mTORC1 directly phosphorylates and regulates human MAF1

Abstract

mTORC1 is a central regulator of growth in response to nutrient availability, but few direct targets have been identified. RNA polymerase (pol) III produces a number of essential RNA molecules involved in protein synthesis, RNA maturation, and other processes. Its activity is highly regulated, and deregulation can lead to cell transformation. The human phosphoprotein MAF1 becomes dephosphorylated and represses pol III transcription after various stresses, but neither the significance of the phosphorylations nor the kinase involved is known. We find that human MAF1 is absolutely required for pol III repression in response to serum starvation or TORC1 inhibition by rapamycin or Torin1. The protein is phosphorylated mainly on residues S60, S68, and S75, and this inhibits its pol III repression function. The responsible kinase is mTORC1, which phosphorylates MAF1 directly. Our results describe molecular mechanisms by which mTORC1 controls human MAF1, a key repressor of RNA polymerase III transcription, and add a new branch to the signal transduction cascade immediately downstream of TORC1.

FIG. 1.

MAF1 is dephosphorylated by treatments that inhibit mTOR. IMR90hTert cells stably expressing MAF1-EGFP were treated as indicated, and the samples were separated on a Phos-tag gel and immunoblotted with an anti-GFP antibody (top panels). The bottom panels show the same samples immunoblotted with an antitubulin antibody (T). (A) Cells were serum starved (ss) for 0 to 60 min (lanes 2 to 6) or 30 min (lanes 9 to 13, 15 to 19, and 21 to 25), after which serum was added back for 0 to 20 min (lanes 10 to 13), 100 nM insulin was added for 0 to 30 min (lanes 16 to 19), or different concentrations of IGF were added for 1 h (lanes 22 to 25). Lanes 1, 14, and 20, nontreated (c) cells; lanes 7 and 8, nontreated cells lysed at 0 and 120 min, respectively. (B) Cells were treated for 1 h with MMS at the indicated concentrations. Lane 1, untreated cells. (C) Cells were treated for 1 h with Torin1 at the indicated concentrations. (D) Cells were treated with rapamycin for 1 h at the indicated concentrations (lanes 2 to 5) or with 1 nM rapamycin for the indicated times (lanes 7 to 11). Lanes 1 and 6, nontreated cells. (E) MAF1-EGFP was immunoprecipitated from untreated cells and incubated with calf intestine phosphatase (CIP) for 30 min at 30°C either with or without phosphatase inhibitors (inh). Lane 1, cell lysate.

FIG. 2.

Pol III inhibition depends on Maf1. Wild-type (wt) or Maf1 KO MEF cells were treated as indicated, and 10 μg of total RNA was analyzed by Northern blotting with probes against mature 7SK and different tRNA Leu precursors (MS3, -4, -5, and -6). (A) Cells were either serum starved (ss) or left untreated (c) for the indicated times; (B and C) cells were left untreated (c) or treated with Torin1 (T) or rapamycin (R) for the indicated times.

FIG. 3.

MAF1 phosphorylation on S60, S68, and S75 depends on mTORC1 activity. (A) MAF1-HT transiently expressed in HeLa cells was affinity purified, electrophoresed on a Phos-tag gel, and revealed with an anti-MAF1 antibody. (B) IMR90hTERT cells stably expressing either wt, S60A, S68A, or S6068A MAF1-EGFP were either left untreated (c), serum starved (ss) for 1 h, or treated with 1 nM rapamycin (R) or 50 nM Torin1 (T) for 1 h and lysed. (Top) Proteins were separated on Phos-tag gels and revealed by immunoblotting with an anti-GFP antibody. Bands corresponding to phosphorylated serine 75 (arrowhead), 68 (arrow), or a group of bands depending on phosphorylated serine 60 (bracket, with the strongest band marked by an asterisk) are indicated. (Bottom) Immunoblot of the same samples probed with an anti-tubulin antibody. α, anti.

FIG. 4.

Expression of 60A68A75A MAF1 decreases the level of tRNA Leu precursors. (A) Total RNA of Maf1 KO MEFs transduced with vectors either empty (lane 1, vector control [ctrl]) or expressing the proteins indicated above the lanes was probed for different tRNA Leu precursors (MS3, -4, -5, and -6) and 7SK RNA. Cells were either left untreated (lanes 1 to 4), serum starved for 8 h (lanes 5 to 8), or treated with 2 nM rapamycin for 8 h (lanes 9 to 12). (B) Quantification of the signals in panel A, with the vector control set at 100%. (C) Quantification of the signals in panel A, with the nontreated control cells set at 100%. 3A, 60A68A75A MAF1; 3D, 60D68D75D MAF1. (D) Immunoblot analysis of affinity-purified MAF1 from the transduced cells, probed with an anti-MAF1 antibody.

FIG. 5.

S6K cannot phosphorylate MAF1. (A) Active myc-S6K (wt) or inactive myc-S6K-GST (KD) was immunoprecipitated from transiently transfected HeLa cells (ctrl, cells transfected with empty vector) and incubated with recombinant MAF1-HT or GST-URI in the presence of Mg²⁺ and [γ-³²P]ATP. The samples were separated by SDS-polyacrylamide gel electrophoresis (PAGE), and the gel was first stained with Coomassie blue (CM) and then analyzed with a phosphorimager (³²P). In the bottom panel, the presence of S6K1 was revealed with an anti-myc antibody. (B) Wt or S6K1 S6K2 KO MEFs were transiently transfected with a vector expressing MAF1-HT and treated or not (c) with 2 nM rapamycin (R) for 1 h. MAF1-HT was affinity purified, separated on a Phos-tag gel, and revealed by immunoblotting with an anti-MAF1 antibody. (C) Wt and S6K1 S6K2 KO MEF cells were probed with anti-S6K1 and anti-tubulin antibodies. (D) The indicated cells were treated or not (c) with 2 nM rapamycin (R) for 4 or 8 h. RNA was analyzed by Northern blotting, with probes against the indicated RNAs.

FIG. 6.

mTORC1 can phosphorylate MAF1 in vitro on S60, S68, and S75. (A) Active HA-mTOR, inactive HA-mTOR (KD), or RAPTOR was immunoprecipitated from transiently transfected HeLa cells (ctrl, empty vector) and incubated with recombinant MAF1-HT in the presence of Mn²⁺ and [γ-³²P]ATP. Lane 8, 100 nM rapamycin and 1 μg of FKBP12 were added to the in vitro kinase reaction. The samples were processed as described in the legend to Fig. 5A. (Bottom) The presence of mTOR was revealed with anti-HA or anti-mTOR antibodies as indicated. (B, top) Wt and mutant MAF1-HT were incubated with anti-HA-mTOR (lanes 3 to 8), anti-RAPTOR (lanes 9 to 12), or anti-RICTOR (lanes 13 to 16) immunoprecipitates in the presence of Mn²⁺ and [γ-³²P]ATP and processed as described in the legend to Fig. 5A, except that after the kinase reaction, the various MAF1-HT proteins were affinity purified and separated on a Phos-tag gel (lanes 3 to 16). Lanes 1 and 2, transiently expressed MAF1-HT wt and 60A were loaded and revealed by anti-MAF1 immunoblotting. (Bottom) Immunoblots performed with an anti-Maf1 antibody showing the input recombinant MAF1 proteins used for the kinase reactions in the top panels separated on a 12% gel.