deficient A4 cell line and expression of pSTAT3 and total STAT3 protein was determined using western blot without (top) and with (bottom) IL-6 (15 minutes, 10 ng/mL). IL-6–treated DLD cells (parental A4 line) are shown as a control. Expression of all mutants led to the detection of STAT3 protein, with the exception of p.A703T, a mutation near the binding site of the antibody used here (clone 79D7). This variant was detectable after IL-6 activation and with a different antibody clone (supplemental Figure 2). Mutant STAT3 proteins were not phosphorylated at baseline, but were phosphorylated after stimulation with IL-6. (C-D) STAT3 binding activity as measured by luciferase assay. A4 cells were transfected with WT or the indicated STAT3 mutants and STAT3-driven firefly luciferase and control renilla reporters. (C) Luciferase was assayed at 48 hours and STAT3 activity is shown as a ratio of firefly/control for each construct. All constructs with the exception of V353F had significantly increased activity at baseline compared with WT. Results represent the mean ± SEM of 5 to 10 independent experiments; the dotted line represents WT (**P < .01, ***P < .001, ****P < .0001). (D) STAT3 activity following 12-hour activation with IL-6 (10 ng/mL, white) or IFN-α (50 ng/mL, black). All mutations demonstrated significantly increased STAT3 activity after at least 1 of the cytokine stimulations (P < .05), typically after both unless indicated. Results shown represent fold-change vs WT for each cytokine stimulus; the dotted line represents a fold-change of 1 (no change from WT). Results represent the mean ± SEM of 5 independent experiments. ns, not significant; pSTAT3, phosphorylated STAT3.

STAT3 mutations confer a GOF. (A) Schematic of human STAT3 protein showing the location of 9 different STAT3 missense mutations and the resulting amino acid changes. Mutations were located throughout the protein in the all alpha, DNA binding, SH2, and C-terminal (transactivation) protein domains. The numbers represent the amino acid location based on STAT3 transcript variant 1 (protein model based on the Pfam protein family database). The p.T716M variant was detected in 2 different families (patient 7 and family 2). (B) Western blot of STAT3 expression. WT, GOF, LOF, and the 9 different mutant STAT3 transcripts were expressed in the STAT3-deficient A4 cell line and expression of pSTAT3 and total STAT3 protein was determined using western blot without (top) and with (bottom) IL-6 (15 minutes, 10 ng/mL). IL-6–treated DLD cells (parental A4 line) are shown as a control. Expression of all mutants led to the detection of STAT3 protein, with the exception of p.A703T, a mutation near the binding site of the antibody used here (clone 79D7). This variant was detectable after IL-6 activation and with a different antibody clone (supplemental Figure 2). Mutant STAT3 proteins were not phosphorylated at baseline, but were phosphorylated after stimulation with IL-6. (C-D) STAT3 binding activity as measured by luciferase assay. A4 cells were transfected with WT or the indicated STAT3 mutants and STAT3-driven firefly luciferase and control renilla reporters. (C) Luciferase was assayed at 48 hours and STAT3 activity is shown as a ratio of firefly/control for each construct. All constructs with the exception of V353F had significantly increased activity at baseline compared with WT. Results represent the mean ± SEM of 5 to 10 independent experiments; the dotted line represents WT (**P < .01, ***P < .001, ****P < .0001). (D) STAT3 activity following 12-hour activation with IL-6 (10 ng/mL, white) or IFN-α (50 ng/mL, black). All mutations demonstrated significantly increased STAT3 activity after at least 1 of the cytokine stimulations (P < .05), typically after both unless indicated. Results shown represent fold-change vs WT for each cytokine stimulus; the dotted line represents a fold-change of 1 (no change from WT). Results represent the mean ± SEM of 5 independent experiments. ns, not significant; pSTAT3, phosphorylated STAT3.

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