Figure 4.

A conserved 14–3–3-binding motif within REDD1 is required for mTORC1 signaling and TSC2/14–3–3 regulation. (A) Endogenous REDD1 is required for hypoxia-induced TSC2/14–3–3 dissociation. MEFs of the indicated genotype were treated with hypoxia (3 h) followed by Western analysis or IP for endogenous 14–3–3. Hypoxia-induced TSC2/14–3–3 dissociation and S6K1 (T389) dephosphorylation are both absent in REDD1^−/− MEFs. (B) Induction of REDD1 but not the REDD1-RPAA mutant inhibits mTORC1 activity and the endogenous TSC2/14–3–3 association. Expression of REDD1 was induced with tetracycline (Tet) for 3 h (hrs) in U2OS cells, followed by Western blot analysis or IP for endogenous 14–3–3. Dephosphorylation of S6K (T389) (lane 1 vs. lane 2) and TSC2/14–3–3 dissociation (lane 9 vs. lane 10) are only induced by wild-type REDD1. (C) Induction of REDD1 but not REDD1-RPAA opposes insulin-induced mTORC1 activation and endogenous TSC2/14–3–3 association. Cells were serum-starved, then treated with insulin (200 nM, 2 h), or were pretreated with wortmannin (100 nM, 30 min) or REDD1 induction (Tet, 60 min) prior to insulin (I) treatment. Lysates were analyzed by Western blot analysis and by IP for endogenous 14–3–3. Only wild-type REDD1 blocks insulin-induced S6K (T389) phosphorylation (lane 2 vs. lane 4) and opposes TSC2/14–3–3 association (lane 10 vs. lane 12). Note that REDD1 induction, unlike wortmannin treatment, does not affect AKT (S473) phosphorylation. (D) REDD1-mediated S6K dephosphorylation and TSC2/14–3–3 dissociation do not involve a change in AKT-dependent TSC2 phosphorylation. Lysates shown in C were probed for the indicated phosphorylated TSC2 residues. (E) Hypoxia replicates tetracycline-induced wild-type REDD1 in opposing insulin effects on TSC2/mTORC1 signaling. Serum-starved MEFs were treated with insulin (200 nM) prior to hypoxia (3 h).