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.2011 Feb 11;286(6):4364-70.
doi: 10.1074/jbc.C110.205401. Epub 2010 Dec 27.

Angiomotin-like proteins associate with and negatively regulate YAP1

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Angiomotin-like proteins associate with and negatively regulate YAP1

Wenqi Wang et al. J Biol Chem..

Abstract

In both Drosophila and mammalian systems, the Hippo pathway plays an important role in controlling organ size, mainly through its ability to regulate cell proliferation and apoptosis. The key component in the Hippo pathway is the Yes-associated protein YAP1, which localizes in nucleus, functions as a transcriptional coactivator, and regulates the expression of several proliferation- and apoptosis-related genes. The Hippo pathway negatively regulates YAP1 transcriptional activity by modulating its nuclear-cytoplasmic localization in a phosphorylation-dependent manner. Here, we describe the identification of several new PY motif-containing proteins, including angiomotin-like protein 1 (AMOTL1) and 2 (AMOTL2), as YAP1-associated proteins. We demonstrate that AMOTL1 and AMOTL2 can regulate YAP1 cytoplasm-to-nucleus translocation through direct protein-protein interaction, which can occur independent of YAP1 phosphorylation status. Moreover, down-regulation of AMOTL2 in MCF10A cells promotes epithelial-mesenchymal transition, a phenotype that is also observed in MCF10A cells with YAP1 overexpression. Together, these data support a new mechanism for YAP1 regulation, which is mediated via its direct interactions with angiomotin-like proteins.

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Figures

FIGURE 1.
FIGURE 1.
Identification of AMOTL1 and AMOTL2 proteins as YAP1iassociated proteins in MCF10A cells.A, mass spectrometry analysis revealed YAP1-associated proteins identified in 293T and MCF10A cells. AMOTL1- and AMOTL2-associated proteins were also revealed by mass spectrometry analysis in MCF10A cells. The number of peptides for each protein identified by mass spectrometry analysis was listed.B, GST-YAP1 fusion proteins immobilized on Sepharose beads were incubated with cell lysates containing exogenously expressed SFB-tagged p130AMOT, p80AMOT (negative control), AMOTL1, or AMOTL2. Immunoblotting was conducted using antibodies as indicated.CBS, Coomassie Blue staining.C, immunoprecipitation (IP) was conducted using anti-YAP1 serum or prebleed serum and lysates prepared from 293T cells. Associated endogenous AMOTL1 and AMOTL2 were revealed by immunoblotting with anti-AMOTL1 and anti-AMOTL2 antibodies, respectively.Pre, prebleed serum control.D, Myc-tagged or SFB-tagged YAP1 was used to precipitate wild-type AMOTL1/AMOTL2 or two PY motifs mutated AMOTL1 (PY1/2m)/PY motif mutated AMOTL2 (PYm) (see “Experimental Procedures”). Immunoblotting was conducted using the indicated antibodies.E, S protein beads were used to pull down SFB-tagged wild-type, S127A mutant, or WW domain mutant YAP1 (see “Experimental Procedures”) from lysates containing exogenously expressed Myc-AMOTL1 or Myc-AMOTL2. Immunoblotting was conducted using antibodies as indicated.
FIGURE 2.
FIGURE 2.
AMOTL1 and AMOTL2 regulate YAP1 subcellular localization.A, the localization of endogenous YAP1 was revealed by anti-YAP1 immunostaining in cells with or without AMOTL1 or AMOTL2 overexpression.B–D, HeLa cells were transfected with plasmids encoding Myc-tagged AMOTL1 or AMOTL2 with plasmids encoding SFB-tagged wild-type YAP1 (B), YAP1 mutant with deletion of its first WW domain (C), or YAP1 S127A mutant (D). Immunostaining was conducted using antibodies as indicated.M, merged.
FIGURE 3.
FIGURE 3.
Down-regulation of AMOTL2 causes EMT in MCF10A cells.A, 293T cells were transfected with the indicated shRNAs together with plasmids encoding FLAG-tagged AMOTL1 or AMOTL2. Cells were collected 72 h later and subjected to Western blotting.Ctrl, control.B, the level of AMOTL1 or AMOTL2 transcripts was revealed by RT-PCR in the indicated stable knockdown cells.C, immunoprecipitation (IP) and immunoblotting were performed using anti-AMOTL2 serum and cell lysates prepared from the indicated cell lines. For each immunoprecipitation, a total of 1 mg of the indicated protein lysates was used. Anti-tubulin immunoblotting was included as a control.D, lentiviral shRNAs were used to infect MCF10A cells, and stable knockdown pools were generated. Bright field pictures were captured to reveal cell morphology in these pools.E, cells with AMOTL2 down-regulation displayed EMT phenotypes. E-cadherin was used as epithelial marker. N-cadherin and vimentin were used as mesenchymal markers.F, cell-cell junction was diminished in AMOTL2 knockdown cells. E-cadherin was used as cell-cell junction marker. Actin filaments were labeled by TRITC-phalloidin.M, merged.G, cell migration capability increased in AMOTL2 knockdown cells as determined by wound healing assay.H, MCF10A cells were infected with the indicated lentiviral shRNAs respectively, and stable pools were used for immunostaining with anti-E-cadherin and anti-vimentin antibodies. The efficiency of YAP1 down-regulation by shRNAs was verified by anti-YAP1 immunoblotting.I, YAP1 retained its dominant nuclear localization in AMOTL2 knockdown (Ri) cells even when cells reached confluence.M, merged.J, YAP1 phosphorylation (p-YAP1) decreased in AMOTL2 knockdown MCF10A cells. AKT and ERK signaling pathways were also activated in AMOTL2 knockdown cells.p-AKT, AKT phosphorylation;p-ERK1/2, ERK1/2 phosphorylation.
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References

    1. Harvey K., Tapon N. (2007) Nat. Rev. Cancer 7, 182–191 - PubMed
    1. Pan D. (2007) Genes Dev. 21, 886–897 - PubMed
    1. Harvey K. F., Pfleger C. M., Hariharan I. K. (2003) Cell 114, 457–467 - PubMed
    1. Dan I., Watanabe N. M., Kusumi A. (2001) Trends Cell Biol. 11, 220–230 - PubMed
    1. Cho E., Feng Y., Rauskolb C., Maitra S., Fehon R., Irvine K. D. (2006) Nat. Genet. 38, 1142–1150 - PubMed

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