AMPK phosphorylation of raptor mediates a metabolic checkpoint

Abstract

AMPK is a highly conserved sensor of cellular energy status that is activated under conditions of low intracellular ATP. AMPK responds to energy stress by suppressing cell growth and biosynthetic processes, in part through its inhibition of the rapamycin-sensitive mTOR (mTORC1) pathway. AMPK phosphorylation of the TSC2 tumor suppressor contributes to suppression of mTORC1; however, TSC2-deficient cells remain responsive to energy stress. Using a proteomic and bioinformatics approach, we sought to identify additional substrates of AMPK that mediate its effects on growth control. We report here that AMPK directly phosphorylates the mTOR binding partner raptor on two well-conserved serine residues, and this phosphorylation induces 14-3-3 binding to raptor. The phosphorylation of raptor by AMPK is required for the inhibition of mTORC1 and cell-cycle arrest induced by energy stress. These findings uncover a conserved effector of AMPK that mediates its role as a metabolic checkpoint coordinating cell growth with energy status.

Figure 1. Peptide library profiling the optimal substrate motif for AMPK and comparison with known and candidate in vivo phosphorylation sites

A. A spatially arrayed PSPL was subjected to in vitro phosphorylation with active AMPKα1 and radiolabelled ATP. Each peptide contained one residue fixed at one of nine positions relative to the centrally fixed phospho-acceptor (an equal mix of serine and threonine). Aliquots of each reaction were spotted onto avidin membrane, which was washed, dried and exposed to a phosphor storage screen, providing the array of spots shown in the figure. AMPK displayed strong selectivity at the −5, −4, −3, −2, +3, and +4 positions. B. The optimal and secondary selections taken from triplicate analyses as in A are displayed. AMPK phosphorylation sites in the best established in vivo substrates of AMPK conform to the substrate motif derived from the peptide library data. All substrates shown were isolated in bioinformatics searches for proteins containing a conserved AMPK phosphorylation motif. These same searches yielded two novel predicted AMPK sites in raptor. C. The predicted AMPK sites in raptor are highly conserved across evolution.

Figure 2. Raptor is phosphorylated in vitro and in vivo by AMPK

A. mTORC1 signaling in TSC2-deficient cells remains responsive to energy stress. TSC2+/+ or TSC2−/− matched murine embryonic fibroblasts (MEFs) were serum-starved overnight (-ser) and replaced with fresh media containing 10% fetal bovine serum (+FBS) or serum-containing media with 2mM AICAR or 5mM phenformin. Cells were lysed one hour after media replacement. Lysates were immunoblotted for the mTORC1-dependent Thr389 phosphorylation in p70 S6K1 and for total S6K1 protein. B. Overexpressed raptor is phosphorylated in HEK293 cells in an LKB1- and AMPK-dependent manner. Left panel: myc-tagged mTOR and myc-tagged raptor were co-expressed in HEK293 cells with empty vector, wild-type LKB1, kinase-dead LKB1, or a constitutively active AMPKα1 allele (1–312 truncation). Raptor phosphorylation was detected using the phospho-14-3-3 motif antibody. Right panel: HEK293 cells expressing mTOR and raptor were treated with 50μM resveratrol for 30 min and phosphorylation of raptor was detected with the Phospho-14-3-3 motif antibody. C. Raptor is phosphorylated at a high level on serine 792 following resveratrol treatment. Mass Spectrometry was performed on raptor protein purified from resveratrol treated HEK293s as in panel B. Commassie-stained raptor protein was isolated from an SDS-polyacrylamide gel and subjected to chymotryptic digestion prior to analysis by LC-MS/MS. Amino acids 761 – 800 of human raptor are shown here. Each recovered peptide is illustrated by a single green line. Phosphorylated residues are shown in magenta. D. A phospho-specific antibody against Serine792 of raptor recognizes raptor phosphorylated in vitro by AMPK (left) as well as wild-type, but not S792A mutant, raptor (right) following treatment with 50μM resveratrol or 5mM phenformin in HEK293 cells.

Figure 3. Raptor Ser722 and Ser792 are phosphorylated by AMPK in cultured MEFs and in murine liver in an AMPK- and LKB1-dependent manner

A. Both Ser722 and Ser792 are phosphorylated in an AMPK-dependent manner in HEK293 cells. HEK293 cells were transfected with wild-type, S722A, S792A, or the double mutant S722A/S792A raptor allele and treated as indicated. Raptor was immunoprecipitated and immunoblotted with the phospho-Ser792, phospho-14-3-3 motif, or anti-myc epitope tag antibody. Phospho-ACC was immunoblotted from the total cell extracts to illustrate the degree of AMPK activation in the cells. B. Endogenous raptor is phosphorylated at Ser792 in wild-type but not AMPK-deficient (AMPKα1−/−, α2−/−) immortalized MEFs. MEFs were treated with 2mM AICAR for 1h (AICAR) or left untreated (NT) and total cell extracts were immunoblotted with the indicated antibodies. C. Endogenous raptor is phosphorylated at Ser792 in wild-type but not LKB1-deficient murine liver following fasting and metformin treatment. 8 week old mice were either fed ad libidum (ad lib) or fasted 18h and treated with either 250mg/kg metformin in saline (Met) or saline alone (Sal) for 1h. Total cell extracts made from harvested livers were immunoblotted with the indicated antibodies.

Figure 4. Phosphorylation of serine 722 and 792 is required to inhibit mTORC1 following energy stress in a variety of cell types

A. C2C12 cells in which endogenous raptor has been knocked down were stably reconstituted with human wild-type or AA raptor (see Fig S3), and were treated with 1mM AICAR or 1mM phenformin for 1 hour as indicated. Total cell extracts were immunoblotted with indicated antibodies to examine mTORC1 signaling. B. TSC2−/−, p53−/−, raptor knockdown MEFs stably reconstituted with wild-type or AA raptor were treated with 2mM AICAR as indicated and immunoblotted with indicated antibodies to examine mTORC1 signaling. C. TSC2−/−, p53−/−, raptor knockdown MEFs stably reconstituted with wild-type or AA raptor were treated with 2mM AICAR as indicated. Raptor was immunoprecipitated in CHAPS buffer and assayed for mTORC1 kinase activity using purified S6K1 as a substrate as previously described (Sancak et al., 2007). Top: IP-kinase assays were immunoblotted for phosphorylation of purified S6K1 substrate using Phospho-Thr389 S6K1 antibody as well as for level of immunoprecipitated raptor, mTOR, and PRAS40. Bottom: 5% of the total cell extracts that raptor was immunoprecipitated from were immunoblotted with indicated antibodies.

Figure 5. AMPK Phosphorylation of Raptor induces 14-3-3 association

A. Wild-type but not AA mutant raptor complexes with 14-3-3 only under energy stress conditions. HEK293 cells were co-transfected with pEBG or pEBG-14-3-3 with wild-type or AA mutant raptor then complexes were precipitated on glutathione beads. Beads or total cell extracts were immunoblotted with the indicated antibodies. Cells were treated with V, vehicle (DMEM) or P, 5mM phenformin for 1h. B. Wild-type but not AA mutant raptor precipitates with recombinant GST-14-3-3 protein in extracts from energy stress treated TSC2−/− MEFs stably reconstituted with human raptor alleles. GST protein pulldowns or total cell extracts were immunoblotted with the indicated antibodies. C. Endogenous raptor binds to immobilized recombinant GST-14-3-3 protein, but not recombinant GST protein, from extracts cells treated with energy stress in an LKB1-dependent manner. GST protein pulldowns or total cell extracts were immunoblotted with the indicated antibodies. D. Myc-tagged raptor immunoprecipitates from phenformin (Ph) or vehicle (v) treated cells were eluted with myc peptide and immunoblotted for endogenous 14-3-3 isoforms as indicated.

Figure 6. Phosphorylation of Raptor at S722 and S792 dictates a metabolic checkpoint controlling growth arrest and apoptosis in response to energy stress

A. TSC2−/−, p53−/− MEFs expressing wild-type raptor undergo G1/S arrest following AICAR treatment, while those expressing AA mutant raptor do not. Cells were left untreated (NT) or treated with 2 mM AICAR, or treated with 2 mM AICAR and 3h later exposed to nocodazole (+nocod) to arrest any cycling cells in G2/M. At 24h after AICAR treatment, all cells were fixed and analyzed for DNA content using propidium iodide and FACS analysis. The percentage of cells in the G2/M phase of the cell cycle are highlighted in each population. B. Cells analogous to those in panel A were plated on coverslips and the next day left untreated (NT) or treated with 2mM AICAR and fixed 18h later. Cells were processed for phospho-histone H3 Ser10 immunocytochemistry to visualize the cells actively going through mitosis at the time the cells were fixed. DAPI was used as a nuclear counterstain. Histogram quantifies Phospho-Histone H3 immunocytochemistry on indicated cells treated with 5mM phenformin, or 2mM AICAR for 18h. At least 300 cells were scored for each condition. C. Cell extracts from parallel plates to the ones analyzed in panel A were immunoblotted for phospho-histone H3 Ser10 as a marker of the percentage of cells in mitosis. D. TSC2+/+, p53−/− MEFs or TSC2−/−, p53−/− MEFs expressing AA mutant raptor undergo apoptosis to a greater extent than those expressing WT raptor at later timepoints following energy stress treatment. Cell populations of indicated genotypes were treated with 5mM phenformin and at 48h the percentage of cells undergoing apoptosis was quantified using AnnexinV staining and FACS analysis. Histograms of cells expressing wild-type raptor (red trace) and AA raptor (blue trace) are overlaid in the right-most panel. The percentage of apoptotic cells in the annexin V-positive population is indicated in red at the upper right hand corner of each histogram. E. Upstream AMPK signals from LKB1 are needed for the protective effect of WT raptor on apoptosis following energy stress. A549 human lung adenocarcinoma cells, which are null for LKB1, were stably reconstituted with wild-type LKB1 (WT) or mutant kinase dead K78I (KD) LKB1 expressing retroviruses. These cells were subsequently stably infected with retroviruses expressing wild-type or AA raptor. Each of the four resulting populations were treated with 5mM phenformin and analyzed for apoptosis as above.

Figure 7. Nutrients and Growth Factors control mTORC1 activity though common (TSC2) and unique (raptor, PRAS40) downstream targets

Strikingly, both AMPK-mediated suppression of raptor and Akt-mediated suppression of PRAS40 involve the phosphorylation sites in each protein binding to 14-3-3, resulting in the inactivation of those targets. Inherited mutations in LKB1, TSC1. TSC2, and PTEN all result in hamartoma syndromes in humans indicating that hyperactivation of mTORC1 is a common biochemical mechanism underlying these genetic disorders.