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. Author manuscript; available in PMC: 2021 Nov 9.
Published in final edited form as: Dev Cell. 2020 Sep 7;55(3):272–288.e5. doi: 10.1016/j.devcel.2020.08.006

Figure 7. Phosphorylation by AKT decreases the lysosomal dynamics of TSC2.

Figure 7.

(A) Immunoblots to detect the level of P-S6K (T389), P-TSC2 (T1462) in WT and WDR24-KO HeLa cells treated with control (NT) or RAGA siRNA, cultured in +AA +FBS, +AA -FBS and AA +dFBS conditions.

(B) Immunoblots show knockdowns of RAGA and RAGC in WDR24-KO cells increase the level of P-AKT (T308) and P-AKT (S473).

(C) Time point images from the Halo-TSC2 FRAP experiment in WT HeLa cells transfected with Halo-TSC2 T1462A, Halo-TSC2 S939A and the Halo-TSC2 T1462A/S939A. Scale bar: 0.5 μm.

(D) Fluorescence recovery versus time curves in C. A total of 30 lysosomes from 30 different cells were used in plotting the curve for each treatment. Error bars represent standard error.

(E) Plot showing the relation between the recovery fraction versus half time (t1/2) from the curves in D. Error bar represents standard error.

(F) Time point images from the Halo-TSC2 FRAP experiment in WDR24-KO HeLa cells transfected with Halo-TSC2 T1462A, Halo-TSC2 S939A and the Halo-TSC2 T1462A/S939A.

(G) Fluorescence recovery versus time curves in F. A total of 30 lysosomes from 30 different cells were used in plotting the curve for each treatment.

(H) Plot showing the relation between the recovery fraction versus half time (t1/2) from the curves in G.

(I) Proposed model: The GATOR complex regulates TORC1 activity by controlling the exchange rate of TSC between lysosome and cytosol through the Rag GTPase and TSC2 phosphorylation. Refer to Discussion for details. See also Figure S5.