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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

A model depicting the phosphorylation-modulated activity regulation of Merlin. (A, B) PAK1 can enhance phosphorylation of Merlin (A), and cause the inactivation of the Hippo-YAP signaling pathway as evidenced by increased expression levels of two YAP target genes CTGF and Cyr61 (B), presumably due to the weakened AMOT/phosphor-Merlin interaction. (C) A model depicting the phosphorylation-mediated activity regulation of Merlin. In this model, WT-Merlin adopts a semi-open conformation via an intra-molecular head-to-tail interaction. Upstream regulators such as AMOT can bind to Merlin-CTD, fully expose the FERM domain and thus activate Merlin. Phosphorylation of Ser518 can weaken AMOT's binding to Merlin-CTD and thus maintain Merlin in its inactive state.
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fig6: A model depicting the phosphorylation-modulated activity regulation of Merlin. (A, B) PAK1 can enhance phosphorylation of Merlin (A), and cause the inactivation of the Hippo-YAP signaling pathway as evidenced by increased expression levels of two YAP target genes CTGF and Cyr61 (B), presumably due to the weakened AMOT/phosphor-Merlin interaction. (C) A model depicting the phosphorylation-mediated activity regulation of Merlin. In this model, WT-Merlin adopts a semi-open conformation via an intra-molecular head-to-tail interaction. Upstream regulators such as AMOT can bind to Merlin-CTD, fully expose the FERM domain and thus activate Merlin. Phosphorylation of Ser518 can weaken AMOT's binding to Merlin-CTD and thus maintain Merlin in its inactive state.

Mentions: Finally, we directly tested the effect of Ser518 phosphorylation of Merlin on its binding to AMOT. As PAK1/2 have been shown to specifically phosphorylate Ser518 of Merlin22,26 (Supplementary information, Figure S6), we assessed the impact of co-expression of a constitutively active form of PAK1 on the interaction between AMOT and Merlin in cultured HEK293 cells. Consistent with our in vitro interaction data, expression of PAK1 led to a decreased association between AMOT and Merlin (Figure 4H). We also assessed the role of the PAK1-mediated phosphorylation of Merlin on AMOT-potentiated Merlin/Lats1/2 interaction. Upon co-expression of PAK1 with GFP-Merlin, the interaction between Merlin and Lats1 was largely diminished even in the presence of an excess amount of AMOT-CC (Figure 5H), indicating that the activation of Merlin by AMOT can be reversed by PAK1-mediated phosphorylation of Merlin on Ser518. Consistent with the above biochemical studies, overexpression of active PAK1 in HEK293 cells led to an increased level of Ser518-phosphorylated Merlin and a concomitant increase of YAP transcriptional activity (Figure 6A and 6B), presumably at least in part due to the weakened AMOT/phosphor-Merlin interaction. We noticed that PAK1-induced Merlin phosphorylation at Ser518 is clearly observable but rather modest, indicating that additional kinases or other regulatory mechanisms may exist to modulate Merlin phosphorylation in vivo. Taken together, our biochemical and structural data presented above not only identify AMOT as an upstream activator of Merlin, but also elucidate a mechanism by which Merlin phosphorylation at Ser518 regulates its activity in the Hippo pathway (Figure 6C).


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)

A model depicting the phosphorylation-modulated activity regulation of Merlin. (A, B) PAK1 can enhance phosphorylation of Merlin (A), and cause the inactivation of the Hippo-YAP signaling pathway as evidenced by increased expression levels of two YAP target genes CTGF and Cyr61 (B), presumably due to the weakened AMOT/phosphor-Merlin interaction. (C) A model depicting the phosphorylation-mediated activity regulation of Merlin. In this model, WT-Merlin adopts a semi-open conformation via an intra-molecular head-to-tail interaction. Upstream regulators such as AMOT can bind to Merlin-CTD, fully expose the FERM domain and thus activate Merlin. Phosphorylation of Ser518 can weaken AMOT's binding to Merlin-CTD and thus maintain Merlin in its inactive state.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig6: A model depicting the phosphorylation-modulated activity regulation of Merlin. (A, B) PAK1 can enhance phosphorylation of Merlin (A), and cause the inactivation of the Hippo-YAP signaling pathway as evidenced by increased expression levels of two YAP target genes CTGF and Cyr61 (B), presumably due to the weakened AMOT/phosphor-Merlin interaction. (C) A model depicting the phosphorylation-mediated activity regulation of Merlin. In this model, WT-Merlin adopts a semi-open conformation via an intra-molecular head-to-tail interaction. Upstream regulators such as AMOT can bind to Merlin-CTD, fully expose the FERM domain and thus activate Merlin. Phosphorylation of Ser518 can weaken AMOT's binding to Merlin-CTD and thus maintain Merlin in its inactive state.
Mentions: Finally, we directly tested the effect of Ser518 phosphorylation of Merlin on its binding to AMOT. As PAK1/2 have been shown to specifically phosphorylate Ser518 of Merlin22,26 (Supplementary information, Figure S6), we assessed the impact of co-expression of a constitutively active form of PAK1 on the interaction between AMOT and Merlin in cultured HEK293 cells. Consistent with our in vitro interaction data, expression of PAK1 led to a decreased association between AMOT and Merlin (Figure 4H). We also assessed the role of the PAK1-mediated phosphorylation of Merlin on AMOT-potentiated Merlin/Lats1/2 interaction. Upon co-expression of PAK1 with GFP-Merlin, the interaction between Merlin and Lats1 was largely diminished even in the presence of an excess amount of AMOT-CC (Figure 5H), indicating that the activation of Merlin by AMOT can be reversed by PAK1-mediated phosphorylation of Merlin on Ser518. Consistent with the above biochemical studies, overexpression of active PAK1 in HEK293 cells led to an increased level of Ser518-phosphorylated Merlin and a concomitant increase of YAP transcriptional activity (Figure 6A and 6B), presumably at least in part due to the weakened AMOT/phosphor-Merlin interaction. We noticed that PAK1-induced Merlin phosphorylation at Ser518 is clearly observable but rather modest, indicating that additional kinases or other regulatory mechanisms may exist to modulate Merlin phosphorylation in vivo. Taken together, our biochemical and structural data presented above not only identify AMOT as an upstream activator of Merlin, but also elucidate a mechanism by which Merlin phosphorylation at Ser518 regulates its activity in the Hippo pathway (Figure 6C).

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