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. Author manuscript; available in PMC: 2021 Jan 8.
Published in final edited form as: Cell Host Microbe. 2019 Dec 31;27(1):129–139.e4. doi: 10.1016/j.chom.2019.11.012

Figure 4. SarA interaction with GSK-3, but not JAK1, is required for STAT3 activation.

Figure 4.

(A) IP-MS identified SarA-STAT3 signaling complex components. Peptide spectral counts from the indicated number of unique peptides. (B-D) JAK inhibitors block gp130-ligands’ induction of STAT3 phosphorylation without affecting SarA-mediated STAT3 phosphorylation in LCL (B) and in HeLa (C, D). LCL GM19154 was pre-treated for 1 hr with 50 μM Midostaurin (Mido), 50 μM JAK Inhibitor I (JAK In), or 50 μM Ruxolitinib (Ruxo) before 1 hr stimulation with IL-6 (50ng/ml) or IL-21 (50ng/ml) or infection with S. Typhimurium (STm). HeLa cells were identically pre-treated before 30 min stimulation with OSM (10ng/mL) or infection with STm. (E) RNAi confirms STAT3 phosphorylation by SarA is JAK independent, unlike OSMR signaling. HeLas were pre-treated with JAK1 and/or JAK2 siRNA for 48 hrs before infection with STm or stimulation with OSM (10ng/mL) for indicated time. qPCR confirmed knockdown exceeded 3-fold each time. (F) Co-immunoprecipitation (coIP) confirms GSK-3α and β, but not SOCS3, interact with SarA. HeLa cells were transfected with codon-optimized pcDNA3.1-sarA or pcDNA3.1-Flag-sarA. (G-I) GSK-3 inhibition demonstrates that GSK-3 enzymatic activity is necessary for SarA-induced STAT3 phosphorylation in HeLa and LCL GM19154. HeLas were pre-treated with 15 μM GSK-3 Inhibitor XIII (XIII) or 15 μM CHIR-98014 (CHIR) for 1 hr before STm infection (MOI 5) and harvested 8 hpi. LCLs were pre-treated for 1 hr with 12 μM GSK-3 Inhibitor XIII or 500 nM CHIR-98014 before STm infection (MOI 30) and lysed 5 hpi. IL-10 in LCL supernatant (I) is eight replicates across three experiments. P value from Sidak’s post-hoc comparison of WT infection after two-way ANOVA (STm genotype, treatment, and interaction term all significant with p<0.0001). Bars are mean ±SEM. (J, K) Knockdown of GSK3A and GSK3B confirms partial dependence of SarA on GSK-3. HeLa were pre-treated identically to D. (L) GSK-3 inhibitor blocks SarA interaction with STAT3 and GSK-3, which prevent STAT3 and SarA phosphorylation. coIP performed like E with 5 μM GSK-3 Inhibitor XIII added 1 h after transfection. (M, N) S130A mutation reduces complementation during STm infection. P value from Welch’s t test. (O) SarA Y167F still associates with GSK-3. Anti-Flag M2 coIP and blotting performed same as figure 2D. (P) Model of the SarA-GSK-3 complex activating STAT3 compared to JAK1-gp130 activation of STAT3. (Q) GSK-3 inhibition reduces IL-6-induced STAT3 phosphorylation in LCLs. GM19154 was pre-treated with 15 μM GSK-3 inhibitor XIII or 50 μM Ruxolitinib for 1 hr before stimulation with IL-6 (100ng/mL) for 1 hr. Blot is representative of three experiments which are quantified relative to uninhibited IL-6 treatment. P value from Welch’s t test on log transformed ratio.