Abstract
The mechanistic Target of Rapamycin complex 1 (mTORC1) senses intracellular amino acid levels through an intricate machinery, which includes the Rag GTPases, Ragulator and vacuolar ATPase (V-ATPase). The membrane-associated E3 ubiquitin ligase ZNRF2 is released into the cytosol upon its phosphorylation by Akt. In this study, we show that ZNRF2 interacts with mTOR on membranes, promoting the amino acid-stimulated translocation of mTORC1 to lysosomes and its activation in human cells. ZNRF2 also interacts with the V-ATPase and preserves lysosomal acidity. Moreover, knockdown of ZNRF2 decreases cell size and cell proliferation. Upon growth factor and amino acid stimulation, mTORC1 phosphorylates ZNRF2 on Ser145, and this phosphosite is dephosphorylated by protein phosphatase 6. Ser145 phosphorylation stimulates vesicle-to-cytosol translocation of ZNRF2 and forms a novel negative feedback on mTORC1. Our findings uncover ZNRF2 as a component of the amino acid sensing machinery that acts upstream of Rag-GTPases and the V-ATPase to activate mTORC1.
Highlights
Mechanistic target of rapamycin is a conserved serine/threonine protein kinase of the phosphatidylinositol 3-kinase (PI3K)-related kinase family (PIKK), which functions as the catalytic subunit of two distinct complexes, mTOR complex 1 (mTORC1) and mTORC2 (Wullschleger et al, 2006)
The interactions of mTOR with ZNRF2 was confirmed by Western blotting, which showed that endogenous mTOR bind to ZNRF2-GFP, but not to the GFP-only control nor to an N-myristoylation-defective mutant (G2A) of ZNRF2 (Hoxhaj et al, 2012), indicating that N-myristoylation of ZNRF2 is important for this interaction (Figure 1a)
We identified a new binding partner of mTOR, ZNRF2, which acts as a positive effector functioning upstream of V-ATPase to mediate amino acid-activation of mTORC1
Summary
Mechanistic target of rapamycin (mTOR) is a conserved serine/threonine protein kinase of the phosphatidylinositol 3-kinase (PI3K)-related kinase family (PIKK), which functions as the catalytic subunit of two distinct complexes, mTORC1 and mTORC2 (Wullschleger et al, 2006). In mTOR complex 1 (mTORC1), mTOR associates with Raptor, mLST8, PRAS40 and Deptor. The second mTOR-containing complex, mTORC2, comprises mTOR, Rictor, mSIN1, mLST8, Deptor and Protor. The functions of the two complexes can be distinguished through the use of the mTOR inhibitor, rapamycin, which acutely and inhibits mTORC1, but has no short-term effect on mTORC2 (Sarbassov et al, 2006). MTORC1 is a central integrator of regulatory inputs from intracellular amino acids, ATP, O2 and extracellular signals such as insulin and growth factors (Dibble and Manning, 2013). Through phosphorylation of its substrates, including ribosomal protein S6 kinase (S6K) and the eIF4E-binding proteins (4-EBP), mTORC1 stimulates mRNA translation and, cell growth and proliferation (Fingar and Blenis, 2004; Showkat et al, 2014)
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