Fig 2: IGF-dependent phosphorylation of STX7 at Ser126 and of STX12 at Ser139 by SGK3.HEK293 stable cells lines expressing (A) GFP-STX7 wild type (WT) or the Ala mutant GFP-STX7 S126A and (B) GFP-STX12 wild type (WT) or the Ala mutant GFP-STX12 S139A were generated in STX7 knock-out (KO) and STX12 KO HEK293 cells, respectively. (A) KO STX7, GFP-STX7 WT and GFP-STX7 S126A HEK293 cells were serum-starved overnight, treated for 1 h with 14H (1 µM) or with DMSO, followed by stimulation with IGF1 (50 ng/ml) for 15 min. STX7 phosphorylation was analyzed by Phos-tag assay (top panels, ° phosphorylated, • non-phosphorylated). Control immunoblots were performed on normal gels with the indicated antibodies. Immunoblots were developed using the LI-COR Odyssey CLx Western Blot imaging system analysis with the indicated antibodies at 0.5–1 µg/ml concentration. (B) KO STX12, GFP-STX12 WT and GFP-STX12 S139A HEK293 cells were serum-starved overnight, treated for 1 h with 14H (1 µM) or with DMSO, followed by stimulation with IGF1 (50 ng/ml) for 15 min. STX12 phosphorylation was analyzed by Phos-tag assay (top panels, ° phosphorylated, • non-phosphorylated, ns; non-specific). Control immunoblots were performed on normal gels with the indicated antibodies. Immunoblots were developed using the LI-COR Odyssey CLx Western Blot imaging system analysis with the indicated antibodies at 0.5–1 µg/ml concentration. (C) WT or STX12 KO HEK293 were serum-starved overnight, treated with IGF1 (50 ng/ml) for 15 min and samples immunoblotted with the indicated antibodies 0.5–1 µg/ml concentration. The immunoblots were developed using enhanced chemiluminescence and X-ray film. (D) HEK293 cells were transfected with plasmids inducing HA-STX12 WT or HA-STX12 S139A. Twenty-four hours post-transfection cells were serum-starved overnight, treated for 1 h with 14H (1 µM), MK2206 (1 µM) or with DMSO, followed by stimulation with IGF1 (50 ng/ml) for 15 min. Samples were analyzed by immunoblot analysis as described in (D). (E) WT or SGK3 KO HEK293 were serum-starved overnight, treated with IGF1 (50 ng/ml) for 15 min and the samples were analyzed as in (D).

Fig 3: Model of the SGK3 signaling pathway.(A) IGF1 stimulates the production of PtdIns(3)P by the Class I and class III Vps34 at the endosomes where SGK3 can be recruited and activated by PDK1 and mTORC2 [22]. Once activated SGK3 phosphorylates its selective substrates STX7 and STX12 that are involved in the endocytic pathway. Phosphorylation of STX12 at Ser 139 by SGK3 that is located just beyond the N-terminal Habc regulatory domain promotes interaction with the t-SNARE proteins syntaxin 6 and Vti1a and the v-SNARE protein VAMP4. Our data suggest that this promotes plasma membrane localization. We propose that assessing phosphorylation of STX7 (Ser126) and STX12 (Ser139) that are specific substrates for SGK2 could be used as key biomarkers for the SGK3 signaling pathway.

Fig 4: Phosphorylation of STX12 at Ser139 by SGK3 enhances the association with the SNARE complex.(A) The indicated knock-out (KO), and KO cells stably re-expressing wild-type or STX[S139A] HEK293 cells were serum-starved overnight, treated for 1 h with 14H (1 µM) or with DMSO, followed by stimulation with IGF1 (50 ng/ml) for 15 min. GFP-STX12 WT and GFP-STX12 S139A were immunoprecipitated using anti-GFP beads and the immunoprecipitates were subjected to western blot analysis using the indicated antibodies. STX12 phosphorylation was analyzed by Phos-tag assay (° phosphorylated, • non-phosphorylated). Control immunoblots were performed on normal gels with the indicated antibodies. Immunoblots were developed using the LI-COR Odyssey CLx Western Blot imaging system analysis with the indicated antibodies at 0.5–1 µg/ml concentration. Quantification of the respective binding is also provided.

Fig 5: SGK3-dependent phosphorylation of endogenous STX7 and STX12 in vivo.(A) Wild-type HEK293 cells were serum-starved overnight, then treated for 1 h with 14H (1 µM), MK2206 (1 µM) or with DMSO, followed by stimulation with IGF1 (50 ng/ml) for 15 min. STX7 and STX12 phosphorylation was analyzed by Phos-tag assays (top panels, ° phosphorylated, • non-phosphorylated, ns; non-specific). Control immunoblots were performed on normal gels with the indicated antibodies. Immunoblots were developed using the LI-COR Odyssey CLx Western Blot imaging system analysis with the indicated antibodies at 0.5–1 µg/ml concentration.(B) Wild-type (WT) and SGK3 knock-out (KO) HEK293 cells were serum-starved overnight, then treated for 1 h with 14H (1 µM) or with DMSO, followed by stimulation with IGF1 (50 ng/ml) for 15 min. STX7 and STX12 phosphorylation was analyzed by Phos-tag assays (top panels, ° phosphorylated, • non-phosphorylated, ns; non-specific). Control immunoblots were performed on normal gels with the indicated antibodies.