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, 290 (32), 19387-401

Phosphorylation of the Hippo Pathway Component AMOTL2 by the mTORC2 Kinase Promotes YAP Signaling, Resulting in Enhanced Glioblastoma Growth and Invasiveness

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Phosphorylation of the Hippo Pathway Component AMOTL2 by the mTORC2 Kinase Promotes YAP Signaling, Resulting in Enhanced Glioblastoma Growth and Invasiveness

Nicholas Artinian et al. J Biol Chem.

Abstract

The mechanistic target of rapamycin (mTOR) and Hippo signaling pathways are two major signaling cascades that coordinately regulate cell growth and proliferation. Dysregulation of these pathways plays a critical role in gliomagenesis. Recent reports have provided evidence of cross-talk between the mTOR and Hippo pathways; however, a complete description of the signaling relationships between these pathways remains to be elucidated. Utilizing a gene-trapping strategy in a mouse glioma model, we report the identification of AMOTL2 as a candidate substrate for mTORC2. AMOTL2 is phosphorylated at serine 760 by mTORC2. Mutation of AMOTL2 mimicking constitutive Ser(760) phosphorylation blocks its ability to bind and repress YAP leading to increased relative expression of known YAP gene targets. Moreover, overexpression of AMOTL2 or a nonphosphorylatable AMOTL2-S760A mutant inhibited YAP-induced transcription, foci formation, growth, and metastatic properties, whereas overexpression of a phosphomimetic AMOTL2-S760E mutant negated these repressive effects of AMOTL2 in glioblastoma (GBM) cells in vitro. Similar effects on xenograft growth were observed in GBM cells expressing these AMOTL2 Ser(760) mutants. YAP was also shown to be required for Rictor-mediated GBM growth and survival. Finally, an analysis of mTORC2/AMOTL2/YAP activities in primary GBM samples supported the clinical relevance of this signaling cascade, and we propose that pharmacological agents cotargeting these regulatory circuits may hold therapeutic potential.

Keywords: AMOTL2; Hippo pathway; glioblastoma; mTOR complex (mTORC); mTORC2; mechanistic target of rapamycin (mTOR); yes-associated protein (YAP).

Figures

FIGURE 1.
FIGURE 1.
Proliferation and invasive properties of gene-trapped Ric0 cells derived from the Gfap-Cre+/Rictor GEM model. A, schematic of the murine Amotl2 transcript expressed in the CNS. Integrations of the retroviral gene-trap vector were identified within the second intron of Amotl2. The number of integrations per site is shown in the second intron. B, XTT proliferation assays of parental, control (EV; empty virus-transduced cells), and gene-trapped 1–7 Ric0 mutant clones. 104 cells were initially plated and assayed 48 h later. The means and +S.D. are shown for three independent experiments (*, p < 0.05, significantly different from Ric0 and Ric0 + EV). C, rescue of AMOTL2 expression in Ric0Δ clones. Parental, control (EV; empty virus transduced cells), Ric0Δ clones 1–7, and Ric0Δ clones 1–7 transfected with a mammalian expression plasmid encoding full-length AMOTL2 were subjected to XTT proliferation assays. 104 cells were initially plated and assayed 48 h later. The means and +S.D. are shown for three independent experiments (*, p < 0.05, significantly different from Ric0 and Ric0 + EV). D, relative migration of parental, control, and Ric0Δ clones. Cells were seeded into Boyden chambers and allowed to migrate toward BSA (white bars), vitronectin (gray bars), or fibronectin (black bars). The means and +S.D. are shown for three independent experiments (*, p < 0.05, significantly different from Ric0 and Ric0 + EV). E, invasive potential of parental, control, and Ric0Δ clones migrating through Matrigel. The means and +S.D. are shown for three independent experiments (*, p < 0.05, significantly different from Ric0 and Ric0 + EV).
FIGURE 2.
FIGURE 2.
mTORC2 signaling and AMOTL2 expression in Ric0Δ gene-trapped clones, effects of siRNA-mediated knockdown of AMOTL2 in U87Rictor cells, and identification of potential mTORC2 phosphorylation site within AMOTL2. A, immunoblot analysis of protein extracts from isolated mature oligodendrocytes, parental Ric0, and gene-trapped Ric0Δ (lanes 1–7) clones using antibodies against the indicated proteins. B, immunoblot analysis of AMOTL2 expression in primary GBM samples and cell lines. NB, normal brain; primary samples GBM lanes 1–4. C, AMOTL2 knockdown following 24 h of incubation with siRNAs targeting AMOTL2 or nontargeting control (Ctrl) in U87 and U87Rictor cells. Expression of AMOTL2 and actin was analyzed by immunoblot at the 24-h time point. D, effects of AMOTL2 knockdown on Rictor-mediated proliferation. The indicated cell lines were treated with control (Ctrl) nontargeting or AMOTL2-targeting siRNAs and growth-assessed by XTT proliferation assays at 48 h. The means and +S.D. are shown for three independent experiments (*, p < 0.05, significantly different from U87Rictor and U87Rictor siCtrl). E, AMOTL2 knockdown stimulates RICTOR-mediated migratory capacity. Cells were seeded into Boyden chambers and allowed to migrate toward BSA (white bars), vitronectin (gray bars), or fibronectin (black bars). The means and +S.D. are shown for three independent experiments (*, p < 0.05, significantly different from U87Rictor and U87RictorsiCtrl). F, effects of AMOTL2 knockdown on RICTOR-mediated invasive properties of U87 and U87Rictor GBM cells migrating through Matrigel. The means and +S.D. are shown for three independent experiments (*, p < 0.05, significantly different from U87Rictor and U87RictorsiCtrl). G, potential conserved mTOR phosphorylation site within AMOTL2. Sequence alignment showing conservation of AMOTL2 Ser760 among vertebrates. Phosphoserine is shown in red and +1 position conserved leucine residue is shown in green.
FIGURE 3.
FIGURE 3.
AMOTL2 is a direct target of mTORC2. A, AMOTL2 interacts with RICTOR in vivo. AMOTL2, RICTOR, or RAPTOR was immunoprecipitated (IP) from U87 cells, and Western blots were subsequently performed on the immunoprecipitates for the indicated proteins. IB, immunoblot. Lane 1, beads without antibody (Ab); lane 2, immunoprecipitation using and irrelevant antibody (actin antibody); lane 3, input U87 cell lysate; lane 4, immunoblot antibody. As a control, in RAPTOR immunoprecipitates AMOTL2 was not detected attesting to the specificity of the interaction. B, schematic diagrams of the deletion mutant constructs of Gal4DBD-RICTOR or Gal4AD-AMOTL2 that were cotransfected into AH109 yeast cells to determine whether an interaction between the proteins was detectable via activation of the HIS3 reporter (+++, strong growth; ++ moderate growth; −, no growth). Colonies that grew were assayed for β-gal activity. C, mTORC2 in vitro kinase assay was performed utilizing RICTOR immunoprecipitates isolated from U87 cells and incubated with purified native human or S760A mutated recombinant HA-tagged AMOTL2 for the indicated time points. Reactions containing native AMOTL2 were performed in the absence or presence of PP242 as shown. Kinase reactions were subsequently immunoblotted using a phospho-specific antibody generated against phospho-Ser760-AMOTL2, HA, or Rictor antibodies for detection. D, U87 and U87Rictor cells were treated in the absence or presence of the mTORC1/2 inhibitor PP242 for 24 h, and lysates were immunoblotted for the indicated proteins. U87Rictor cells harboring hyperactivated mTORC2 express elevated levels of phospho-Ser760-AMOTL2, which is abrogated by treatment with the mTORC1/2 inhibitor PP242. E, U87Rictor cells were treated with siRNAs targeting AMOTL2 or nontargeting control (Ctrl) for 24 h, and cell lysates were analyzed by immunoblotting for the indicated proteins. F, effects of insulin stimulation in U87 cells. Cells were stimulated with insulin (10 nm, 5 h), lysed, and extracts immunoblotted for the indicated proteins.
FIGURE 4.
FIGURE 4.
Ser760 phosphorylation of AMOTL2 blocks YAP binding and results in increased expression of YAP target genes. A, YAP was cotransfected with human AMOTL2, AMOTL2-S760A, or AMOTL2-S760E into U87 cells. YAP was immunoprecipitated (IP) with anti-FLAG antibody, and coimmunoprecipitated AMOTL2 proteins were analyzed by anti-HA immunoblotting. IB, immunoblot. B, YAP was cotransfected with human AMOTL2, AMOTL2-S760A, or AMOTL2-S760E into U87Rictor cells and treated with PP242 (100 nm, 2.5 h) as indicated. YAP was immunoprecipitated with anti-FLAG antibody, and coimmunoprecipitated AMOTL2 proteins were analyzed by anti-HA immunoblotting. C, YAP target genes CTGF and CYR61 are induced in Ric0Δ clones in which AMOTL2 is disrupted. RNA was extracted from the indicated cells (NMO, normal mature oligodendrocytes), and mRNA levels of CTGF and CYR61 were examined by quantitative RT-PCR. RT-PCR measurements were done in quadruplicate, and the means and +S.D. are shown (*, p < 0.05, significantly different from normal mature oligodendrocytes and Ric0). D, relative YAP expression levels in Ric0 and Ric0Δ clones 1–7 (left panel). Inset shows immunoblot of YAP expression from Ric0 and Ric0Δ1 cells. Nuclear/cytoplasmic ratio (N/C ratio) of YAP in Ric0 and Ric0Δ clones 1–7 (right panel). Inset shows immunoblot of nuclear (N) and cytoplasmic (C) fractions from Ric0 and Ric0Δ#1 cells. α-Lamin A/C and α-tubulin were immunoblotted as markers for nuclear and cytoplasmic fractions, respectively. E, U87 cells coexpressing YAP and native AMOTL2, nonphosphorylatable AMOTL2-S760A, or phosphomimetic AMOTL2-S760E were assayed for CTGF and CYR61 expression by quantitative RT-PCR. RT-PCR measurements were done in quadruplicate, and the means and +S.D. are shown (*, p < 0.05, significantly different from control (ctrl) and AMOTL2–760E). F, lysates from the indicated cell lines were immunoblotted using anti-HA and actin antibodies as shown.
FIGURE 5.
FIGURE 5.
AMOTL2 Ser760 phosphorylation is essential for YAP-induced transformation, proliferation, and migration. A, YAP-induced foci formation in MCF10A cells cotransfected with YAP and native AMOTL2, AMOTL2-S760A, or AMOTL2-S760E. The numbers of foci were determined, and the means and +S.D. are shown (*, p < 0.05, significantly different from control (ctrl) and AMOTL2-S760E). B, XTT proliferation assays of U87 cells cotransfected with YAP and AMOTL2, AMOTL2-S760A, or AMOTL2-S760E. 104 cells were plated and assayed 48 h later. The means and +S.D. are shown for three independent experiments (*, p < 0.05, significantly different from control (ctrl) and AMOTL2-S760E). C, YAP-induced migration of U87 cells cotransfected with YAP and native AMOTL2, AMOTL2-S760A, or AMOTL2-S760E. Cells were seeded into Boyden chambers and allowed to migrate toward BSA (white bars), vitronectin (gray bars), or fibronectin (black bars). The means and +S.D. are shown for three independent experiments (*, p < 0.05, significantly different from control (ctrl) and AMOTL2-S760E). D, YAP-induced invasive properties of U87 cells coexpressing YAP and native AMOTL2, AMOTL2-S760A, or AMOTL2-Ser760E migrating through Matrigel. The means and +S.D. are shown for three independent experiments. (*, p < 0.05, significantly different from control (ctrl) and AMOTL2-S760E).
FIGURE 6.
FIGURE 6.
AMOTL2 Ser760 phosphorylation is required for in vivo GBM tumor growth. A, growth of parental U87 (♦), U87AMOTL2 (▴), U87AMOTL2-S760A (●), or U87AMOTL2-S760E (■) subcutaneous xenografts in SCID mice (n = 4–5 per group; *, p < 0.05, significantly different from U87, U87-AMOTL2, and U87-AMOTL2-S760E). B, average tumor weight at sacrifice (*, p < 0.05, significantly different from U87, U87-AMOTL2, and U87-AMOTL2-S760E). C, immunoblot of lysates from the indicated cells after tumor harvest at autopsy. Protein levels of HA-tagged AMOTL2, phospho-Ser127-YAP, and actin are shown. Values in parentheses are fold increases in phospho-Ser127-YAP expression relative to parental U87 expression levels as determined by densitometric measurements. D, mRNA levels of CTGF and CYR61 isolated from the indicated cells after tumor harvest at autopsy. Levels were determined by quantitative RT-PCR. Measurements were done in quadruplicate, and the means and +S.D. are shown (*, p < 0.05, significantly different from U87, U87-AMOTL2, and U87-AMOTL2-S760E).
FIGURE 7.
FIGURE 7.
YAP is required for RICTOR-mediated GBM growth and survival. A, mTORC2 and AMOTL2 signaling in U87, U87Rictor, H4, and H4Rictor cell lines. Lysates from the indicated cell lines were immunoblotted for the proteins shown. B, YAP knockdown following 24 h of incubation with siRNAs targeting YAP or nontargeting control (ctrl). Expression of YAP and actin were analyzed by immunoblot at the 24-h time point. C, effects of YAP knockdown on RICTOR-mediated growth. The indicated cell lines were treated with control (Ctrl) or YAP-targeting siRNAs, and growth was assessed by XTT proliferation assays at the indicated time points. The means and +/−S.D. are shown for three independent experiments (left panel, *, p < 0.05, significantly different from U87Rictor siCtrl; right panel, *, p < 0.05, significantly different from H4Rictor siCtrl). D, apoptotic cells were identified by TUNEL assays at the 48-h time point for the indicated lines and treatment groups. The percentage of TUNEL+ cells was determined by counting TUNEL+ nuclei among at least 500 cells in distinct fields of each experiment. Three independent experiments were performed and analyzed. Means and +S.D. are shown (left panel, *, p < 0.05, significantly different from U87Rictor siCtrl; right panel, *, p < 0.05, significantly different from H4Rictor siCtrl). E, effects of YAP knockdown on growth of U87Rictor and U87Rictor AMOTL2-S760A cells (left panel) or H4Rictor and H4Rictor AMOTL2-S760A cells (right panel). The indicated cell lines were treated with control (Ctrl) or YAP-targeting siRNAs and growth assessed by XTT proliferation assays at the indicated time points. The means and +/−S.D. are shown for three independent experiments. F, TUNEL assays of U87Rictor and U87Rictor AMOTL2-S760A cells (left panel) or H4Rictor and H4Rictor AMOTL2-S760A cells (right panel). Three independent experiments were performed and analyzed. Means and +S.D. are shown. G, effects of verteporfin exposure on Rictor-mediated proliferation. The indicated cell lines were treated with control (Ctrl) or YAP-targeting siRNAs and growth was assessed by XTT proliferation assays at the indicated time points. The means and +S.D. are shown for three independent experiments (left panel, *, p < 0.05, significantly different from U87Rictor DMSO; right panel, *, p < 0.05, significantly different from H4Rictor DMSO). H, effects of YAP knockdown or verteporfin exposure on RICTOR-induced YAP target gene expression. CTGF and CYR61 mRNA levels were determined by quantitative RT-PCR. Measurements were done in quadruplicate, and the means and +S.D. are shown (left panel, *, p < 0.05, significantly different from U87RictorsiCtrl and U87RictorDMSO groups; right panel, *, p < 0.05, significantly different from H4RictorsiCtrl and H4RictorDMSO groups).
FIGURE 8.
FIGURE 8.
Expression of YAP in primary GBM samples and model of mTORC2/AMOTL2/YAP signaling in GBM. A, representative images of GBM sections immunohistochemically stained for YAP1. GBM1 demonstrated nuclear staining (arrows) associated with activated YAP, and GBM2 displayed YAP immunoreactivity within the cytoplasmic compartment (arrows) where YAP is sequestered. Bar, 10 μm. B, in this model, mTORC2 inhibits the YAP negative regulator AMOTL2 by phosphorylating serine 760. This phosphorylation event blocks AMOTL2 binding to YAP, permitting nuclear translocation and subsequent activation of YAP target genes.

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