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Angiomotin binding-induced activation of Merlin/NF2 in the Hippo pathway.

Li Y, Zhou H, Li F, Chan SW, Lin Z, Wei Z, Yang Z, Guo F, Lim CJ, Xing W, Shen Y, Hong W, Long J, Zhang M - Cell Res. (2015)

Bottom Line: Phosphorylation of Ser518 outside the Merlin's auto-inhibitory tail does not obviously alter Merlin's conformation, but instead prevents angiomotin from binding and thus inhibits Hippo pathway kinase activation.Cancer-causing mutations clustered in the angiomotin-binding domain impair angiomotin-mediated Merlin activation.Our findings reveal that angiomotin and Merlin respectively interface cortical actin filaments and core kinases in Hippo signaling, and allow construction of a complete Hippo signaling pathway.

View Article: PubMed Central - PubMed

Affiliation: Division of Life Science, State Key Laboratory of Molecular Neuroscience, Hong Kong, China.

ABSTRACT
The tumor suppressor Merlin/NF2 functions upstream of the core Hippo pathway kinases Lats1/2 and Mst1/2, as well as the nuclear E3 ubiquitin ligase CRL4(DCAF1). Numerous mutations of Merlin have been identified in Neurofibromatosis type 2 and other cancer patients. Despite more than two decades of research, the upstream regulator of Merlin in the Hippo pathway remains unknown. Here we show by high-resolution crystal structures that the Lats1/2-binding site on the Merlin FERM domain is physically blocked by Merlin's auto-inhibitory tail. Angiomotin binding releases the auto-inhibition and promotes Merlin's binding to Lats1/2. Phosphorylation of Ser518 outside the Merlin's auto-inhibitory tail does not obviously alter Merlin's conformation, but instead prevents angiomotin from binding and thus inhibits Hippo pathway kinase activation. Cancer-causing mutations clustered in the angiomotin-binding domain impair angiomotin-mediated Merlin activation. Our findings reveal that angiomotin and Merlin respectively interface cortical actin filaments and core kinases in Hippo signaling, and allow construction of a complete Hippo signaling pathway.

No MeSH data available.


Related in: MedlinePlus

Merlin-AmBD is rich in disease-related mutations found in Neurofibromatosis 2 and other cancer patients. (A) Combined surface and stick representation of disease-related missense mutations located in Merlin-AmBD, with (A1) showing the positions of the mutation sites and their interaction networks for the C-terminal half of α1CTD, and (A2) showing that the two mutation sites at the beginning of α1CTD are spatially close to Ser518 and thus expected to disturb Merlin's binding to AMOT. (B, D, F) Even with the presence of an excess amount of AMOT-CC, the cancer-related mutations such as L517P (B), L535P and Q538P (D) of Merlin have only the background level of binding to Lats1, and mutations like E463K (B) and M514V (F) have much weakened binding to Lats1. To ensure that the binding data are directly comparable and quantifiable, all binding experiments in each panel were performed in parallel with freshly purified proteins and ran in one single SDS-PAGE. The bindings of WT-Merlin to GST-Lats1 in each panel serve as the cross-panel control for the quantifications. (C, E, G) Quantifications of the bindings of various Merlin mutants to Lats1 shown in B, D and F. ***P < 0.001. (H) Membrane localization and protein stability of WT-Merlin and several of its mutants when expressed in polarized MDCK cells. (I) HEK293 cells overexpressing Merlin mutants with weakened AMOT binding abilities displayed increased YAP activation (i.e., decreased phosphorylation of YAP).
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fig7: Merlin-AmBD is rich in disease-related mutations found in Neurofibromatosis 2 and other cancer patients. (A) Combined surface and stick representation of disease-related missense mutations located in Merlin-AmBD, with (A1) showing the positions of the mutation sites and their interaction networks for the C-terminal half of α1CTD, and (A2) showing that the two mutation sites at the beginning of α1CTD are spatially close to Ser518 and thus expected to disturb Merlin's binding to AMOT. (B, D, F) Even with the presence of an excess amount of AMOT-CC, the cancer-related mutations such as L517P (B), L535P and Q538P (D) of Merlin have only the background level of binding to Lats1, and mutations like E463K (B) and M514V (F) have much weakened binding to Lats1. To ensure that the binding data are directly comparable and quantifiable, all binding experiments in each panel were performed in parallel with freshly purified proteins and ran in one single SDS-PAGE. The bindings of WT-Merlin to GST-Lats1 in each panel serve as the cross-panel control for the quantifications. (C, E, G) Quantifications of the bindings of various Merlin mutants to Lats1 shown in B, D and F. ***P < 0.001. (H) Membrane localization and protein stability of WT-Merlin and several of its mutants when expressed in polarized MDCK cells. (I) HEK293 cells overexpressing Merlin mutants with weakened AMOT binding abilities displayed increased YAP activation (i.e., decreased phosphorylation of YAP).

Mentions: Interestingly, Merlin-AmBD is enriched with missense mutations found in Neurofibromatosis type 2 patients39. It is further noted that α1CTD (part of Merlin-AmBD), but not the rest of auto-inhibitory CTD (α2CTD-α4CTD), contains at least 8 different mutations (Figures 2F and 7A). All these mutations are not expected to affect Merlin-FERM-mediated binding to Lats1/2, and thus we tested the impact of these mutations on AMOT-mediated activation of Merlin. Among the five of these eight Merlin mutants characterized in this study, four (M514V, L517P, L535P, and Q538P; Figure 7A) failed to bind to Lats1 even in the presence of AMOT (Figure 7B-7G), indicating the critical role of α1CTD for Merlin to bind to AMOT. Based on the structure shown in Figure 7A, these mutations may disrupt/destabilize the helical structure of α1CTD, and thus compromise Merlin's binding to AMOT. One Neurofibromatosis type 2 mutation (E463K) is located in the “Helical” region N-terminal to α1CTD, and this mutation also significantly weakens AMOT-potentiated Lats1 binding to Merlin (Figure 7B and 7C), further supporting that the “Helical” region and “α1CTD” together form the Merlin-AmBD.


Angiomotin binding-induced activation of Merlin/NF2 in the Hippo pathway.

Li Y, Zhou H, Li F, Chan SW, Lin Z, Wei Z, Yang Z, Guo F, Lim CJ, Xing W, Shen Y, Hong W, Long J, Zhang M - Cell Res. (2015)

Merlin-AmBD is rich in disease-related mutations found in Neurofibromatosis 2 and other cancer patients. (A) Combined surface and stick representation of disease-related missense mutations located in Merlin-AmBD, with (A1) showing the positions of the mutation sites and their interaction networks for the C-terminal half of α1CTD, and (A2) showing that the two mutation sites at the beginning of α1CTD are spatially close to Ser518 and thus expected to disturb Merlin's binding to AMOT. (B, D, F) Even with the presence of an excess amount of AMOT-CC, the cancer-related mutations such as L517P (B), L535P and Q538P (D) of Merlin have only the background level of binding to Lats1, and mutations like E463K (B) and M514V (F) have much weakened binding to Lats1. To ensure that the binding data are directly comparable and quantifiable, all binding experiments in each panel were performed in parallel with freshly purified proteins and ran in one single SDS-PAGE. The bindings of WT-Merlin to GST-Lats1 in each panel serve as the cross-panel control for the quantifications. (C, E, G) Quantifications of the bindings of various Merlin mutants to Lats1 shown in B, D and F. ***P < 0.001. (H) Membrane localization and protein stability of WT-Merlin and several of its mutants when expressed in polarized MDCK cells. (I) HEK293 cells overexpressing Merlin mutants with weakened AMOT binding abilities displayed increased YAP activation (i.e., decreased phosphorylation of YAP).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4493278&req=5

fig7: Merlin-AmBD is rich in disease-related mutations found in Neurofibromatosis 2 and other cancer patients. (A) Combined surface and stick representation of disease-related missense mutations located in Merlin-AmBD, with (A1) showing the positions of the mutation sites and their interaction networks for the C-terminal half of α1CTD, and (A2) showing that the two mutation sites at the beginning of α1CTD are spatially close to Ser518 and thus expected to disturb Merlin's binding to AMOT. (B, D, F) Even with the presence of an excess amount of AMOT-CC, the cancer-related mutations such as L517P (B), L535P and Q538P (D) of Merlin have only the background level of binding to Lats1, and mutations like E463K (B) and M514V (F) have much weakened binding to Lats1. To ensure that the binding data are directly comparable and quantifiable, all binding experiments in each panel were performed in parallel with freshly purified proteins and ran in one single SDS-PAGE. The bindings of WT-Merlin to GST-Lats1 in each panel serve as the cross-panel control for the quantifications. (C, E, G) Quantifications of the bindings of various Merlin mutants to Lats1 shown in B, D and F. ***P < 0.001. (H) Membrane localization and protein stability of WT-Merlin and several of its mutants when expressed in polarized MDCK cells. (I) HEK293 cells overexpressing Merlin mutants with weakened AMOT binding abilities displayed increased YAP activation (i.e., decreased phosphorylation of YAP).
Mentions: Interestingly, Merlin-AmBD is enriched with missense mutations found in Neurofibromatosis type 2 patients39. It is further noted that α1CTD (part of Merlin-AmBD), but not the rest of auto-inhibitory CTD (α2CTD-α4CTD), contains at least 8 different mutations (Figures 2F and 7A). All these mutations are not expected to affect Merlin-FERM-mediated binding to Lats1/2, and thus we tested the impact of these mutations on AMOT-mediated activation of Merlin. Among the five of these eight Merlin mutants characterized in this study, four (M514V, L517P, L535P, and Q538P; Figure 7A) failed to bind to Lats1 even in the presence of AMOT (Figure 7B-7G), indicating the critical role of α1CTD for Merlin to bind to AMOT. Based on the structure shown in Figure 7A, these mutations may disrupt/destabilize the helical structure of α1CTD, and thus compromise Merlin's binding to AMOT. One Neurofibromatosis type 2 mutation (E463K) is located in the “Helical” region N-terminal to α1CTD, and this mutation also significantly weakens AMOT-potentiated Lats1 binding to Merlin (Figure 7B and 7C), further supporting that the “Helical” region and “α1CTD” together form the Merlin-AmBD.

Bottom Line: Phosphorylation of Ser518 outside the Merlin's auto-inhibitory tail does not obviously alter Merlin's conformation, but instead prevents angiomotin from binding and thus inhibits Hippo pathway kinase activation.Cancer-causing mutations clustered in the angiomotin-binding domain impair angiomotin-mediated Merlin activation.Our findings reveal that angiomotin and Merlin respectively interface cortical actin filaments and core kinases in Hippo signaling, and allow construction of a complete Hippo signaling pathway.

View Article: PubMed Central - PubMed

Affiliation: Division of Life Science, State Key Laboratory of Molecular Neuroscience, Hong Kong, China.

ABSTRACT
The tumor suppressor Merlin/NF2 functions upstream of the core Hippo pathway kinases Lats1/2 and Mst1/2, as well as the nuclear E3 ubiquitin ligase CRL4(DCAF1). Numerous mutations of Merlin have been identified in Neurofibromatosis type 2 and other cancer patients. Despite more than two decades of research, the upstream regulator of Merlin in the Hippo pathway remains unknown. Here we show by high-resolution crystal structures that the Lats1/2-binding site on the Merlin FERM domain is physically blocked by Merlin's auto-inhibitory tail. Angiomotin binding releases the auto-inhibition and promotes Merlin's binding to Lats1/2. Phosphorylation of Ser518 outside the Merlin's auto-inhibitory tail does not obviously alter Merlin's conformation, but instead prevents angiomotin from binding and thus inhibits Hippo pathway kinase activation. Cancer-causing mutations clustered in the angiomotin-binding domain impair angiomotin-mediated Merlin activation. Our findings reveal that angiomotin and Merlin respectively interface cortical actin filaments and core kinases in Hippo signaling, and allow construction of a complete Hippo signaling pathway.

No MeSH data available.


Related in: MedlinePlus