Nutrient sensing and signalling
Changes in cellular amino acid availability exert powerful effects on signalling pathways regulating cell growth and differentiation. We are exploring the role of membrane amino acid transporters - not only in terms of their capacity to relay nutrients to the intracellular compartment, but as molecular sensors of amino acid availability that can regulate the activity of key intracellular molecules (e.g. mTOR) involved in nutrient signalling. In particular we are interested in defining the sensing/signalling functions associated with the SNAT2 (System A) transporter. Our recent work indicates that SNAT2 substrates can induce activation of the mTOR/S6K1 signalling axis and that System A activity may be important for supporting growth and proliferation of cells. However, this transporter is also important during periods of amino acid insufficiency. Cellular amino acid deprivation induces a marked increase in SNAT2 expression and activity, but the mechanisms that underlie this effect remain poorly understood. The mammalian or mechanistic target of rapamycin complex 1 (mTORC1) pathway is known to be responsive to changes in amino acid availability, but we have no evidence to support the idea that this pathway is responsible for up-regulating SNAT2 function during amino acid limitation. In fact, during periods of amino acid lack the activity of the mTOR pathway is very low, and the mTOR inhibitor, rapamycin, does not antagonise SNAT2 induction under these conditions. This excludes a role for mTORC1 and implicates another amino acid-responsive pathway. Intriguingly, repression of SNAT2 can be achieved by re-supply of any single System A substrate, including synthetic substrates. The substrate amino acid concentration required to repress SNAT2 expression reflects that required for extracellular binding to the transporter, raising the strong possibility that SNAT2 itself may function as a “transceptor” capable of not only transporting amino acids, but sensing their availability. Our current work aims to specifically assess (i) how, under amino acid sufficient conditions, SNAT2 signals to the mTOR pathway, (ii) how, during amino acid insufficiency, the transporter “senses” reduced extracellular amino acid availability and how this is transduced to promote an increase in SNAT2 gene expression. We are particularly keen to identify whether specific SNAT2 domains are important for sensing/signalling functions, which we may gain insight into through domain swapping experiments with structurally related transporters (e.g. System N) that do not exhibit the capacity to adapt to an altered nutrient environment or signal in response to substrate binding.
Whilst there is considerable ongoing interest in trying to elucidate how macronutrients such as amino acids (AA) regulate the mTOR/S6K1 signalling axis little has been done to establish whether this signalling pathway is also modulated by changes in availability of micronutrients, such as iron. Growth of mammalian cells is sensitive to iron availability and, as such, this may be an important determinant of the proliferative capacity of cells that normally “turn-over” very rapidly, such as the absorptive epithelial cells lining the intestinal mucosa. Reduced mTORC1 signalling has been reported in cells and tissues of animals rendered iron deficient, although the mechanism(s) underlying these observations remains unclear.
mTORC1 integrates mitogenic and nutrient signals to ensure that growth and proliferation of cells only occurs under nutritionally favourable conditions – a role made possible by the fact that mTORC1 is activated under AA sufficient conditions (thus promoting phosphorylation of downstream effectors, such as p70S6 kinase 1 (S6K1) and 4E-BP1 that play important roles in the regulation of protein synthesis) but is dramatically repressed upon AA withdrawal. Activation of mTORC1 is crucially dependent upon a small G-protein called Rheb, which in its GTP-loaded “on” form is a potent activator of mTORC1. The relative amounts of Rheb in the GTP “on” or GDP “off” form depend upon its intrinsic GTPase activity, which is a target for the GTPase-activating protein (GAP) activity of the tuberous sclerosis complex (TSC1/2). TSC2 is a physiological substrate for PKB/Akt, whose activation by insulin and growth factors induces phosphorylation of TSC2 and inhibition of its GAP activity, which then aids accumulation of active Rheb and a consequential increase in mTORC1 activity. Activation of mTORC1 is also dependent on small G proteins of the Rag family, which operate as heterodimers (RagA or RagB with RagC or RagD) to promote redistribution of mTORC1 to lysosomal membranes in response to AA provision. Rags are tethered to the lysosomal surface by interactions with two heteromeric protein complexes; (i) the Ragulator (Rag regulator) complex and (ii) the vacuolar H+-ATPase resident in the lysosomal membrane. AA-dependent modulation of these interactions appears to facilitate binding of mTORC1 to Rag complexes, placing it in close proximity to its activator Rheb.
In contrast, inactivation of mTOR may, in part, be driven by regulating the localisation of the TSC complex. Insulin and AAs have recently been shown to promote dissociation of TSC1/TSC2 from lysosomal membranes, whereas the absence of these stimuli induces greater lysosomal association of the complex where it facilitates conversion of Rheb to its inactive GDP-form and thus a reduction in mTOR activity. mTORC1 can also be negatively regulated by REDD1 (regulated in DNA damage and development 1), a small 25 kDa protein whose expression is induced in response to environmental stresses, such as hypoxia. Precisely how REDD1 inhibits mTORC1 activity is unclear although it has been suggested to sequester 14-3-3 proteins away from TSC2, which may then permit TSC2 to target its GAP activity towards Rheb. More recent work has shown that ectopic over-expression of REDD1 in HEK293 cells induces association of protein phosphatase 2A (PP2A) with Akt and that this promotes dephosphorylation and inactivation of the kinase on one of its key regulatory sites (Thr308) that in turn reduces its capacity to phosphorylate and inhibit TSC2 and consequently downstream activation of Rheb. However, it remains unclear if such a mechanism may account for the reduction in Akt and mTORC1 signalling observed in cells and tissues of animals rendered iron deficient. We have recently nvestigated the effect of iron deficiency on the growth and proliferative potential of intestinal epithelial cells. We have found that iron depletion induced in human intestinal Caco-2 cells by treatment with the iron chelator deferoxamine (DFO) results in the induction of REDD1 which is associated with not only a fall in Akt and TSC2 phosphorylation, but reduced mTORC1 signalling and a marked suppression in protein synthesis and cellular proliferation. Strikingly, we find that the increase in REDD1 expression initiated by DFO treatment can be attenuated by PP2A inhibition and that this is associated with retention of mTORC1 signalling in otherwise iron-deficient cells. Our work identifies REDD1 and PP2A as potential therapeutic targets whose modulation may help mitigate loss in mTORC1 activity and thereby potentially limit intestinal mucosal atrophy associated with chronic or recurrent iron deficiency disorders.
For more information on our work in this area click on the links to following Journals.