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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 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.
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fig5: 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.

Mentions: The FERM domain-mediated target bindings of Merlin require the release of the CTD-mediated auto-inhibition (e.g., a frequently used but rather physiologically artificial approach is truncating a part of CTD, see13 for an example). The α1CTD segment, a core element of Merlin-AmBD, is central for the Merlin head-to-tail auto-inhibition, as well as for directly blocking the binding of Lats1/2-FBD to Merlin-FERM (Figure 3G). Therefore, the binding of AMOT-CC to Merlin-AmBD may release the inhibitory CTD from Merlin-FERM, thereby facilitating Merlin to bind to its downstream targets such as Lats1/2. Consistent with this prediction, addition of AMOT-CC fragment or the full-length AMOT (either the p80 or the p130 isoform) can significantly potentiate the binding of Lats1-FBD to WT-Merlin (Figure 5A-5D). This biochemical result qualifies AMOT as an upstream activator of Merlin in the Hippo pathway. Interestingly, the fully closed mutant Merlin (A585W-Merlin) can no longer be activated by AMOT-CC (Figure 5A and 5B), pointing to the functional importance of the semi-open 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)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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fig5: 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.
Mentions: The FERM domain-mediated target bindings of Merlin require the release of the CTD-mediated auto-inhibition (e.g., a frequently used but rather physiologically artificial approach is truncating a part of CTD, see13 for an example). The α1CTD segment, a core element of Merlin-AmBD, is central for the Merlin head-to-tail auto-inhibition, as well as for directly blocking the binding of Lats1/2-FBD to Merlin-FERM (Figure 3G). Therefore, the binding of AMOT-CC to Merlin-AmBD may release the inhibitory CTD from Merlin-FERM, thereby facilitating Merlin to bind to its downstream targets such as Lats1/2. Consistent with this prediction, addition of AMOT-CC fragment or the full-length AMOT (either the p80 or the p130 isoform) can significantly potentiate the binding of Lats1-FBD to WT-Merlin (Figure 5A-5D). This biochemical result qualifies AMOT as an upstream activator of Merlin in the Hippo pathway. Interestingly, the fully closed mutant Merlin (A585W-Merlin) can no longer be activated by AMOT-CC (Figure 5A and 5B), pointing to the functional importance of the semi-open 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