YAP1-AMOTL1 binding requires the AMOTL1 motifs LPTY and the PPEY.

<p>Either of Flag-tagged AMOTL1^188–191A (LY: LPTY, amino acids 188–191), AMOTL1 ^{188–191A/310–313A} (LY/PY1: LPTY, amino acids 188–191 and PPEY, amino acids 310–313) or AMOTL1 ^{188–191A/367–370A} (LY/PY2: LPTY, amino acids 188–191 and PPEY, amino acids 367–370) mutants were used to precipitate YAP1. Immunoblotting was carried out using the indicated antibodies.</p

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.</p

Nedd4.2 interacts with AMOTL1 and mislocalizes AMOTL1 from tight junctions.

<p><i>A</i>, Nedd4.2 reduces AMOTL1 protein levels. Co-expression of myc-tagged Nedd4.2 (myc.Nedd4.2) decreased Flag-tagged AMOTL1 (F.AMOTL1), occasionally to very low protein levels (lane 2). <i>B</i>, AMOTL1 interacts with Nedd4.2. HEK 293T cells were transfected with constructs encoding F.AMOTL1, myc.GFP, or myc.Nedd4.2. Cell lysates were probed with anti-myc and anti-Flag antibodies. Precipitation of F.AMOTL1 immobilized myc.Nedd4.2, but not myc.GFP, while precipitated F.GFP did not bind myc.Nedd4.2. <i>C</i>, Binding of endogenous AMOTL1 to Nedd4.2 was detected with anti-AMOTL1 and anti-Nedd4.2 antibodies, respectively. CEP164 was used as a negative control. <i>D</i>, AMOTL1 co-localizes mostly at the plasma membrane with Nedd4.2 in HEK293T cells. <i>E</i>, MDCK cells were transfected either with empty vector or Nedd4.2. Cells were then stained for endogenous AMOTL1 (grayscale) and the tight junction marker, ZO-1 (red). AMOTL1 is localized to tight junctions together, with ZO-1. Note that in dividing cells (yellow arrowheads) less ZO-1 as well as AMOTL1 are observed at junctions. When Nedd4.2 is added, endogenous ZO-1 as well as AMOTL1 are mislocalized from tight junctions and appear mostly in the cytoplasm. Scale bars represent 20 µm.</p

Phosphorylation of Nedd4.2 by c-Abl at tyrosine 71 and 457 inhibits its E3 ligase activity.

<p><i>A</i>, Myc-Nedd4.2 proteins purified from HEK293T cells, co-expressing either wild type or kinase-dead (KD) c-Abl, were analyzed by nanoLC-MS/MS after in-gel digestion and phosphopeptide enrichment. Two singly Y-phosphorylated peptides (Y71 and Y457) were identified. <i>B</i>, Nedd4.2 proteins with Y71F and Y457F substitutions exhibit moderately reduced levels of c-Abl-mediated tyrosine phosphorylation. HEK293T cells were transfected with plasmids encoding myc-tagged wild type (WT) Nedd4.2 or mutants (Y71F, Y457F, or Y71F/Y457F), together with Flag-tagged AMOTL1 and WT or kinase-dead c-Abl, as indicated. Nedd4.2 proteins were precipitated with anti-myc antibodies and blotted with antibodies, as indicated. c-Abl and AMOTL1 were detected in the lysates with anti-Flag antibody. <i>C</i>, c-Abl restricts the ability of Nedd4.2 to extend the poly-ubiquitin chains of AMOTL1. Flag-tagged AMOTL1 (F.AMOTL1) and HA.Ubiquitin were co-expressed with either myc.Nedd4.2 alone, or with myc.Nedd4.2 and V5.cAbl. Ubiquitylated AMOTL1 species, detected by anti-HA antibodies, were shifted from higher molecular weights (poly-Ubi-AMOTL1) above 220 kDa towards lower molecular weight complexes between 100 kDa and 220 kDa (oligo-Ubi-AMOTL1) which are no longer degraded (see text for details). Nedd4.2^Y71F/Y457F is resistant to c-Abl phosphorylation. While c-Abl reduced the molecular weight of ubiquitylated AMOTL1 (left panel), there was no detectable change in molecular weight of ubiquitylated AMOTL1 (right panel) when c-Abl was co-expressed with the double tyrosine Nedd4.2 (Nedd4.2^Y71F/Y457F) mutant. <i>D</i>, The dual functions of YAP1. Upper panel: AMOTL1 recruits cytoplasmic YAP1 and c-Abl to tight junctions to curtail the ability of Nedd4.2 to poly-ubiquitylate AMOTL1. Lower panel: In cells primed to undergo proliferation, YAP1 translocates to the nucleus. Now, Nedd4.2 poly-ubiquitylates AMOTL1 and targets it for degradation.</p

c-Abl phosphorylates Nedd4.2 which forms a triple complex with AMOTL1 and YAP1.

<p><i>A</i>, Flag-tagged c-Abl (F.cAbl), immobilized on M2 sepharose beads, precipitates Nedd4.2. Immunoprecipitates were blotted with anti-myc antibody. AIP4, E3 ligase was used as a negative control. <i>B</i>, Protein-protein interactions among AMOTL1, Nedd4.2 and YAP1. HEK293T cells transfected with Flag-tagged AMOTL1, myc.Nedd4.2, V5.YAP1 and V5.cAbl plasmids were lysed and immunoprecipitation was performed using M2 beads. Precipitates were blotted with the antibodies indicated. Increased levels of AMOTL1 were detected in the presence of YAP1 and c-Abl. <i>C</i>, HEK293T cells were co-transfected with myc.Nedd4.2 and either wild type or kinase-dead (KD) c-Abl. Immunoprecipitation was performed with anti-myc antibody. Samples were blotted with anti-phosphotyrosine antibody to test for Nedd4.2 phosphorylation. <i>D</i>, Tyrosine phosphorylation of Nedd4.2 by c-Abl inhibits AMOTL1 degradation. Flag-tagged AMOTL1 (F.AMOTL1) and myc-tagged Nedd4.2 (myc.Nedd4.2) were co-expressed with either wild type or kinase-dead Flag-tagged c-Abl (F.cAbl). Precipitates, immobilized with anti-myc antibody, were probed with anti-phosphotyrosine and with anti-myc antibody show equal amounts of Nedd4.2; staining with anti-Flag revealed AMOTL1 protein levels.</p

Nedd4.2 targets AMOTL1 for proteasomal degradation.

<p>HEK 293T cells were transfected with AMOTL1, Nedd4.2 and YAP1, as indicated. Cells were treated with 5 µM MG132 for 120 min. Lysates were analyzed by Western blotting. AMOTL1, Nedd4.2 and YAP1 were detected with anti-Flag, anti-myc and anti-V5 antibodies, respectively. Actin was used as a loading control.</p

Depletion of endogenous YAP1 or Nedd4.2 affects endogenous AMOTL1 levels.

<p>HEK293T cells were transiently transfected with either YAP1 or Nedd4.2 shRNA. Even a small reduction of YAP1 results in almost complete absence of AMOTL1. However depletion of Nedd4.2 maintains AMOTL1 levels. Actin was used as a loading control.</p

The WW3 domain of Nedd4.2 interacts with AMOTL1.

<p><i>A</i>, The two PPxY motifs of AMOTL1 (PY1: PPEY, amino acid 310–313, and PY2: PPEY, amino acid 367–370) are important for the interaction with Nedd4.2. Precipitation of wild type F.AMOTL1 immobilized both myc.Nedd4.2 and V5.YAP1 (left panel), while precipitation of mutant F.AMOTL1PY1/2, lacking both PPxY motifs, immobilized V5.YAP1, but only small amounts of myc.Nedd4.2 (right panel). <i>B</i>, The WW1, WW2, WW3 and WW4 domains of Nedd4.2 were mutated, and co-expressed with Flag-tagged AMOTL1. Only the interaction between the myc.Nedd4.2 WW3 and AMOTL1 was decreased, indicating that this domain interacts with AMOTL1.</p

Glis2 interacts with PIAS4 and SUMO-3.

<p>(A) FLAG-tagged PIAS4 (F.PIAS4) and V5-tagged Glis2 (V5.Glis2) were transiently co-expressed in HEK 293T cells together with F.CD2AP and V5.Diversin as controls. Precipitation of F.PIAS4 immobilized only V5.Glis2 but not V5.Diversin, while V5.Glis2 was not detectable in F.CD2AP precipitates. (B) Immunofluorescence analysis of HEK 293T cells expressing YFP.PIAS4 (green) and RFP.Glis2 (red) showed nuclear co-localisation of both proteins. Nuclei were stained with Hoechst blue. (C) His-tagged SUMO proteins (SUMO-1, SUMO-2, and 3) were transiently co-transfected with V5.Glis2, V5.NPHP3 and V5.NPHP2 in HEK 293T cells. After 36 hours of transfection, SUMO proteins were precipitated using Ni-NTA beads. Glis2 and other nephronophthisis gene products (NPHP) were detected using an anti-V5 antibody.</p

Lysine 195 is required for Glis2 stability.

<p>(A) HEK 293T cells, transfected with Glis2 wild type (WT) or Glis2.K195R along with V5-tagged GFP, were treated with MG132 (12 μM) for 2 hours to inhibit proteasomal degradation. DMSO was used as a vehicle control. The cell lysates were resolved on SDS-PAGE, and the protein levels of Glis2 were detected using anti-V5 antibody. (B) Bars represent the quantification of Glis2.WT and Glis2.K195R protein levels normalised to V5-tagged GFP levels from three independent experiments (n = 3). (C) <i>In vivo</i> ubiquitylation assays demonstrate the increased ubiquitylation of F.Glis2.K195R in comparison to Glis2 wild type (WT). (D) <i>In vivo</i> ubiquitylation assays were performed with the plasmids as indicated to compare the SUMO-3 mediated inhibition of ubiquitylation of F.Glis2 wild type (WT) and F.Glis2.K195R.</p