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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

Mammalian Merlin adopts a semi-open conformation. (A) Schematic diagrams showing the domain organizations of Merlin and EBP50. The Merlin-FERM/EBP50-CT interaction is indicated by a two-way arrow. The S518 site is indicated by a red asterisk and used throughout the rest of the figures. The color coding scheme of Merlin is used throughout the rest of the paper unless specified otherwise. (B-D) ITC-based measurements of the bindings between EBP50-CT and Merlin-FERM (B), WT-Merlin and various mutant forms of full-length Merlin (C, D). (E) Sedimentation velocity analysis showing the hydrodynamic properties of WT Merlin and A585W-Merlin. The fitted molecular weight of A585W-Merlin is indicated. The green dashed line scales the y-axis of the WT-Merlin profile by 10 folds to show that the broader peak at S value ∼2.5 when compared to that of A585W-Merlin. The loading concentrations of the two proteins for the experiment are the same. (F, G) Differential scanning fluorimetry-based thermal denaturation assay showing the temperature-dependent denaturation profiles of WT-Merlin, S518D-Merlin, A585W-Merlin, and S518D/A585W-Merlin.
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fig1: Mammalian Merlin adopts a semi-open conformation. (A) Schematic diagrams showing the domain organizations of Merlin and EBP50. The Merlin-FERM/EBP50-CT interaction is indicated by a two-way arrow. The S518 site is indicated by a red asterisk and used throughout the rest of the figures. The color coding scheme of Merlin is used throughout the rest of the paper unless specified otherwise. (B-D) ITC-based measurements of the bindings between EBP50-CT and Merlin-FERM (B), WT-Merlin and various mutant forms of full-length Merlin (C, D). (E) Sedimentation velocity analysis showing the hydrodynamic properties of WT Merlin and A585W-Merlin. The fitted molecular weight of A585W-Merlin is indicated. The green dashed line scales the y-axis of the WT-Merlin profile by 10 folds to show that the broader peak at S value ∼2.5 when compared to that of A585W-Merlin. The loading concentrations of the two proteins for the experiment are the same. (F, G) Differential scanning fluorimetry-based thermal denaturation assay showing the temperature-dependent denaturation profiles of WT-Merlin, S518D-Merlin, A585W-Merlin, and S518D/A585W-Merlin.

Mentions: Despite more than two decades of extensive research on Merlin, the precise mechanism by which Merlin functions to regulate Hippo pathway kinases or any other targets are poorly understood. Most of the current working models for Merlin are inferred based on the action mechanisms of the related Ezrin, Radixin, and Moesin (ERM) family proteins and biochemical and cell biology studies of Merlin, as the family of FERM domain proteins share high amino acid sequence homology (FERM domains in particular) and similar domain organizations (Figure 1A). The C-terminal tail domain (CTD) of Merlin is assumed to bind to its FERM domain intra-molecularly, resulting in a conformationally closed and functionally dormant form of Merlin17,18,19, although a recent study suggests that mammalian Merlin adopts a partially open conformation20. Unique to Merlin, Ser518 at the beginning of the proposed inhibitory CTD of Merlin (Figure 1A), instead of Thr577 which is the equivalent of Thr558 in Moesin, can undergo p21-activated kinase (PAK)- or cyclic AMP-dependent protein kinase (PKA)-dependent phosphorylation4,21,22. It is generally accepted that phosphorylation of Ser518 acts to convert Merlin into a less active state in suppressing cell growth20,22,23,24,25,26. However, whether phosphorylation of Ser518 leads to a more closed or more open conformation of Merlin remains a topic of debate. A very recent small-angle scattering-based study has shown that the Ser518Asp substitution does not significantly alter the overall closed conformation of the full-length Merlin27. In contrast, in another study the Ser518Ala substitution has been shown to cause Merlin to be more functionally active and interpreted to release Merlin's auto-inhibited conformation20, although it is rather stretching to imagine how the subtle Ala substitution for Ser might open the auto-inhibited conformation of Merlin.


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)

Mammalian Merlin adopts a semi-open conformation. (A) Schematic diagrams showing the domain organizations of Merlin and EBP50. The Merlin-FERM/EBP50-CT interaction is indicated by a two-way arrow. The S518 site is indicated by a red asterisk and used throughout the rest of the figures. The color coding scheme of Merlin is used throughout the rest of the paper unless specified otherwise. (B-D) ITC-based measurements of the bindings between EBP50-CT and Merlin-FERM (B), WT-Merlin and various mutant forms of full-length Merlin (C, D). (E) Sedimentation velocity analysis showing the hydrodynamic properties of WT Merlin and A585W-Merlin. The fitted molecular weight of A585W-Merlin is indicated. The green dashed line scales the y-axis of the WT-Merlin profile by 10 folds to show that the broader peak at S value ∼2.5 when compared to that of A585W-Merlin. The loading concentrations of the two proteins for the experiment are the same. (F, G) Differential scanning fluorimetry-based thermal denaturation assay showing the temperature-dependent denaturation profiles of WT-Merlin, S518D-Merlin, A585W-Merlin, and S518D/A585W-Merlin.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig1: Mammalian Merlin adopts a semi-open conformation. (A) Schematic diagrams showing the domain organizations of Merlin and EBP50. The Merlin-FERM/EBP50-CT interaction is indicated by a two-way arrow. The S518 site is indicated by a red asterisk and used throughout the rest of the figures. The color coding scheme of Merlin is used throughout the rest of the paper unless specified otherwise. (B-D) ITC-based measurements of the bindings between EBP50-CT and Merlin-FERM (B), WT-Merlin and various mutant forms of full-length Merlin (C, D). (E) Sedimentation velocity analysis showing the hydrodynamic properties of WT Merlin and A585W-Merlin. The fitted molecular weight of A585W-Merlin is indicated. The green dashed line scales the y-axis of the WT-Merlin profile by 10 folds to show that the broader peak at S value ∼2.5 when compared to that of A585W-Merlin. The loading concentrations of the two proteins for the experiment are the same. (F, G) Differential scanning fluorimetry-based thermal denaturation assay showing the temperature-dependent denaturation profiles of WT-Merlin, S518D-Merlin, A585W-Merlin, and S518D/A585W-Merlin.
Mentions: Despite more than two decades of extensive research on Merlin, the precise mechanism by which Merlin functions to regulate Hippo pathway kinases or any other targets are poorly understood. Most of the current working models for Merlin are inferred based on the action mechanisms of the related Ezrin, Radixin, and Moesin (ERM) family proteins and biochemical and cell biology studies of Merlin, as the family of FERM domain proteins share high amino acid sequence homology (FERM domains in particular) and similar domain organizations (Figure 1A). The C-terminal tail domain (CTD) of Merlin is assumed to bind to its FERM domain intra-molecularly, resulting in a conformationally closed and functionally dormant form of Merlin17,18,19, although a recent study suggests that mammalian Merlin adopts a partially open conformation20. Unique to Merlin, Ser518 at the beginning of the proposed inhibitory CTD of Merlin (Figure 1A), instead of Thr577 which is the equivalent of Thr558 in Moesin, can undergo p21-activated kinase (PAK)- or cyclic AMP-dependent protein kinase (PKA)-dependent phosphorylation4,21,22. It is generally accepted that phosphorylation of Ser518 acts to convert Merlin into a less active state in suppressing cell growth20,22,23,24,25,26. However, whether phosphorylation of Ser518 leads to a more closed or more open conformation of Merlin remains a topic of debate. A very recent small-angle scattering-based study has shown that the Ser518Asp substitution does not significantly alter the overall closed conformation of the full-length Merlin27. In contrast, in another study the Ser518Ala substitution has been shown to cause Merlin to be more functionally active and interpreted to release Merlin's auto-inhibited conformation20, although it is rather stretching to imagine how the subtle Ala substitution for Ser might open the auto-inhibited conformation of Merlin.

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