Summary Proliferating mammalian cells use glutamine as a source of nitrogen and as a key anaplerotic source to provide metabolites to the tricarboxylic acid cycle (TCA) for biosynthesis. Recently, mTORC1 activation has been correlated with increased nutrient uptake and metabolism, but no molecular connection to glutaminolysis has been reported. Here, we show that mTORC1 promotes glutamine anaplerosis by activating glutamate dehydrogenase (GDH). This regulation requires transcriptional repression of SIRT4, the mitochondrial-localized sirtuin that inhibits GDH. Mechanistically, mTORC1 represses SIRT4 by promoting the proteasome-mediated destabilization of cAMP response element binding-2 (CREB2). Thus, a relationship between mTORC1, SIRT4 and cancer is suggested by our findings. Indeed, SIRT4 expression is reduced in human cancer, and its overexpression reduces cell proliferation, transformation and tumor development. Finally, our data indicate that targeting nutrient metabolism in energy-addicted cancers with high mTORC1 signaling may be an effective therapeutic approach.

Summary mTORC1 is a signal integrator and master regulator of cellular anabolic processes linked to cell growth and survival. Here we demonstrate that mTORC1 promotes lipid biogenesis via SRPK2, a key regulator of RNA-binding SR proteins. mTORC1-activated S6K1 phosphorylates SRPK2 at Ser494, which primes Ser497 phosphorylation by CK1. These phosphorylation events promote SRPK2 nuclear translocation and phosphorylation of SR proteins. Genome-wide transcriptome analysis reveals that lipid biosynthetic enzymes are among the downstream targets of mTORC1-SRPK2 signaling. Mechanistically, SRPK2 promotes SR protein binding to U1-70k to induce splicing of lipogenic pre-mRNAs. Inhibition of this signaling pathway leads to intron retention of lipogenic genes which triggers nonsense-mediated mRNA decay. Genetic or pharmacological inhibition of SRPK2 blunts de novo lipid synthesis, thereby suppressing cell growth. These results thus reveal a novel role of mTORC1-SRPK2 signaling in post-transcriptional regulation of lipid metabolism and demonstrate that SRPK2 is a potential therapeutic target for mTORC1-driven metabolic disorders.

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Our paper reported that mTORC1 promotes glutamine consumption through the TCA cycle by activating glutamate dehydrogenase (GDH). After being contacted about apparent similarities between the streptavidin-and GDH-probed blots in Figure 2A, we determined that the data collection approach was not clearly described in the Experimental Procedures. Mono-ADP ribosylated GDH was detected first and the nitrocellulose membrane was then probed with streptavidin. The authors apologize for any confusion; the Experimental Methods subsection ''ADP-Ribosylation Assay'' should have been as follows: ''Mono-ADP-ribosylation levels were determined as previously described (Mao et al., 2011) with slight modifications. DLD-1 cells stably expressing either SIRT4-HA or empty vector (EV) were transfected with 6-Biotin-NAD+ (Lonza transfection protocol). Twenty-four hours after transfection, cells were treated with rapamycin for 24 hr and harvested with IP buffer. Mitochondria were purified using the isolation kit from Pierce. Poly-ADP ribosylated proteins from mitochondrial samples were cleared using PAR antibody (1:200) with protein A beads for 1 hr. Avidin coated beads (1:10) (Sigma) were added and incubated for 1 hr at 4C. Samples were run in SDS-PAGE gels and proteins were transferred into nitrocellulose membranes. Mono-ADP ribosylated GDH was detected using the GDH antibody (Abcam). Following GDH detection, the membrane was incubated with Streptavidin-HRP (Abcam) to evaluate the levels of avidin control. Lysates to measure endogenous GDH and HA-SIRT4 over-expression were run in parallel.'' ll

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