INTERACTION OF THE TMEM127 TUMOR SUPPRESSOR WITH THE MTORC1 LYSOSOMAL ACTIVATING COMPLEX
The mTORC1 pathway regulates multiple cellular processes to promote a switch from catabolic toanabolic metabolism and is thus under tight regulatory control by growth factor signaling and nutrientsensing pathways. Dysregulation of this complex machinery is implicated in many cancers, so definingthe key mechanisms by which mTORC1 senses changes in cellular homeostasis to activate growthsignals is of great relevance. Spatial regulation of mTORC1 signaling has been recognized as a majormechanism that influences the cellular response to nutrients and the lysosome is central to thisprocess. We found that the tumor suppressor TMEM127, a poorly characterized lysosomal protein, is acomponent of the lysosome-anchored multiprotein complex involved in the mTORC1 response toamino acids. In this proposal we seek to define the mechanisms through which the interaction betweenTMEM127 and the mTORC1 regulatory machinery is regulated and how this can impact on mTORC1inhibition.We previously identified TMEM127 as a tumor suppressor gene mutated in hereditary neuroendocrinetumors, pheochromocytomas, and in renal cancers, and found that mutant tumors display increasedmTORC1 signaling. Our earlier studies revealed that TMEM127 loss leads to lysosomal expansion withredistribution of mTOR toward the lysosome. The lysosome functions as a docking platform formTORC1 signaling in response to amino acids through the assembly of a multi?protein complexinvolving Rag GTPases, Ragulator (LAMTOR1?5) and vacuolar ATPase (v?ATPase). Using multiple invivo and in vitro approaches developed in our lab, we found that TMEM127 physically associates withragulator, vATPase and Rags, and in its absence the interaction between ragulator and Rags isenhanced and mTOR recruitment by Rags is increased. Furthermore, our preliminary data support ofan effect of TMEM127 in nutrient sensing. Based on these observations, our general hypothesis is thatTMEM127 disrupts mTOR recruitment to the lysosomal?centered protein complex throughinhibition of the ragulator-Rag interaction in response to amino acids. To test this hypothesis, wepropose to define the signals that regulate the TMEM127-ragulator-Rag-mTORC1 interaction, includingits response to individual amino acids, to other nutrients (e.g. glucose), to cellular stresses includingstarvation, osmolality, oxidative stress, and growth factor signaling. Furthermore, we propose tosystematically define the components of the lysosomal multiprotein assembly that are required forTMEM127 binding, and conversely, which domains of TMEM127 are necessary for this interaction.Finally, we will explore the emerging notion that the nutrient sensing and the growth-factor signalingbranches of mTORC1 activation are integrated at the lysosome by defining TMEM127's contribution tothe mTOR inhibitory actions by TSC2 at the lysosomal surface.To carry out these experiments we willtake advantage of tools and models that were developed in our lab, i.e. an in vivo mouse strain oftargeted Tmem127 deletion, cell models of human TMEM127 knockout (by CRISPR-Cas9-basedgenome editing), knockdown (by siRNA), as well as a cohort of primary tumor samples with TMEM127mutations, along with mutant constructs and genome edited cells that mimic naturally-occurringTMEM127 mutations identified in patients with cancer. In addition, genetic models of activation or lossof ragulator, Rags and mTOR components will complement our analysis.Given its tumor suppressor role in humans, understanding the contribution of TMEM127, a novellysosomal protein that participates in the mTORC1 lysosomal assembly should provide insights intohow nutrient and growth signals are integrated and can be coopted by cancer cells to promoteuncontrolled proliferation.