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Abstract
Ragulator is a pentamer composed of p18, MP1, p14, C7orf59, and hepatitis B virus X‐interacting protein (HBXIP; LAMTOR 1—5) which acts as a lysosomal scaffold of the Rag GTPases in the amino acid sensitive branch of TORC1 signaling. Here, we present the crystal structure of human HBXIP‐C7orf59 dimer (LAMTOR 4/5) at 2.9 Å and identify a phosphorylation site on C7orf59 which modulates its interaction with p18. Additionally, we demonstrate the requirement of HBXIP‐C7orf59 to stabilize p18 and allow further binding of MP1‐p14. The structure of the dimer revealed an unfolded N terminus in C7orf59 (residues 1–15) which was shown to be essential for p18 binding. Full‐length p18 does not interact stably with MP1‐p14 in the absence of HBXIP‐C7orf59, but deletion of p18 residues 108–161 rescues MP1‐p14 binding. C7orf59 was phosphorylated by protein kinase A (PKA) in vitro and mutation of the conserved Ser67 residue to aspartate prevented phosphorylation and negatively affected the C7orf59 interaction with p18 both in cell culture and in vitro. C7orf59 Ser67 was phosphorylated in human embryonic kidney 293T cells. PKA activation with forskolin induced dissociation of p18 from C7orf59, which was prevented by the PKA inhibitor H‐89. Our results highlight the essential role of HBXIP‐C7orf59 dimer as a nucleator of pentameric Ragulator and support a sequential model of Ragulator assembly in which HBXIP‐C7orf59 binds and stabilizes p18 which allows subsequent binding of MP1‐p14.
Highlights
The MIT Faculty has made this article openly available
Ragulator is a pentamer composed of p18, MP1, p14, C7orf59, and hepatitis B virus X-interacting protein (HBXIP; LAMTOR 1—5) which acts as a lysosomal scaffold of the Rag GTPases in the amino acid sensitive branch of TORC1 signaling
In order to survive in nutrient insufficient/deprived conditions, eukaryotic organisms have a signaling pathway controlled by the protein kinase mammalian/ mechanistic target of rapamycin
Summary
The MIT Faculty has made this article openly available Please share how this access benefits you. We present the crystal structure of human HBXIP-C7orf dimer (LAMTOR 4/5) at 2.9 A and identify a phosphorylation site on C7orf which modulates its interaction with p18. C7orf was phosphorylated by protein kinase A (PKA) in vitro and mutation of the conserved Ser residue to aspartate prevented phosphorylation and negatively affected the C7orf interaction with p18 both in cell culture and in vitro. Abbreviations cAMP, cyclic adenosine monophosphate; Ego-TC, Ego ternary complex; FPLC, fast performance liquid chromatography; GST, glutathione Stransferase; HBXIP, hepatitis B virus X-interacting protein; HDX, hydrogen-deuterium exchange; HEK293, human embryonic kidney 293; MS, mass spectrometry; mTOR, mammalian/mechanistic target of rapamycin; MWCO, molecular weight cutoff; PDB, Protein Data Bank; PKA, protein kinase A; RB, Roadblock domain; SAXS, small-angle X-ray scattering; UPLC, ultra-performance liquid chromatography. Deregulation of mTOR can be observed in cancer, type 2 diabetes, obesity, and various neurodegenerative diseases [4,5,6,7]
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