Rag Proteins Regulate Amino Acid-Induced mTORC1 Signaling

Abstract

The serum and nutrient-sensitive protein kinase mTOR is a master regulator of cell growth and survival. The mechanisms through which nutrients regulate mTOR have been one of the major unanswered questions in the mTOR field. Identification of rag family of GTPases as mediators of amino acid signaling to mTOR is an important step towards understanding this mechanism.

The mTOR (mammalian target of rapamycin) kinase is an evolutionarily conserved protein that regulates cell growth, survival and metabolism. mTO[1]R participates in two biochemically and functionally distinct protein complexes: mTORC1 and 2 (mTOR complex 1 and 2). mTOR and mLST8 are common members of both complexes; raptor and pras40 participate in mTORC1 specifically; and rictor, sin1 and protor are unique members of mTORC2 [1–13]. mTORC1 is downstream of both growth factor and nutrient signaling (glucose and amino acids), and integrates the two so as to ensure cell growth only when conditions are ideal. Given the central role of mTORC1 in cell growth, it is not surprising that many upstream regulators of mTORC1 are involved in disease, and understanding how it is regulated is of great interest.

The identification of the small GTPase rheb (ras homolog enriched in brain) as a potent mTORC1 activator, and the finding that TSC1/2 (tuberous sclerosis complex 1/2) is the GAP (GTPase activating) protein for rheb advanced our understanding of mTORC1 regulation significantly[14–20]. Both growth factors and cellular energy levels regulate TSC1/2 activity, which in turn modulates the level of Rheb-GTP, which binds to and directly activates mTORC1[7, 21–27]. In addition, two mTORC1 members, pras40 and raptor, are phosphorylated in response to mitogenic stimuli and cellular stress, leading to mTORC1 activation and inhibition, respectively[6, 7, 28, 29].

Although rheb is necessary for amino acid-induced mTORC1 activation and rheb over-expression can overcome amino acid-starvation induced mTORC1 inhibition, TSC2 null MEFs are sensitive to amino acid starvation[30]. This observation suggested that there are additional important players in the amino acid regulation of mTORC1. Earlier this year, the finding that rag proteins mediate amino acid signaling to mTORC1 provided new insights into this problem[31, 32].

The rag proteins are a unique family of GTPases with a canonical N-terminal ras-like GTPase domain and a unique C-terminal ragA conserved region. In mammals, there are four rag genes (ragA, B, C and D) whereas yeast and fly have one ragA-like gene and one ragC-like gene.

We identified ragC by mass spectrometric analysis as a raptor-interacting protein. Kim et al. found that the rag proteins are important for amino acid-induced TORC1 activation in fly cells using an RNAi screen. Both groups showed that knocking down the rag proteins impairs amino acid signaling to TORC1, and that over-expression of constitutively GTP-bound ragA-like mutant makes TORC1 insensitive to amino acid deprivation. Moreover, Kim et al. – monitoring cell and organ size, as well as autophagy – observed that rag over-expression or deletion in flies directly parallels the effects of dTOR activation or inhibition, respectively.

Although we do not completely understand how rag proteins activate mTORC1 in detail, the critical observation that amino acid stimulation induces a change in mTORC1 localization prompted us to hypothesize that rag proteins may regulate mTORC1 localization. Supporting our hypothesis, when rag proteins are knocked down, amino acid- induced mTORC1 localization change is ablated. Similarly, when a constitutively GTP-bound ragB mutant is expressed, mTORC1 localization resembles the amino acid- induced state even in the absence of amino acids. We also showed that after amino acid stimulation, mTORC1 moves to rab7 containing vesicles, where its activator rheb is thought to reside. Based on these observations, we proposed a model: upon amino acid stimulation, rag proteins initiate a localization change of mTORC1, taking it to rheb-contaning vesicles, leading to its activation. This model explains (1) how over-expressed and thus mislocalized Rheb can overcome amino acid starvation, (2) why TSC2 null MEFs are sensitive to amino acid starvation: even though Rheb is always in a GTP-bound state, mTORC1 is not in the same subcellular compartment as its activator when amino acids are not present.

The identification of rag proteins as members of the mTORC1 pathway is an important first step towards deciphering the molecular events that signal nutrient availability to mTORC1, and evokes many interesting questions. How amino acid availability is sensed and communicated to the rag proteins, how mTOR localization contributes to its activity, and whether rag-related signaling can be targeted in disease are some of the exciting questions for which mTOR biologists will be seeking answers.

Abbreviations used

GTP

guanosine triphosphate

mTORC1

mammalian target of rapamycin complex 1

Rag

Ras-related GTPase

Rheb

Ras homolog enriched in brain

TSC

tuberous sclerosis complex

MEF

mouse embryonic fibroblast

RNAi

RNA interference