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.