Control of mTORC1 signaling by the Opitz syndrome protein MID1

Abstract

Mutations in the MID1 gene are causally linked to X-linked Opitz BBB/G syndrome (OS), a congenital disorder that primarily affects the formation of diverse ventral midline structures. The MID1 protein has been shown to function as an E3 ligase targeting the catalytic subunit of protein phosphatase 2A (PP2A-C) for ubiquitinmediated degradation. However, the molecular pathways downstream of the MID1/PP2A axis that are dysregulated in OS and that translate dysfunctional MID1 and elevated levels of PP2A-C into the OS phenotype are poorly understood. Here, we show that perturbations in MID1/PP2A affect mTORC1 signaling. Increased PP2A levels, resulting from proteasome inhibition or depletion of MID1, lead to disruption of the mTOR/Raptor complex and downregulated mTORC1 signaling. Congruously, cells derived from OS patients that carry MID1 mutations exhibit decreased mTORC1 formation, S6K1 phosphorylation, cell size, and cap-dependent translation, all of which is rescued by expression of wild-type MID1 or an activated mTOR allele. Our findings define mTORC1 signaling as a downstream pathway regulated by the MID1/PP2A axis, suggesting that mTORC1 plays a key role in OS pathogenesis.ubiquitin ligase | proteasome-mediated degradation M utations in the MID1 gene are causally linked to X-linked Opitz BBB/G syndrome (OS), a congenital disorder that primarily affects the formation of ventral midline structures. The clinical manifestations of Opitz syndrome are characterized by a diverse spectrum of symptoms, including hypertelorism and hypospadias, cleft lip/palate, dysphagia, heart defects, and mental retardation (1).The MID1 protein is a member of the RBCC/TRIM family, which contains a conserved module of the RING domain, followed by B-boxes and a coiled-coil domain (2). MID1 also contains fibronectin type III and B30.2/SPRY domains. The RING domain has been well characterized in ubiquitin-mediated protein degradation, whereas the other domains mediate proteinprotein interactions (3-6). Opitz syndrome-derived mutations in MID1 have been identified throughout the protein and show several functional consequences such as compromised association with microtubules and/or transport along microtubules (4, 7-9). In addition to its microtubule-binding function, MID1 also functions as an E3 ligase that targets the microtubule-associated pool of the catalytic subunit of protein phosphatase 2A (PP2A-C) for ubiquitin-mediated degradation through an interaction with the protein α4 (3). Perturbation of the E3 ligase function of MID1 in OS cells leads to the accumulation of PP2A-C and the dramatic dephosphorylation of microtubule-associated proteins, which is postulated to contribute to OS pathogenesis (3).Signaling from the mammalian target of rapamycin (mTOR) controls diverse cellular processes such as growth, autophagy, stress responses, cytoskeletal reorganization, cell motility, metabolism, and aging (10-12). mTORC1 consists of mTOR and the interacting proteins, Raptor, and mLST8. In particular, Raptor plays an essential scaffolding...

Translation of HTT mRNA with expanded CAG repeats is regulated by the MID1–PP2A protein complex

“…MID1 mediates binding of the protein complex to the CAG repeat mRNA and acts as a negative regulator of PP2A. MID1 also regulates mTOR activity: in the absence of MID1 PP2Ac binds to its subunits A and Ba (instead of binding MID1 and a4) and then inhibits binding of mTOR to its interacting proteins for example, raptor, which leads to inhibition of mTOR in this protein complex (called TORC1) 19 . Therefore, increased binding of MID1 and S6K to HTT mRNA with elongated CAG repeats leads to an increased protein translation.…”

“…MID1 recruits PP2Ac and the translation regulator S6K to these mRNAs. MID1 is a negative regulator of PP2Ac, thereby stimulating mTOR activity, the phosphorylation of S6K, and protein translation 19 . This then would lead to an overproduction of aberrant protein coded by the mRNA and its expanded CAG repeat stretch (Fig.…”

“…Through this negative regulatory influence on PP2A activity, MID1 also controls the activity of the mTOR kinase. High levels of PP2A activity in MID1-deficient cells abolish the interaction between mTOR and raptor, which is an essential cofactor of mTOR activity 19 . Accordingly, a significant reduction of mTOR activity is found in these cells.…”

X-linked microtubule-associated protein, Mid1, regulates axon development

“…Indeed, subsequent studies have shown that in OS the normal regulated turnover of PP2Ac along the microtubules is perturbed, leading to other microtubule-associated proteins being caught in an abnormal phosphorylation state (13), which may affect microtubule polymerization and stability. Recently, mTORC1 signaling, which is also involved in cytoskeletal remodeling, was identified as a downstream component of the Mid1-PP2Ac pathway (35). Further investigation should therefore be focused on elucidating the downstream molecular events triggered by Mid1-directed PP2Ac regulation.…”

Novel role of Rac-Mid1 signaling in medial cerebellar development

“…A recent study demonstrated that inhibition/depletion of Mid1 results in the downregulation of mTORC1 signaling (Liu et al, 2011). This report prompted us to examine a new signaling pathway: Rac1-Mid1-mTORC1.…”