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. 2010 Jul 15;29(28):4068-79.
doi: 10.1038/onc.2010.177. Epub 2010 May 24.

Proton-assisted amino-acid transporters are conserved regulators of proliferation and amino-acid-dependent mTORC1 activation

S Heublein  1 , S Kazi, M H Ogmundsdóttir, E V Attwood, S Kala, C A R Boyd, C Wilson, D C I Goberdhan
Affiliations

Proton-assisted amino-acid transporters are conserved regulators of proliferation and amino-acid-dependent mTORC1 activation

S Heublein et al. Oncogene. .

Abstract

The phosphoinositide3-kinase (PI3K)/Akt and downstream mammalian target of rapamycin complex 1 (mTORC1) signalling cascades promote normal growth and are frequently hyperactivated in tumour cells. mTORC1 is also regulated by local nutrients, particularly amino acids, but the mechanisms involved are poorly understood. Unexpectedly, members of the proton-assisted amino-acid transporter (PAT or SLC36) family emerged from in vivo genetic screens in Drosophila as transporters with uniquely potent effects on mTORC1-mediated growth. In this study, we show the two human PATs that are widely expressed in normal tissues and cancer cell lines, namely PAT1 and PAT4, behave similarly to fly PATs when expressed in Drosophila. Small interfering RNA knockdown shows that these molecules are required for the activation of mTORC1 targets and for proliferation in human MCF-7 breast cancer and HEK-293 embryonic kidney cell lines. Furthermore, activation of mTORC1 in starved HEK-293 cells stimulated by amino acids requires PAT1 and PAT4, and is elevated in PAT1-overexpressing cells. Importantly, in HEK-293 cells, PAT1 is highly concentrated in intracellular compartments, including endosomes, wherein mTOR shuttles upon amino-acid stimulation. Therefore our data are consistent with a model in which PATs modulate the activity of mTORC1 not by transporting amino acids into the cell but by modulating the intracellular response to amino acids.

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Figures

Figure 1
Figure 1
Human PAT1 and PAT4 have similar activities to Drosophila PATs in flies. GMR-GAL4 was used to overexpress the following UAS-coupled transgenes in the differentiating cells of the eye: the fly PAT path from a transposable element insertion in the gene (pathGS13857; B), human PAT1 (C) and human PAT4 (D). All produced modest, but significant, increases in ommatidial size relative to non-expressing controls (A). The ommatidial loss induced by overexpressing foxo from a transposable element insertion in the gene, foxoGS9928, using GMR-GAL4 is most clearly seen at the ventro-posterior edge of the eye (arrow in E). It is enhanced by co-overexpression of both fly and human PAT transporters (F-H). Scale bar is 100 μm and applies to all panels. Ommatidial size measurements shown at bottom of panels A-D, mean ± s.d. relative to control; * P<0.001, n=6.
Figure 2
Figure 2
PAT1 and PAT4 are both required for normal proliferation of MCF-7 breast cancer cells. The proliferation of MCF-7 breast cancer cells after transfection with siRNAs for mTOR (si825, si826 and si827; A), PAT1 (si158, si159 and si160; B) and PAT4 (si435, si436 and si437; C) was compared to control cells treated with a scrambled siRNA control (sc), lipofectamine™ only (lp) and non-transfected (nt). A combination of the most effective PAT1 and PAT4 siRNAs did not reduce cell number further (B). Bar chart (D) shows cell number at 120 h for samples treated with the most effective siRNAs for mTOR (si827), PAT1 (si159) and PAT4 (si437) normalised to lp control. Error bars represent SEM. *P<0.0002: reduced versus lp control. All measurements were made on triplicate samples and the experiment was repeated three times with similar results.
Figure 3
Figure 3
PAT1 and PAT4 modulate the activity of the mTORC1 signalling cascade in MCF-7 breast cancer cells. Lysates from MCF-7 cells grown in medium containing 10% serum treated for 120 h with siRNA against PAT1 (si158, si159 and si160), PAT4 (si435, si436 and si437) and mTOR (si825) were compared to control lysates from cells treated with a scrambled siRNA (sc) or lipofectamine™ only (lp) by probing with anti-Phospho-T389-p70 S6K1 (P-T389-S6K1) and anti-Phospho-T24-FoxO1/T32-FoxO3a (P-FoxO1/3a; A), anti-Phospho-S240/244-S6 (P-S240/244-S6) and anti-Phospho-S65-4E-BP1 (P-S65-4E-BP1; B), as well as non-Phospho-specific antisera against S6K1 and 4E-BP1, and an anti-α-tubulin antibody to confirm equal loading. All experiments were repeated at least three times with similar results.
Figure 4
Figure 4
PAT1 is located inside MCF-7 and HEK-293 cells from where it regulates cell proliferation. An antiserum against human PAT1 reveals a cytoplasmic localisation of endogenous PAT1 protein in both MCF-7 (A) and HEK-293 (B) cells. HEK-293 cells overexpressing PAT1 produce a much stronger signal with this antibody (C; the detection gain is reduced compared to B in this confocal image), but the protein distribution is very similar to the endogenous protein pattern. PAT1-containing structures are frequently surrounded by (top arrow), overlapping (lower arrows) or adjacent to regions containing a GFP-Rheb fusion protein, which was previously shown to reside around late endosomal compartments in HEK-293 cells (see low and high magnification views in D and E respectively). Other PAT1-containing intracellular structures may represent early endosomal or lysosomal compartments, where PATs are located in other cell types. The proliferation of HEK-293 human embryonic kidney cells after transfection with siRNAs for mTOR (si825 and si827; F), PAT1 (si158 and si159; G) and PAT4 (si435 and si437; H) was compared to control cells treated with a scrambled siRNA (sc), or with the MATra reagent only (MATra). Bar chart (I) shows cell number at 72 h for samples treated with each siRNA normalised to sc control. Error bars represent SEM. *P<0.01; **P<0.001; reduced versus MATra control. All cell number measurements were made on triplicate samples and the experiment was repeated three times with similar results.
Figure 5
Figure 5
PAT1 and PAT4 are required for normal mTORC1 signalling in HEK-293 cells. A. Lysates from HEK-293 cells grown in medium containing 10% serum for 72 h after transfection with siRNAs against PAT1 (si158 and si159), PAT4 (si435 and si437) and mTOR (si825 and si826) were compared to control lysates from cells treated with a scrambled siRNA (sc) or with the magnet-assisted transfection protocol without siRNA (MATra). Blots were probed with anti-Phospho-Thr-389-p70 S6K1 (P-T389-S6K1), anti-Phospho-Ser-240/244-S6 (P-S240/244-S6), anti-Phospho-S65-4E-BP1 (P-S65-4E-BP1), anti-Phospho-T24-FoxO1/T32-FoxO3a (P-FoxO1/3a), anti-Phospho-S473-Akt (P-S473-Akt) antibodies, non-Phospho-specific antisera against S6K1, S6 and 4E-BP1, and an anti-α-tubulin antibody to confirm equal loading. All experiments were repeated at least three times with similar results. Intensity measurements made from blots produced in three independent experiments reveal significant reduction in S6K1 (B), S6 (C) and 4E-BP1 (D) activation in response to PAT1, PAT4 and mTOR knockdown. *P<0.5; **P<0.001.
Figure 6
Figure 6
PAT1 and PAT4 are required for normal activation of the mTORC1 signalling cascade by amino acids in starved HEK-293 cells. A. HEK-293 cells were grown in medium containing 10% serum for 72 h after transfection with siRNA against PAT1 (si158 and si159), PAT4 (si435 and si437), mTOR (si825 and si826), a scrambled siRNA (sc) or after exposure to MATraA without siRNA (MATra). They were starved of serum and amino acids for 50 min and then exposed to amino acids for 30 min. Extracts from these cells and cells that were not exposed to amino acids after starvation were analysed by western blot probed with anti-Phospho-Thr-389-p70 S6K1 (P-T389-S6K1), anti-Phospho-Ser-240/244-S6 (P-S240/244-S6), anti-Phospho-Ser-65-4E-BP1 (P-S65-4E-BP1), anti-Phospho-Ser-473-Akt (P-S473-Akt) antibodies, non-Phospho-specific antisera against S6K1, S6 and 4E-BP1, and an anti-α-tubulin antibody to confirm equal loading. All experiments were repeated at least three times with similar results. Intensity measurements made from blots produced in three independent experiments reveal significant reduction in S6K1 (B), S6 (C) and 4E-BP1 (D) activation in response to PAT1, PAT4 and mTOR knockdown. *P<0.05; **P<0.001. (E) HEK-293 cells stably transfected with empty vector and a PAT1 overexpression vector were grown in medium containing 10% serum for 72 h, starved of serum and amino acids. Cultures were then exposed either to medium containing amino acids or to starvation medium for a further 30 min. Extracts from these cells were analysed by western blot with the antibodies described above. All experiments were repeated at least three times with similar results. (F) The proliferation of PAT1-overexpressing cells was compared to non-overexpressing cells over a 48 h time course (n=6). *P<0.001, proliferation significantly greater than non-expressing cells.
Figure 7
Figure 7
A model to explain the effects of the PATs on mTORC1 signalling and proliferation. We have shown that PATs modulate the response of mTORC1 to extracellular amino acids in HEK-293 cells, even though one of the critical PATs in this process, PAT1, is concentrated intracellularly. We propose that these intracellular PATs are likely to affect mTORC1 signalling in late endosomes to which mTOR is shuttled upon amino acid stimulation. They may act via a transport-dependent or transceptor mechanism, forming a complex with other mTOR regulatory proteins, or they may be components of one of a series of endosomal complexes involved in this process. Cytoplasmic leucine, which activates S6K1, may bind to the cytoplasmic face of the PATs to enhance their ability to activate mTORC1. Rag GTPases are required to shuttle mTOR to Rheb-containing endosomal compartments upon amino acid stimulation, but evidence in yeast suggests they may also bind to amino acid transporters and promote their shuttling. Rag-dependent shuttling of PATs to the endosomes may therefore be a critical aspect of mTOR regulation.
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References

    1. Akins MB, Yasothan U, Kirkpatrick P. Everolimus. Nat Rev Drug Discov. 2009;8:535–536. - PubMed
    1. Avruch J, Long X, Ortiz-Vega S, Rapley J, Papageorgiou A, Dai N. Amino acid regulation of TOR complex 1. Am J Physiol Endocrinol Metab. 2009;296:E592–602. - PMC - PubMed
    1. Bermingham JR, Jr, Pennington J. Organization and expression of the SLC36 cluster of amino acid transporter genes. Mamm Genome. 2004;15:114–125. - PubMed
    1. Beugnet A, Tee AR, Taylor PM, Proud CG. Regulation of targets of mTOR (mammalian target of rapamycin) signalling by intracellular amino acid availability. Biochem J. 2003;372:555–566. - PMC - PubMed
    1. Boll M, Foltz M, Rubio-Aliaga I, Kottra G, Daniel H. Functional characterization of two novel mammalian electrogenic proton-dependent amino acid cotransporters. J Biol Chem. 2002;277:22966–22973. - PubMed

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