The mTORC1 complex is a master controller of cell growth. During intracellular energy stress AMPK is known to suppress the mTORC1 pathway by interacting with TSC2. Gwinn et al. 1 have now discovered that raptor on mTOR is a novel substrate of AMPK and that the phosphorylation of raptor is required for the full engagement of an AMPK-mediated metabolic checkpoint. Just like musicians in an orchestra utilising musical cues and direct signals from the conductor to form a piece of music, proteins often utilise environmental cues and direct signals from other molecules to be able to integrate and form cellular pathways. One of those proteins is mammalian target of Rapamycin (mTOR). mTOR is part of two complexes; mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). Found in all eukaryotic cells, mTOR interprets a multitude of factors that influence cell growth (stressors, nutrients and transduction outputs) by targeting substrates that drive processes critical to cell growth.2 One of those processes; protein translation, is controlled through the mTORC1 pathway. Composed of five known subunits, -mTOR, mLST8, PRAS40, Raptor and DEPTOR 2- mTORC1 works as a switch to ensure cells have adequate amounts of nutrients and oxygen before undergoing protein translation.3 In cases where adequate conditions are absent (energy stress), AMP-activated protein Kinase (AMPK) inhibits mTORC1.4 Figure 1: Mechanism of AMPK-mediated mTORC1 inhibition.In response to energy stress, -where there are low levels of intracellular ATP and high levels of intracellular ATP- LKB1 phosphorylates the critical activation loop, threonine, in the alpha subunit of AMPK, activating AMPK. Meanwhile, AMP directly binds to tandem repeats of crystathionine-?-synthase (CBS) domains in the AMPK ? subunit, preventing the dephosphorylation of the critical activation loop threonine. The activated AMPK binds to phosphorylate raptor and also directly phosphorylates TSC2. The phosphoryation of TSC2 renders it unable to use its GAP domain to activate Rheb GTPase. Rheb GTPase normally activates TORC1, so upon inactivation of Rheb GTPase, mTORC1 is essentially inhibited.3,4 Pointed arrows indicate the phosphorylation is activating the target protein and flat-ended arrows indicate that the phosphorylation is inhibiting the target protein.AMPK is a protein kinase consisting of a catalytic (?) subunit and two regulatory (? and ?) subunits. Like all protein kinases, AMPK’s phosphorylation abilities make it a key regulator in every aspect of biological functions. In the case of AMPK-mTORC1 interactions, AMPK acts as an energy status sensor.4 (See fig. 1)A commonly accepted paradigm holds the idea that during energy stress, AMPK phosphorylation of Tuberous Sclerosis Complex 2 (TSC2) inhibits mTORC1 and that this is the hallmark of cell protein-translation and hence cell growth control.4 However, this simplistic scheme has been challenged by the demonstration of TSC2-deficient cells being responsive to energy stress.5 Furthermore, despite AMPK inactivation, mTOR activation is observed in all eukaryotes, including those that do not possess TSC2 orthologs.5 These two separate observations suggest that either additional mechanisms exist to coordinate these two master regulators of cell growth, or, that AMPK utilises additional components of the mTORC1 pathway. In this issue, Gwinn et al. reveal that AMPK acts to inhibit mTORC1 by the direct phosphorylation of Regulatory Associated Protein Of MTOR (raptor), and that this facilitates a metabolic checkpoint.Despite the fact that kinase activity may modify up to 30% of all human proteins,6 the mechanisms involved in substrate specificity of kinases are still poorly understood. Interactions between substrates and the active sites of enzymes are momentary, and substrates often bind to kinases via more than one binding site. Moreover, kinases can also utilise other adaptor proteins, therefore for a kinase-protein interaction to be understood each individual part of the interaction must be understood. Several studies have come to light over the past years on a new proteonomic, high throughput method of detecting optimum substrates to kinases. These positional scanning peptide libraries (PSLP’s) are based on the idea of the consensus sequence, in which key residues proximal to the phospho-acceptor residue are crucial for efficient phosphorylation.7In the study, Gwinn et al. utilised PSLP’s to show that Raptor has a high degree of compatibility to the optimum phosphorylation motif of AMPK and show that Raptor contains two conserved (see fig. 2) serine sites (serine 722 and serine 792 of human raptor) that match the AMPK consensus, pointing to a potential relationship between AMPK and raptor. Figure 2: Conservation of predicted AMPK sites in raptorInterestingly, the critical residues Ser792 in human raptor are highly conserved through Drosophila, C. elegans, and Dictyostelium, as well as in both budding and fission yeast. Such a high degree of conservation is rare among phosphorylation sites. Only two of ten other well established AMPK substrates (ACC1 Ser1216 and HMG CoR Ser862) are conserved across eukaryotes and more than half do not have orthologs in primitive eukaryotes. On another spectrum, AMPK may have been one of the earliest signalling pathways to have arisen during eukaryotic evolution as AMPK orthologs are found in essentially all eukaryotes (excluding Encephalitozoon cuniculi, a eukaryote that lives as an obligate parasite stealing ATP from its host cell).8 The outstanding conservation found in Ser 792 in raptor suggests it could represent an ancestral interaction with AMPK (Not shown in diagram).Gwinn et al. found that stimuli that activate AMPK (resveratrol or phenformin) induced in vivo phosphorylation of Ser792 at high stoichometry, lining up to the theory of raptor being a substrate for AMPK. Ser722 could not be well represented in Gwinn et al.’s analysis; however Gwinn et al. examined two separate large-scale phospho-protein analyses that revealed phosphorylation of endogenous raptor at Ser722. This phosphorylation event seems to suggest Ser722 is indeed an authentic phosphorylation site in vivo. Gwinn et al. then examined the extent of control AMPK-raptor interactions have on mTORC1. Results found AMPK-Raptor interaction to restrict mTORC1 activity. Experimentation using phospho-motif antibodies found 14-3-3 motif recognizing raptor in HEK293 cells (See fig. 3). Site-specific phospho-motif antibodies are useful in monitoring specific signalling events between protein kinases and substrates 9 pointing to the idea that Raptor interacts or binds with 14-3-3 proteins. Mass spectrometry showed that upon activation of AMPK by phenformin, (amongst others) two endogenous isoforms (zeta and gamma) of 14-3-3 proteins were precipitated. A common mechanism for phosphorylation-based inactivation of target proteins is through binding to the 14-3-3 family of proteins.1 Indeed, cells subjected to AICAR showed 14-3-3 co-transfected in proteins (glutathione S-transferases) co-immunoprecipitate with raptor, compared to non AICAR-subjected cells and LKB1 deficient cells, leading to the idea that raptor phosphorylation of serine 722 and 792 may induce 14-3-3 binding. However, 14-3-3 isoforms usually form heterodimers with each other1 and little specificity was found for isoforms other than 14-3-3 sigma, meaning that the 14-3-3 isoforms that bind AMPK-phosphorylated raptor may vary between cell types.In various large scale studies1, the regulatory roles of 14-3-3 binding mechanisms have been established; with allosteric stabilization to unfavourable states (anvil hypothesis) being the key mechanism in protein-protein interactions. Gwinn et al. used an immunoprecipitation-kinase (IP-Kinase) assay in conditions to find that raptor immunoprecipitates in a time-resolved relationship with suppression of mTORC1 kinases. Confirming that AMPK phosphorylation requirement on raptor for the inhibition of mTORC1 and thereby possibly pointing to the idea raptor 13-3-3 binding caused by AMPK phosphorylation inhibits mTORC1 due to being stabilizing in an unfavourable state.In the last and perhaps most striking experiment of all, Gwinn et al found raptor to be required for the full activation of the AMPK-mediated checkpoint. Previous studies onto AMPK lead to find that AMPK activation by energy stress formed a metabolic checkpoint; cells defective for AMPK activation continue cycling and subsequently undergo apoptosis, whilst, AMPK-activated cells resulted in cell cycle arrest.1 Therefore if energy stress is present but there is a failure in down regulation of mTORC1 (due to defective components in the AMPK mechanism) apoptosis is increased. Gwinn et al. used a non-posphorylable mutant raptor (AA) in cells absent of other apoptosis effectors (TSC2 and p53) to find that with AMPK activation, apoptosis rates almost doubled in comparison to cells lacking TSC2 and expressing the human wild-type raptor.The exhaustive studies by Gwinn et al. have proved successful in establishing the crucial contribution of raptor on AMPK-mediated mTORC1 inhibition and thus cell growth control. The striking conservation of raptor found may be an ancestral mechanism for coupling cell growth to nutrient status. More studies would be appropriate on the premises of how 14-3-3 raptor binding causes mTORC1 inhibition.Excitement about this study stems from the potential to target raptor signalling to treat nutrient-dependent diseases and perhaps the pathology of ageing. It will be interesting to further study whether AMPK phosphorylation sites in raptor orthologs play a role in these nutrient diseases in lower organisms. Over activation of mTOR signalling is not only linked to cancer but also to type 2 diabetes. 10 Therapeutics like metformin who activate AMPK are already being utilised in treating diabetes10 . The requirement of raptor to fully suppress mTORC1 suggests a therapeutic window for the use of AMPK agonists to treat tumours associated with TSC or tumours exhibiting hyper-activation of mTOR.