Research ResourceBiochemistry

Proximity biotinylation identifies a set of conformation-specific interactions between Merlin and cell junction proteins

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Science Signaling  23 Apr 2019:
Vol. 12, Issue 578, eaau8749
DOI: 10.1126/scisignal.aau8749
  • Fig. 1 Proximity biotinylation constructs.

    (A) A schematic diagram depicting the six BirAR118G fusion constructs expressed from Dox-inducible plasmids in immortalized NF2−/− mouse Schwann cells. Constructs include a negative control (BirAR118G–A-fos), two wild-type Merlin constructs with BirA fused to either the N or C terminus (BirAR118G-Merlin and Merlin-BirAR118G), a PIP2 binding–deficient mutant (Merlin-6N–BirAR118G), a C-terminal deletion mutant intended to mimic the open, FERM-accessible conformation (Merlin-FH–BirAR118G), and a mutant intended to mimic a closed, FERM-inaccessible conformation (Merlin-AR–BirAR118G). (B and C) Immunoblotting (IB) of cell lysates from Dox-induced cells, probed with antibodies directed against BirA (B) or with streptavidin (C) to visualize biotinylated proteins. The three prominent bands on the streptavidin-probed blot correspond to the endogenous biotinylated proteins pyruvate carboxylase, propionyl CoA carboxylase, and acetyl-CoA carboxylase. (D) Immunofluorescence of individual immortalized NF2−/− Schwann cells expressing the BirA constructs and stained for biotin (green) and BirA (red) in the context of an unlabeled confluent monolayer. Scale bar, 10 μm.

  • Fig. 2 Proteins biotinylated by Merlin-BirAR118G.

    (A) Venn diagram showing the 52 proteins from cells expressing Merlin-BirAR118G (pink) and the 12 proteins from cells expressing BirAR118G-Merlin (green) that met the selection criteria for Merlin interaction by proximity biotinylation followed by mass spectroscopy. (B) Venn diagram showing the 55 proteins from cells expressing Merlin-FH–BirAR118G, 81 proteins from cells expressing Merlin-AR-BirAR118G, and 27 proteins from cells expressing Merlin-6N–BirAR118G that met the selection criteria for Merlin interaction by proximity biotinylation followed by mass spectroscopy. Proteins shown in blue were also identified in the Merlin-BirAR118G dataset in (A).

  • Fig. 3 Merlin-BirAR118G biotinylates cell junction proteins.

    (A) Venn diagram showing the results of GO analysis performed on Merlin-associated proteins identified by proximity biotinylation mass spectroscopy that are components of cell junctions, bind to actin, or are members of the Hippo pathway. (B) Venn diagram of Merlin-associated cell junction proteins that are components of adherens junctions, tight junctions, and focal adhesions, based on GO analysis. (C) Proximity map of proteins biotinylated by Merlin-BirAR118G connected by lines to previously published proximity biotinylation datasets (blue boxes) generated for focal adhesions [Kindlin-2–BirAR118G and Paxillin-BirAR118G (55)], tight junctions [ZO-1–BirAR118G and BirAR118G–ZO-1 (57)], adherens junctions [E-cadherin– BirAR118G (56)], and the Hippo pathway [Lats1 and Lats2 (46)]. Actin-binding proteins are highlighted in pink (55).

  • Fig. 4 Indirect and direct Merlin binding assays.

    (A) Schematic diagram of the basic RFP pull-down assay with extracts of HEK 293T cells coexpressing RFP fused to bait proteins and a Merlin-Nanoluciferase probe (Merlin-NLuc) immunoprecipitated using a nanobody directed against RFP bound to magnetic beads. Data are expressed as % luciferase activity bound and normalized to red fluorescence on the bead (excitation 565 nm, emission 590). (B) Indirect interaction assays showing the relative luciferase activity that coimmunoprecipitated with the indicated RFP-tagged bait proteins from HEK 293T cells coexpressing the bait and Merlin-NLuc. RFP alone (no bait) was a negative control, and RFP-Angiomotin was a positive control. The dotted line represents the luciferase activity in the RFP-only negative control. Data are means relative to control ± SD; n = 3 independent experiments. *P > 0.05 by Student’s t test relative to control. (C) Direct binding assays showing the luciferase activity bound to purified RFP bait proteins that had been incubated with purified Merlin-NLuc. The dotted line represents the luciferase activity in the RFP-only negative control. Representative data of n > 3 independent biological experiments relative to control. (D) Relative binding of ASPP2 to the Merlin mutants used in the proximity biotinylation experiments (Merlin-FH, Merlin-AR, and Merlin-6N), the phosphorylation-deficient mutant S518A, the phosphomemetic mutant S518A, patient-derived mutations Δ39-121 and L360P, and the 18–amino acid N-terminal deletion mutant ΔN18. The dotted line represents the luciferase activity with wild-type (WT) Merlin. Data are means relative to wild type ± SD; n = 3 biological replicates.

  • Fig. 5 ASPP2-Merlin binding.

    (A) Schematic diagram of ASPP2 deletion mutants used to map the Merlin-binding domain. The regions of known interactions with Ras, Merlin, Ddx42p, YAP, PP1, p53, and Bcl and the known structural domains are noted in the diagram. Gln, Gln-rich domain; Pro, proline-rich domain; Ank, ankyrin repeat region; SH3, SH3 domain; AA, amino acid. Quantification of Merlin-NLuc binding to RFP-ASPP2 deletion mutants using purified proteins. Data are means relative to full-length ASPP2 ± SD; n = 3 biological replicates. (B) Schematic diagram of the domain structure of Merlin and the Merlin-NLuc deletion mutants used to map ASPP2-binding regions. WT, full-length wild-type; FH, FERM-helix (CTD deleted); HC, helix-CTD (FERM deleted); H, helix (CTD and FERM deleted). Quantification of Merlin-NLuc deletion mutants binding to RFP-Angiomotin, RFP-Lats1, and RFP-ASPP2 using purified proteins; n = 3 biological replicates. (C) Lysates of HEK 293T cells cotransfected with Merlin-NLuc and RFP-angiomotin (RFP-AM) and either GFP alone or GFP-ASPP2 (GFP-AS) were subjected to Western blotting, RFP immunoprecipitation (RFP IP), or GFP immunoprecipitation (GFP IP) and probed with a combination of antibodies directed against Merlin, GFP, and RFP. (D) Schematic diagram depicting the proposed protein-protein interactions of different Merlin conformations. We propose that Angiomotin binds to the closed conformation of Merlin at the CTD, ASPP2 binds to the closed conformation of Merlin through the FERM domain, and Lats1 binds to the open conformation of Merlin through the FERM domain.

  • Table 1 Merlin-proximal proteins.

    The proteins that met the selection criteria from proximity biotinylation assays with Merlin-BirAR118G are arranged by the average number of MS peptide reads normalized to protein molecular weight, along with the number of peptides from these proteins identified in all other samples. n = 3 independent experiments. Merlin and known Merlin-binding proteins Angiomotin (Amot), angiomotin-like protein 1 (Amotl1), Erbin (Erbb2ip), and moesin (Msn) are highlighted in bold.

    GeneDescriptionMWCtrlWTBMFHAR6N
    Nf2Neurofibromin 2 (Merlin)69,7764.7187.381.6220.8241.7124.7
    Rai14Retinoic acid induced 14108,85211.28019.249.36126.3
    Epb41l2Erythrocyte membrane protein band 4.1-like 2109,94010.948.718.58154.719.4
    UtrnUtrophin392,70613.9101.512.1137.810023.7
    Mllt4Afadin206,4991.948.35.948.129.36.8
    Amotl1Angiomotin-like protein 1107,9500.624.49.13.626.98.5
    Ruvbl1RuvB-like AAA ATPase 150,2142.211.21914.614
    Kank2KN motif and ankyrin repeat domains 290,2454.419.63.521.216.69.1
    Epb41l3Erythrocyte membrane protein band 4.1-like 3103,3382.820.35.433.329.47.9
    Epb41l1Erythrocyte membrane protein band 4.1-like 198,315019.37.318.714.62.2
    Tjp1ZO-1, tight junction protein 1194,7423.437.36.521.4265.1
    Tp53bp2ASPP2, tumor protein p53 binding protein 2125,3011.821.50.413.317.82.9
    Rassf8Ras association domain family member 848,10308.20.85.89.91.7
    VclVinculin116,7171.217.11.26.516.98
    PrunePrune exopolyphosphatase50,239071.72.46.42.9
    MpripMyosin phosphatase Rho-interacting protein116,4082.516.22.424.422.85
    Erc1ELKS/RAB6-interacting/CAST family member 1128,3313.117.81.714.22610.3
    AmotAngiomotin120,915016.22014.44.3
    Phldb2Pleckstrin homology-like domain, family B member 2141,4861.318.93.412.2148.1
    Dlg5Discs, large homolog 5214,386028.50526.96.4
    Erbb2ipErbb2-interacting protein157,248019.33.423.614.90.5
    Myh10Myosin heavy chain 10, nonmuscle228,9967.525.643.49.921.135
    LppLipoma-preferred partner65,89117.12.16.111.76.6
    Cobll1Cordon-bleu WH2 repeat protein-like 1137,3820.614.80.423.78.91.6
    Stk38Serine/threonine kinase 3854,1740.35.71.14.54.43.9
    MsnMoesin54,17405.73.57.915.26.5
    Cep55Centrosomal protein of 55 kDa53,93005.30.201.80
    Actn1Actinin, a1103,068010.14.30.4125.2
    ScribScribbled planar cell polarity protein174,0590.616.42.515.616.73.8
    Dlg1Discs, large homolog 1100,12009.31.58.612.80
    Phldb1Pleckstrin homology-like domain, family B, member 1150,0700.613.81.218.311.31.4
    Shroom4Shroom family member 4163,2381.814.81.219.38.62.1
    Ptpn13Protein tyrosine phosphatase, type 13270,3341.324.47.823.8225.1
    MpdzMultiple PDZ domain protein218,711019.20.72.419.62.7
    Tnks1bp1Tankyrase 1 binding protein 1, 182 kDa181,8251.514.92.631.319.87
    Tjp2Tight junction protein 2131,2800.310.61.243.93.2
    Phactr4Phosphatase and actin regulator 476,63205.80.66.140.5
    Fam129bFamily with sequence similarity 129, member B84,819063.72.662.5
    Ctnnd1Catenin (cadherin-associated protein), d1104,92507.31.65.85.31.4
    Peak1Pseudopodium-enriched atypical kinase 1191,0970.612.75.617.413.14.8
    Tln2Talin 2253,6210.315.107.68.30.5
    TriobpTRIO and F-actin binding protein223,3680.913.2216.411.61.6
    Sipa1Signal-induced proliferation-associated 1112,0660.660.44.583.5
    Sipa1l1Signal-induced proliferation-associated 1-like 1197,03109.91.94.160.9
    Magi3Membrane associated guanylate kinase 3161,67206.72.303.50
    Ehbp1l1EH domain binding protein 1-like 1184,83407.4019.97.81.6
    CgnCingulin136,44705.3002.30
    Rrbp1Ribosome binding protein 1172,8790.65.711.72.210.15.8
    Magi1Membrane associated guanylate kinase 1161,97405.33.81.620
    Atg2bAutophagy-related 2B231,39907.11.42.43.55.5
    Itsn1Intersectin 1194,29705.608.55.10
    SpegSPEG complex locus354,3430.98.719.111.213.41
    TrioTrio Rho guanine nucleotide exchange factor347,8610.66.31.49.650.7

Supplementary Materials

  • stke.sciencemag.org/cgi/content/full/12/578/eaau8749/DC1

    Fig. S1. Characterization of Nf2−/− Schwann cells.

    Fig. S2. Validation of proximity biotinylation reagents.

    Fig. S3. Merlin-NLuc interaction analysis.

    Table S1. All proteins above threshold.

    Data file S1. Merlin proximity biotinylation data.

  • The PDF file includes:

    • Fig. S1. Characterization of Nf2−/− Schwann cells.
    • Fig. S2. Validation of proximity biotinylation reagents.
    • Fig. S3. Merlin-NLuc interaction analysis.
    • Table S1. All proteins above threshold.
    • Legend for data file S1

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Data file S1 (Microsoft Excel format). Merlin proximity biotinylation data.

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