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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

AMOT specifically binds to Merlin. (A) Domain organizations of Merlin, AMOT-p130, and AMOT-p80. The interaction between Merlin and AMOT-CC is indicated by a two-way arrow. (B-F) Analytical gel filtration chromatography-based mapping of the minimal binding domains of AMOT and Merlin for their mutual interaction. The elution profiles of various forms of Merlin, AMOT, and their mixtures are shown in red, green, and black, respectively. (G) Analytical gel filtration chromatography showing that AMOT-CC does not bind to a Moesin fragment containing both the helical region and CTD. (H) Immunoprecipitation of overexpressed HA-tagged AMOT and endogenous Merlin showing that the AMOT/Merlin interaction occurs in cultured HEK293 cells, and this interaction is weakened by PAK1-mediated phosphorylation (right panel). The inputs are shown on the left. It is noted that in the AMOT-bound fraction of Merlin, no phosphor-Merlin is detected.
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fig4: AMOT specifically binds to Merlin. (A) Domain organizations of Merlin, AMOT-p130, and AMOT-p80. The interaction between Merlin and AMOT-CC is indicated by a two-way arrow. (B-F) Analytical gel filtration chromatography-based mapping of the minimal binding domains of AMOT and Merlin for their mutual interaction. The elution profiles of various forms of Merlin, AMOT, and their mixtures are shown in red, green, and black, respectively. (G) Analytical gel filtration chromatography showing that AMOT-CC does not bind to a Moesin fragment containing both the helical region and CTD. (H) Immunoprecipitation of overexpressed HA-tagged AMOT and endogenous Merlin showing that the AMOT/Merlin interaction occurs in cultured HEK293 cells, and this interaction is weakened by PAK1-mediated phosphorylation (right panel). The inputs are shown on the left. It is noted that in the AMOT-bound fraction of Merlin, no phosphor-Merlin is detected.

Mentions: AMOT, a recently identified Hippo pathway component, was proposed to act downstream of Merlin and regulate Ras-MAPK activities30,42. The relationship between AMOT and Merlin in the Hippo pathway is unclear. Recently, the predicted coiled-coil (CC) domain of AMOT was shown to interact with the helical region between the FERM domain and the inhibitory CTD of Merlin30. We reasoned that AMOT might function as a candidate of our hypothesized, Ser518 phosphorylation-modulated Merlin activity regulatory factor. To test this hypothesis, we first verified the AMOT/Merlin interaction and mapped their precise binding domains using high-quality, purified recombinant proteins. Consistent with the reported finding30, an elongated α-helix-rich region of AMOT (residues 404-728 of AMOT-p130, Figure 4A) robustly binds to the entire C-terminal half of Merlin, forming a stable complex with 1:1 stoichiometry (Figure 4B). Further truncation experiments demonstrated that the predicted CC domain of AMOT-p130 (AMOT-CC, residues 404-633) is sufficient for binding to Merlin (Figure 4E and 4F). Interestingly, the helical region of Merlin previously predicted to interact with AMOT shows weak binding to AMOT (Figure 4C and Supplementary information, Figure S5) and the CC domain N-terminal to this helical region is not required for AMOT binding (Figure 4D). The above biochemical data indicate that at least part of the inhibitory Merlin-CTD is also required for AMOT binding. Further boundary mapping revealed that truncation of the α2CTD-α4CTD portion of Merlin-CTD has no impact on the Merlin/AMOT binding (Figure 4E and 4F), indicating that, in addition to the helical region, the α1CTD segment is also critical for Merlin's binding to AMOT. Therefore, the minimal and complete AMOT-binding domain of Merlin (Merlin-AmBD) encompasses residues 401-550 (Figure 4A). Importantly, the AMOT-CC fails to bind to the corresponding “Helical-α1CTD” region in Moesin (Figure 4G), indicating that the AMOT/Merlin interaction is very specific. It is important to note that the amino acid sequences of the “Helical-α1CTD” regions are the most diverse ones between Merlin and ERMs (Supplementary information, Figure S2), providing a possible mechanistic explanation to the specificity of the AMOT/Merlin interaction.


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)

AMOT specifically binds to Merlin. (A) Domain organizations of Merlin, AMOT-p130, and AMOT-p80. The interaction between Merlin and AMOT-CC is indicated by a two-way arrow. (B-F) Analytical gel filtration chromatography-based mapping of the minimal binding domains of AMOT and Merlin for their mutual interaction. The elution profiles of various forms of Merlin, AMOT, and their mixtures are shown in red, green, and black, respectively. (G) Analytical gel filtration chromatography showing that AMOT-CC does not bind to a Moesin fragment containing both the helical region and CTD. (H) Immunoprecipitation of overexpressed HA-tagged AMOT and endogenous Merlin showing that the AMOT/Merlin interaction occurs in cultured HEK293 cells, and this interaction is weakened by PAK1-mediated phosphorylation (right panel). The inputs are shown on the left. It is noted that in the AMOT-bound fraction of Merlin, no phosphor-Merlin is detected.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig4: AMOT specifically binds to Merlin. (A) Domain organizations of Merlin, AMOT-p130, and AMOT-p80. The interaction between Merlin and AMOT-CC is indicated by a two-way arrow. (B-F) Analytical gel filtration chromatography-based mapping of the minimal binding domains of AMOT and Merlin for their mutual interaction. The elution profiles of various forms of Merlin, AMOT, and their mixtures are shown in red, green, and black, respectively. (G) Analytical gel filtration chromatography showing that AMOT-CC does not bind to a Moesin fragment containing both the helical region and CTD. (H) Immunoprecipitation of overexpressed HA-tagged AMOT and endogenous Merlin showing that the AMOT/Merlin interaction occurs in cultured HEK293 cells, and this interaction is weakened by PAK1-mediated phosphorylation (right panel). The inputs are shown on the left. It is noted that in the AMOT-bound fraction of Merlin, no phosphor-Merlin is detected.
Mentions: AMOT, a recently identified Hippo pathway component, was proposed to act downstream of Merlin and regulate Ras-MAPK activities30,42. The relationship between AMOT and Merlin in the Hippo pathway is unclear. Recently, the predicted coiled-coil (CC) domain of AMOT was shown to interact with the helical region between the FERM domain and the inhibitory CTD of Merlin30. We reasoned that AMOT might function as a candidate of our hypothesized, Ser518 phosphorylation-modulated Merlin activity regulatory factor. To test this hypothesis, we first verified the AMOT/Merlin interaction and mapped their precise binding domains using high-quality, purified recombinant proteins. Consistent with the reported finding30, an elongated α-helix-rich region of AMOT (residues 404-728 of AMOT-p130, Figure 4A) robustly binds to the entire C-terminal half of Merlin, forming a stable complex with 1:1 stoichiometry (Figure 4B). Further truncation experiments demonstrated that the predicted CC domain of AMOT-p130 (AMOT-CC, residues 404-633) is sufficient for binding to Merlin (Figure 4E and 4F). Interestingly, the helical region of Merlin previously predicted to interact with AMOT shows weak binding to AMOT (Figure 4C and Supplementary information, Figure S5) and the CC domain N-terminal to this helical region is not required for AMOT binding (Figure 4D). The above biochemical data indicate that at least part of the inhibitory Merlin-CTD is also required for AMOT binding. Further boundary mapping revealed that truncation of the α2CTD-α4CTD portion of Merlin-CTD has no impact on the Merlin/AMOT binding (Figure 4E and 4F), indicating that, in addition to the helical region, the α1CTD segment is also critical for Merlin's binding to AMOT. Therefore, the minimal and complete AMOT-binding domain of Merlin (Merlin-AmBD) encompasses residues 401-550 (Figure 4A). Importantly, the AMOT-CC fails to bind to the corresponding “Helical-α1CTD” region in Moesin (Figure 4G), indicating that the AMOT/Merlin interaction is very specific. It is important to note that the amino acid sequences of the “Helical-α1CTD” regions are the most diverse ones between Merlin and ERMs (Supplementary information, Figure S2), providing a possible mechanistic explanation to the specificity of the AMOT/Merlin interaction.

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