YAP1 inhibits AMOTL1 degradation via its first WW domain.

<p><i>A</i>, Nedd4.2 facilitates the ubiquitylation of AMOTL1. HEK293T cells were transfected with AMOTL1, Nedd4.2 and YAP1, as indicated. AMOTL1 was precipitated and probed for incorporated HA-tagged ubiquitin. AMOTL1 levels were determined by re-probing the blot with anti-Flag antibody. A catalytically inactive (DN) Nedd4.2 was used as a control for ligase activity. <i>B</i>, Wild type YAP1 and the YAP1WW2 mutant, but not the YAP1WW1 mutant, reduce ubiquitylation of AMOTL1. HA-tagged ubiquitin (HA.Ubiquitin) and expression vectors, as indicated, were co-expressed in HEK293T cells. AMOTL1 was precipitated by M2 sepharose beads, and then probed with antibody to HA to identify ubiquitylated AMOTL1. <i>C</i>, YAP1 protects both single and double AMOTL1 PPxY mutants against Nedd4.2-mediated ubiquitylation. Either of AMOTL1^310–313A (F.AMOTL1PY1), AMOTL1^367–370A (F.AMOTL1PY2) or AMOTL1^{310–313A/367–370A} (F.AMOTL1PY1/2) were co-expressed with different combinations of HA.Ubiquitin, myc.Nedd4.2, V5.YAP1 and V5.YAP1 WW1, as indicated. Cell extracts were precipitated with M2 beads and blotted with anti-HA antibody. In blots for ubiquitin, brackets indicate molecular weights that correspond to poly- or oligo-ubiquitylation of AMOTL1. Poly-ubiquitylation results in complexes with molecular weights above 220 kDa which signal degradation of the protein by the 26S proteasome. Complexes between 100 kDa and 220 kDa indicate oligo-ubiquitylated peptides that are generally not degraded by the 26S proteasome.