Editors' ChoiceCell Biology

Activating mTOR with Ral Instead of Rheb

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Sci. Signal.  04 Feb 2014:
Vol. 7, Issue 311, pp. ec37
DOI: 10.1126/scisignal.2005150

The kinase mechanistic (or mammalian) target of rapamycin (mTOR) participates in two complexes: mTORC1 and mTORC2. Activation of mTORC1 by amino acids and growth factors promotes anabolic processes, such as protein synthesis, and inhibits catabolic processes, such as autophagy. One of the best-characterized pathways for mTORC1 activation is its recruitment to the lysosomal surface, where it is stimulated by the guanosine triphosphatase (GTPase) Rheb. Rheb is inhibited by the GTPase-activating protein (GAP) complex TSC1/TSC2. Upon examination of the genome of the model organism Caenorhabditis elegans, Martin et al. noted that this nematode lacks the genes encoding TSC1 and TSC2 but has those encoding the related proteins RalGAPα (HGAP-1) and RalGAPβ (HGAP-2), as well as RHEB-1 and the related GTPase RAL-1. In various species, including C. elegans, increased activity of mTORC1 is associated with reduced life span, and reduced function, either by knockdown or genetic mutation, of either HGAP-1 or HGAP-2 in worms reduced life span. Life span of worms with reduced HGAP function was restored by knockdown of RAL-1. To determine whether a similar pathway functioned in mammalian cells, the authors used mouse embryonic fibroblasts (MEFs) deficient in RalGAPβ (and thus the RalGAP complex, because RalGAPα and RalGAPβ form an obligatory dimer). RalGAPβ-deficient MEFs had increased GTP loading of RalA and RalB but not Rheb, increased phosphorylation of an mTORC1 target, and reduced autophagic flux. mTORC1 isolated from these cells had increased catalytic activity. Phosphorylation of the mTORC1 target was diminished in RalGAPβ-deficient MEFs by knockdown of RalB, but not Rheb. In human embryonic kidney 293 (HEK293) cells, constitutively active RalB, but not RalA, coimmunoprecipitated with mTOR, and the interaction between endogenous proteins was stimulated by serum. Recombinant RalB and mTOR interacted in vitro, indicating that the interaction was direct. RalB, mTOR, and Raptor (an mTORC1-specific component) colocalized at perinuclear structures in the absence of serum, and serum increased the abundance of Rab1, mTOR, and Raptor at the plasma membrane. Endogenous mTOR coimmunoprecipitated with tagged versions of components of the exocyst complex, a known Ral effector, and knockdown of one of these components, Sec5, reduced the interaction between RalB and mTOR and reduced the phosphorylation of an mTORC1 target. Using a fluorescent sensor of RalB activity, the addition of serum or knockdown of RalGAPβ increased RalB activity at the plasma membrane. Analysis of phosphorylation of mTORC1 targets in cells expressing Raptor fused to various membrane-targeting sequences showed that forced localization of Raptor to either the lysosomal surface or the plasma membrane was sufficient to stimulate mTORC1 activity in the absence of serum. Only expression of an inhibitor of Ral targeted to the plasma membrane, but not the lysosome, reduced mTORC1 signaling in response to serum. These data suggested that RalB activity at the plasma membrane contributes to activation of mTORC1 in response to growth factors. From a medically relevant standpoint, RalB is implicated in the invasive phenotype and metastatic potential of pancreatic ductal adenocarcinoma (PDAC). RalGAPα and RalGAPβ were present in PDAC cell lines, and knockdown of either increased the invasiveness of the cells in Matrigel, which was prevented by inhibition of mTOR with rapamycin. Thus, Ral-dependent activation of mTORC1 at the plasma membrane may contribute to the metastasis of some cancers.

T. D. Martin, X.-W. Chen, R. E.W. Kaplan, A. R. Saltiel, C. L. Walker, D. J. Reiner, C. J. Der, Ral and Rheb GTPase activating proteins integrate mTOR and GTPase signaling in aging, autophagy, and tumor cell invasion. Mol. Cell 53, 209–220 (2014). [PubMed]