C7orf59/LAMTOR4 phosphorylation and structural flexibility modulate Ragulator assembly

Author(s):Show less -

Rasheed, Nadia ^{[1, 2]} ; Lima, Tatiani B. ^{[3, 2]} ; Mercaldi, Gustavo F. ^[2] ; Nascimento, Andrey F. Z. ^[4] ; Silva, Ana L. S. ^[2] ; Righetto, Germanna L. ^[2] ; Bar-Peled, Liron ^[5] ; Shen, Kuang ^{[6, 7, 8, 9, 10]} ; Sabatini, David M. ^{[6, 7, 8, 9, 10]} ; Gozzo, Fabio C. ^[3] ; Aparicio, Ricardo ^[3] ; Smetana, Juliana H. C. ^[2]

Affiliation

^[1] Univ Estadual Campinas, Inst Biol, Campinas, SP - Brazil

^[2] Brazilian Ctr Res Energy & Mat CNPEM, Brazilian Biosci Natl Lab LNBio, BR-13083970 Campinas, SP - Brazil

^[3] Univ Estadual Campinas, Inst Chem, Campinas, SP - Brazil

^[4] Brazilian Ctr Res Energy & Mat CNPEM, Brazilian Synchrotron Light Lab LNLS, Campinas, SP - Brazil

^[5] Massachusetts Gen Hosp, Canc Ctr, Boston, MA 02114 - USA

^[6] Whitehead Inst Biomed Res, 9 Cambridge Ctr, Cambridge, MA 02142 - USA

^[7] MIT, Dept Biol, Cambridge, MA - USA

^[8] Howard Hughes Med Inst, Cambridge, MA - USA

^[9] Koch Inst Integrat Canc Res, Cambridge, MA - USA

^[10] Broad Inst Harvard & Massachusetts Inst Technol, Cambridge, MA - USA

• Document type: Journal article
• Source: FEBS OPEN BIO; v. 9, n. 9 JULY 2019.
• Web of Science Citations: 0

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 angstrom 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. (AU)

• FAPESP's process: 17/21455-7 - Structure and function of proteins involved in amino acid-mediated regulation mTORC1 pathway
• Grantee: Juliana Helena Costa Smetana
• Support type: Regular Research Grants
• FAPESP's process: 14/17264-3 - New frontiers in structural proteomics: characterizing protein and protein complex structures by mass spectrometry
• Grantee: Fabio Cesar Gozzo
• Support type: Research Projects - Thematic Grants
• FAPESP's process: 14/12445-0 - Structural characterization of the ragulator complex, a mediator of the amino acid signaling to mTORC1
• Grantee: Juliana Helena Costa Smetana
• Support type: Regular Research Grants