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. 2020 Jul 15;133(14):jcs241356. doi: 10.1242/jcs.241356

Fig. 6.

Fig. 6.

BRSK2 suppresses protein translation. (A) BRSK2 induces AMPK substrate phosphorylation. Western blot analysis of AMPK consensus phosphorylation sites in HEK293T cells transfected with hcRED, BRSK2 or the indicated BRSK2 mutants (representative of n=3). (B) BRSK2 and BRSK1 downregulate mTOR signaling. Western blot analysis of BRSK1 and BRSK2 expression in HEK293T cells for mTOR targets S6K (T389) and 4E-BP1 (T37/46 and S65), which increases 4E-BP1 stability (representative of n=3). (C) BRSK2 and BRSK1 downregulate protein translation. Global protein translation assay performed using 35S-methionine labeling in cells expressing hcRED (control), BRSK2, BRSK2 K48A or BRSK1. (D) BRSK2 reduced polyribosome formation. Polyribosome fraction of HEK293T cells overexpressing hcRED control, BRSK2, BRSK2 K48A or BRSK1 performed on 50-10% sucrose gradient. Experiments represent biological triplicates, unless indicated otherwise. (E) mTOR inhibition reduced NRF2 protein. HEK293T cells were treated with DMSO, INK-128 (100 nM) or TORIN2 (100 nM) for 7 h followed by western blot analysis. (F) Quantification of NRF2 blots using DMSO as control. (G) BRSK2 overexpression recapitulates phenotypes observed with mTOR inhibitors. NS, nonspecific. (H) Signaling model: BRSK2-mediated mTOR downregulation leads to decreased protein translation. Two-way ANOVA with multiple comparison was performed comparing controls to other conditions. *P<0.05, **P<0.01, ***P<0.001; ns, not statistically significant.