Spatial organization of Hippo signaling at the plasma membrane mediated by the tumor suppressor Merlin/NF2

Abstract

Although Merlin/NF2 was discovered two decades ago as a tumor suppressor underlying Neurofibromatosis type II, its precise molecular mechanism remains poorly understood. Recent studies in Drosophila revealed a potential link between Merlin and the Hippo pathway by placing Merlin genetically upstream of the kinase Hpo/Mst. In contrast to the commonly depicted linear model of Merlin functioning through Hpo/Mst, here we show that in both Drosophila and mammals, Merlin promotes downstream Hippo signaling without activating the intrinsic kinase activity of Hpo/Mst. Instead, Merlin directly binds and recruits the effector kinase Wts/Lats to the plasma membrane. Membrane recruitment, in turn, promotes Wts phosphorylation by the Hpo-Sav kinase complex. We further show that disruption of the actin cytoskeleton promotes Merlin-Wts interactions, which implicates Merlin in actin-mediated regulation of Hippo signaling. Our findings elucidate an important molecular function of Merlin and highlight the plasma membrane as a critical subcellular compartment for Hippo signal transduction.

Figure 1. Merlin promotes Hippo signaling without stimulating the intrinsic kinase activity of Hpo/Mst. See also Figure S1

(A-C) Mer/Mer^1-600 promotes Wts and Yki phosphorylation without affecting Hpo phosphorylation. S2R+ cell lysates expressing the indicated constructs were probed for P-Wts (A), P-Yki (B) or P-Hpo (C). Relative P-Wts and Wts levels were quantified in Figure S1B. (D) In vitro kinase assay. Myc-Hpo was expressed alone or together with HA-Mer^1-600 in S2R+ cells, immunoprecipitated, and incubated with GST-Wts, and the reaction products were probed for P-Wts. (E) S2R+ cells transfected with the indicated plasmids along with control or hpo dsRNA were probed with the indicated antibodies. Note reduction of P-Wts levels by hpo RNAi. (F) RNAi of mer leads to decreased P-Yki and P-Wts but increased P-Hpo levels in S2R+ cells. Also note comparable levels of total Yki, Wts or Hpo in control and RNAi^mer cells. (G) NF2 promotes Lats1 phosphorylation without affecting Mst1 phosphorylation. HEK293 cells expressing the indicated constructs were probed for P-Lats and P-Mst. (H) Control (Nf2^flox/flox) and Nf2 (Alb-Cre; Nf2^flox/flox) livers from 2-month old littermates were probed with the indicated antibodies. Note the comparable levels of Lats1 and YAP but diminished P-Lats and P-YAP levels in Nf2 mutant livers. Also note the increased P-Mst levels in Nf2 mutants.

Figure 2. Merlin promotes plasma membrane association of Wts/Lats in Drosophila and mammalian cells. See also Figure S2

(A) Mer^1-600, but not Mer or Ex, induces Wts membrane translocation. S2R+ cells expressing the indicated constructs were fractioned into membrane (M) and cytosolic (C) fraction and probed with the indicated antibodies. T: total cell lysates. (B) Mer^1-600 induces membrane association of Wts and Wts^T1077A in S2R+ cells. Subcellular fractions were analyzed as in (A). (C) RNAi of mer results in a significant increase of endogenous Wts in the cytoplasmic fraction relative to the membrane fraction. Subcellular fractions were probed with α–Wts antibody. Nervana, a plasma membrane-localized Na+/K+ ATPase, serves as a control for fractionation. (D) NF2 promotes membrane association of Lats1 and Lats1^T1079A in HEK293 cells, as revealed by subcellular fractionation. (E) Loss of NF2 results in increased cytosolic accumulation of endogenous Lats1 and Lats2. Subcellular fractions of confluent FH-912 and FC-912 cells were probed. (F) FC-912 cells were transfected with empty vector (pcDNA), NF2 or NF2 mutants along with Myc-Lats1. Subcellular fractions were probed for Myc-Lats1. FC-912 cells expressing empty vector or NF2 mutants had higher Myc-Lats1 levels in cytosolic than membrane fraction, but FC-912 cells expressing wildtype NF2 showed similar cytosolic and membrane Myc-Lats1 levels. The cell lysates were also probed for the expression of NF2 mutants (bottom gel). (G) Liver sections from 8-day-old control and Nf2-deficient littermates, stained with Lats2 (red) and the plasma membrane marker Pan-cadherin (green). Lats2 is concentrated at the cell cortex immediately on the inner face of plasma membrane in wildtype hepatocytes, but shows a uniform cytoplasmic localization in Nf2-deficient hepatocytes.

Figure 3. Active Merlin binds to Wts/Lats through its FERM domain. See also Figure S3

(A) Wts associates with Mer^1-600, but not Mer or Ex, as revealed by immunoprecipitation in S2R+ cells as indicated. (B) Mer^1-600 exhibits an open conformation. S2R+ cells expressing Mer-N fragment (1-375) and wildtype (375-635) or truncated (375-600) Mer-C fragment were subjected to immunoprecipitation. (C) Lats1 associates with N-terminal half of NF2, as revealed by immunoprecipitation in HEK293 cells. (D) Co-immunoprecipitation between endogenous NF2 and Lats1/2 in HEK293 cells. Endogenous Lats1 or Lats2 was detected in α-NF2, but not control, immunoprecipitates. (E) NF2-Lats1 binding in vitro. HEK293 cell lysates containing Myc-Lats1 or Myc-Lats1^3m were incubated with glutathione Sepharose beads containing GST (as a control) or GST-NF2. GST-NF2 bound to Myc-Lats1 but not Myc-Lats1^3m. (F-H) C-terminal truncation of NF2 results in a more open conformation and enhances Lats1 binding and phosphorylation. HEK293 cells expressing the indicated constructs were probed for association between NF2-N and NF2-C fragments (F), NF2-Lats1 association (G) and Lats1 hydrophobic motif phosphorylation (H). (I) Wildtype NF2, but not disease-associated missense mutants, associates with Lats1, as revealed by immunoprecipitation in HEK293 cells. (J) Wildtype NF2, but not disease-associated missense mutants, promotes membrane association of Lats1 in HEK293 cells, as revealed by subcellular fractionation.

Figure 4. Identification of a conserved N-terminal motif in Wts/Lats that mediates Merlin-Wts/Lats interactions. See also Figure S4

(A) HEK293 cells expressing HA-NF2 and various truncations of Lats1 were subjected to immunoprecipitation as indicated. (B) Alignment of the conserved N-terminal motif (CNM) in Wts/Lats protein from different species. (C) Myc-Lats1, but not Myc-Lats1^3m, was co-immunoprecipitated with HA-NF2 in HEK293 cells. (D-E) The CNM is required for NF2-induced membrane association of Lats1 in HEK293 cells. Subcellular fractionation (D) and immunofluorescent staining (E) of Myc-Lats1 or Myc-Lats1^3m in the presence or absence of NF2. (F-G) The CNM is required for Mer^1-600-mediated membrane association of Wts in Drosophila S2R+ cells. Subcellular fractionation (F) and immunofluorescent staining (G) of V5-Wts or V5-Wts^3m in the presence or absence of Mer^1-600. (H) UAS-Wts and UAS-Wts^3m transgenes inserted at identical chromosomal locus were crossed to nub-Gal4 driver. Note the small-wing phenotype induced by Wts, but not Wts^3m.

Figure 5. Constitutive membrane targeting promotes Hpo-mediated hydrophobic motif phosphorylation and tumor suppressor activity of Wts. See also Figure S5

(A) S2R+ cells expressing the indicated constructs were probed for P-Yki and P-Wts. (B) Enhanced growth-suppressive activity of membrane-targeted Wts and dominant negative activity of membrane-targeted Wts^T1077A. All UAS Wts transgenes were inserted at the same locus. Note the complete loss of adult wings in nub: Myr-Wts flies, and the suppression of small-wing phenotype of nub:Hpo in flies that co-expressed Myr-Wts^T1077A. (C-E) Partial rescue of hpo mutant clones by Myr-Wts. Eye discs containing clones of the indicated genotype (GFP+, arrowheads) were stained for Diap1 (red). Note elevated Diap1 levels in hpo clones without or with Myr-Wts expression. Also note the smaller clone size and less rounded shape of the latter genotype. Clones size is quantified in (E).

Figure 6. Sav-mediated membrane association of Hpo and regulation of Merlin-Wts binding by actin cytoskeleton. See also Figure S6

(A) S2R+ cells expressing Myc-Hpo together with HA-Mer^1-600 or FLAG-Sav were fractioned into membrane (M) and cytosolic (C) fraction. T: total cell lysates. Sav, but not Mer^1-600, promoted Hpo membrane association. (B) S2R+ cells expressing V5-Wts together with HA-Mer^1-600 or FLAG-Sav were analyzed by subcellular fractionation. Mer^1-600, but not Sav, promoted Wts membrane association. (C) RNAi of sav results in decreased membrane localization of endogenous Hpo. Subcellular fractions were probed with α–Hpo antibody. (D) Sav^shrp6 mimics a sav allele that deletes just the SARAH domain (Kango-Singh et al., 2002). Unlike wildtype Sav, Sav^shrp6 failed to induce membrane recruitment of Hpo. (E) S2R+ cells expressing the indicated constructs were analyzed for P-Wts. Wildtype Sav, but not Sav^shrp6 (Kango-Singh et al., 2002) or Sav^ΔFBM (Yu et al., 2010), greatly potentiated Mer^1-600-induced Wts phosphorylation. (F-G) S2R+ cells expressing the indicated constructs were treated with DMSO (0.1%, lane 1), LatB (1 μg/ml, lane 2) or Fosk (30 μM, lane 3) for 1 hr, followed by immunoprecipitation. LatB, but not Fosk, promoted Mer-Wts interaction (F). Neither drug affected Sav-Hpo interaction (G). (H) S2R+ cells expressing the indicated constructed were treated with DMSO (0.1%, lane 1), LatB (1 μg/ml, lane 2) or C3 (2 μg/ml, lane 3), followed by immunoprecipitation. (I) S2R+ cells were treated with DMSO (0.1%, lane 1), LatB (1 μg/ml, lane 2) or C3 (2 μg/ml, lane 3), followed by immunoprecipitation with anti-Mer antibody and detection with anti-Wts antibody. (J) S2R+ cells expressing HA-Mer^1-600 and V5-Wts were treated with DMSO, LatB or C3 as in (H-I) followed by immunoprecipitation. Neither treatment affected interactions between Mer^1-600 and Wts. (K) Schematic models of Merlin function in Hippo signaling. For simplicity, only proteins relevant to the current study are illustrated in the schematic diagrams. Left: the prevailing linear model placing Merlin upstream of Hpo activation. Right: a new model based on the current study highlighting a dual mechanism in which Merlin and Sav promotes the membrane association of Wts and Hpo, respectively.

Figure 7. Loss of Mer/NF2 and Sav/Sav1 leads to synergistic defects in Hippo signaling in Drosophila and mice

(A) Synergistic effect of mer and sav mutations in Drosophila. Mid-pupal retina of the indicated genotypes was stained for Discs-Large to highlight cell outlines. Note the significantly increased number of interommatidial cells in mer; sav double mutant eyes. (B-E) Synergistic effect of Nf2 and Sav1 mutations in mice. Livers from 8 day-old pups of the following genotype were analyzed: control (Alb-Cre), Nf2 mutant (Alb-Cre; Nf2^flox2/flox2), Sav1 mutant (Alb-Cre; Sav1^flox/flox), and double mutant (Alb-Cre; Nf2^flox2/flox2; Sav1^flox/flox). Whole amount liver images (B), H&E staining (C) and Pan-CK staining (D) were shown, along with quantification of liver weight (E). While Nf2 and Sav1 livers displayed mild or no overproliferation of the CK-positive BECs, Nf2; Sav1 double mutant livers were predominantly occupied by the BECs. Also note the unique external appearance and overgrowth of Nf2; Sav1 double mutant livers. (F) Western blot analysis of liver lysates from mouse livers analyzed in (B-E). Liver lysates from three mice of each genotype were probed. Note the further decrease in YAP and Lats phosphorylation in Nf2; Sav1 double mutant livers compared to the single mutant. Also note the comparable level of Lats1 in control and Nf2 mutant livers.