AMOT potentiates WT but not the Ser518-phosphorylated Merlin to bind to Lats1. (A) Binding of the semi-open WT-Merlin, but not the closed A585W-Merlin, to Lats1-FBD can be significantly enhanced in the presence of AMOT-CC. (B) Lats1 binding to WT-Merlin and A585W-Merlin in the presence or absence of AMOT-CC was quantified. Values are mean ± SD from three independent experiments (as with the rest of the binding experiments shown in this figure and Figure 7). ***P < 0.001, and n.s. stands for non-significant. (C) Addition of the full-length AMOT (AMOT-p80 or AMOT-p130) potentiates Merlin's binding to Lats1. (D) Quantification of the binding experiments shown in C. (E) Phosphorylation-mimic S518D-Merlin shows only a background level of binding to Lats1 both in the presence and absence of AMOT-CC. In contrast, S518A-Merlin displays an AMOT-CC-dependent binding to Lats1 as WT-Merlin does. (F) Quantification of the binding experiments shown in E. (G) Analytical gel filtration-based assay shows that deletion of a 9-residue fragment surrounding Ser518 (Δ513-521) of Merlin-AmBD essentially disrupts its binding to AMOT-CC. (H) Phosphorylation at Ser518 of Merlin by PAK1 prevents its AMOT-CC-potentiated binding to Lats1. In this assay, we co-transfected GFP-tagged full-length Merlin and Myc-tagged constitutively active form of PAK1 (empty Myc-tagged vector as the control) into the HEK293 cells, and compared AMOT-potentiated binding between Merlin and Lats1. In the upper panel, the presence of PAK1 eliminated AMOT's capacity in potentiating Merlin/Lats1 interaction. The bottom panel shows the input of GST and GST-Lats1 by Ponceau S staining.