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. 2024 Sep 9;15:7873. doi: 10.1038/s41467-024-52284-8

Fig. 3. Merlin dephosphorylation at Serine 13 activates meningioma Wnt signaling.

Fig. 3

a Full-length structural model of Merlin, based on the crystal template 4RZJ. Red shows cancer-associated missense mutations. Cyan shows the NTD, which is not conserved in other FERM family members, containing a consensus PP1A/PKC phosphorylation motif and phosphorylation site (S13) in orange. b the 4RZJ X-ray structure of Merlin shows L46 and A211 in hydrophobic pockets that may be destabilized by charged cancer-associated missense mutations. c TOP-Flash Tcf/Lef luciferase reporter assay in M10GdCas9-KRAB meningioma cells expressing sgRNAs suppressing NF2 (sgNF2) with or without rescue of Merlin constructs or overexpression of the FERM family member Moesin. Data are representative of four biological replicates. d Immunoblots for FLAG (Merlin), β-catenin, or GAPDH before versus after FLAG immunoprecipitation from M10G meningioma cells with or without doxycycline-inducible MerlinFLAG overexpression (1 μg/ml). e TOP-Flash Tcf/Lef luciferase reporter assay in M10GdCas9-KRAB meningioma cells expressing sgNF2 with or without rescue of Merlin wildtype or S13 unphosphorylatable (S13A) or phospho-mimetic (S13D) constructs. Representative of 3 experimental repeats. f CH-157MN meningioma cell MTT assays for cell proliferation with or without doxycycline-inducible Merlin rescue (100 ng/ml). Representative of four experimental repeats. g CH-157MN xenograft measurements in NU/NU mice with or without doxycycline-inducible Merlin rescue (20 μg/ml) as in (f). h Kaplan-Meier survival curve for CH-157MN xenograft overall survival in NU/NU mice as in g (log-rank test). i, j Immunoblots for HA (Merlin) or Merlin with phosphorylated S13 (MerlinpS13) after Merlin immunoprecipitation from M10G cells with or without Merlin overexpression and concurrent expression of non-targeted control siRNAs (siNTC) or siRNAs suppressing PP1A or PKC isoforms. GAPDH immunoblots are shown from immunoprecipitation inputs as a loading control. Representative of four experimental repeats. k TOP-Flash Tcf/Lef luciferase reporter assay in M10GdCas9-KRAB meningioma cells expressing non-targeted control sgRNAs (sgNTC) or sgNF2 with concurrent siNTC or siRNA suppression of PP1A or PKC isoforms. Data are representative of four biological replicates. Lines represent means, and error bars represent standard error of the means. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.0001 (Student’s t test, one sided). Source data are provided as a Source Data file.