RESUMEN
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.
Asunto(s)
Factores de Intercambio de Guanina Nucleótido/química , Factores de Intercambio de Guanina Nucleótido/metabolismo , Complejos Multiproteicos/química , Complejos Multiproteicos/metabolismo , Células Cultivadas , Cristalografía por Rayos X , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Células HEK293 , Humanos , Modelos Moleculares , Fosforilación , Conformación ProteicaRESUMEN
The phosphoinositide-3-kinase (PI3K)/protein kinase B (Akt) axis plays a central role in attenuating inflammation upon macrophage stimulation with toll-like receptor (TLR) ligands. The mechanistic target of rapamycin complex 2 (mTORC2) relays signal from PI3K to Akt but its role in modulating inflammation in vivo has never been investigated. To evaluate the role of mTORC2 in the regulation of inflammation in vivo, we have generated a mouse model lacking Rictor, an essential mTORC2 component, in myeloid cells. Primary macrophages isolated from myeloid-specific Rictor null mice exhibited an exaggerated response to TLRs ligands, and expressed high levels of M1 genes and lower levels of M2 markers. To determine whether the loss of Rictor similarly affected inflammation in vivo, mice were either fed a high fat diet, a situation promoting chronic but low-grade inflammation, or were injected with lipopolysaccharide (LPS), which mimics an acute, severe septic inflammatory condition. Although high fat feeding contributed to promote obesity, inflammation, macrophage infiltration in adipose tissue and systemic insulin resistance, we did not observe a significant impact of Rictor loss on these parameters. However, mice lacking Rictor exhibited a higher sensitivity to septic shock when injected with LPS. Altogether, these results indicate that mTORC2 is a key negative regulator of macrophages TLR signalling and that its role in modulating inflammation is particularly important in the context of severe inflammatory challenges. These observations suggest that approaches aimed at modulating mTORC2 activity may represent a possible therapeutic approach for diseases linked to excessive inflammation.