Proteomic analysis reveals GIT1 as a novel mTOR complex component critical for mediating astrocyte survival

Abstract

As a critical regulator of cell growth, the mechanistic target of rapamycin (mTOR) protein operates as part of two molecularly and functionally distinct complexes. Herein, we demonstrate that mTOR complex molecular composition varies in different somatic tissues. In astrocytes and neural stem cells, we identified G-protein-coupled receptor kinase-interacting protein 1 (GIT1) as a novel mTOR-binding protein, creating a unique mTOR complex lacking Raptor and Rictor. Moreover, GIT1 binding to mTOR is regulated by AKT activation and is essential for mTOR-mediated astrocyte survival. Together, these data reveal that mTOR complex function is partly dictated by its molecuflar composition in different cell types.

Keywords: GIT1; astrocytes; brain; mTOR.

Figure 1.

mTOR complex composition is cell type-specific. (A) Representative immunoblots demonstrate little Deptor and Protor-1 expression in wild-type mouse astrocytes and fibroblasts relative to HEK293T (293T) cells. Immunoblots from mTOR immunoprecipitations reveal Rictor, Raptor, mSIN1, PRAS40, and mLST8 in wild-type astrocytes (B) and fibroblasts (C). Rb IgG was included as an internal control for nonspecific binding. (D) While Raptor (mTORC1) immunoprecipitations contained mTOR, Raptor, PRAS40, and mLST8 (but not Rictor), Rictor (mTORC2) immunoprecipitations contained mSIN1 and mLST8 but lacked Raptor and PRAS40 in wild-type astrocytes.

Figure 2.

GIT1 is a novel mTOR-binding protein in astrocytes. (A) Proteomic analysis of mTOR immunoprecipitations from wild-type astrocytes reveals several potential mTOR-binding proteins. (B) Validation of mTOR-binding proteins with spectral counts of ≥10 by immunoblot reveals that endogenous GIT1, but not TGM2 or RhoC, associates with mTOR. Rb IgG was included as an internal control for nonspecific binding. (C) GIT1 and mTOR binding is also observed in GIT1 immunoprecipitations from wild-type astrocytes. (D) Full-length Flag-tagged GIT1 (Flag-GIT1) and myc-tagged mTOR (myc-mTOR) associate in HEK293 cells. Flag-pCMVg (empty vector) was used as a control. (E) GIT1 is not found in Raptor (mTORC1) or Rictor (mTORC2) immunoprecipitations by immunoblot. Raptor, Rictor, and mLST8 are not contained in GIT1 immunoprecipitations. (F) Full-length myc-mTOR binding to Flag-GIT1 requires GIT1 residues 420–770 (dotted outline) containing the paxillin-binding domain (PBD) and a portion of the synaptic localization domain (SLD). ANK denotes the ankyrin repeats, while SHD denotes the Spa2 homology domain. (G) Full-length Flag-GIT1 binding to myc-mTOR requires mTOR residues 1482–2008, encompassing the mTOR FRAP, ATM, and TRAP (FAT) domains.

Figure 3.

Neurofibromin loss decreases GIT1 binding to mTOR. (A) Neurofibromin loss results in increased astrocyte growth and mTOR activation (phospho-S6^S240/244) relative to wild-type controls. Reduced GIT1 binding to mTOR is observed in Nf1^−/− relative to wild-type astrocytes. Scatter plots illustrate independently generated mTOR immunoprecipitations. Treatment with the either 10 nM rapamycin (B) or 50 nM MK2206 (C) increases GIT1 binding to mTOR in Nf1^−/− astrocytes. mTOR and AKT inhibition were confirmed by decreased S6 (phospho-S6^Ser240/244), AKT (phospho-AKT^Thr308), PRAS40 (phospho-PRAS40^Thr246), and GSK-3β (phospho-GSK-3β^Ser9) phosphorylation. (*) P < 0.05; (**) P < 0.005.

Figure 4.

Neurofibromin-mediated astrocyte survival operates in an mTOR/GIT1-dependent manner. (A) Git1 knockdown using two independently generated mouse-specific RNAi (shGit1) lentiviruses reduces GIT1 expression in wild-type and Nf1-deficient (Nf1^−/−) astrocytes. (B) shGit1 knockdown decreases Nf1^−/−, but not wild-type, astrocyte proliferation relative to (vector) controls. (C) shGit1 knockdown increases apoptosis (“% TUNEL⁺ cells”; arrows denote TUNEL⁺ cells). (D) Proposed mechanism for GIT1-mediated neurofibromin/mTOR-regulated astrocyte survival. While neurofibromin inhibits AKT/mTOR/GIT1-mediated astrocyte growth (left panel), neurofibromin loss leads to an AKT/mTOR-dependent reduction in mTOR/GIT1 binding, which results in increased astrocyte survival (middle panel). (Right panel) Genetic and pharmacological inhibition of AKT, mTOR, or GIT1 reduces astrocyte survival. (*) P < 0.05; (**) P < 0.005; (***) P < 0.0005.