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

Abstract

Neurofibromatosis type 2 is an inherited, neoplastic disease associated with schwannomas, meningiomas, and ependymomas and that is caused by inactivation of the tumor suppressor gene NF2 The NF2 gene product, Merlin, has no intrinsic catalytic activity; its tumor suppressor function is mediated through the proteins with which it interacts. We used proximity biotinylation followed by mass spectrometry and direct binding assays to identify proteins that associated with wild-type and various mutant forms of Merlin in immortalized Schwann cells. We defined a set of 52 proteins in close proximity to wild-type Merlin. Most of the Merlin-proximal proteins were components of cell junctional signaling complexes, suggesting that additional potential interaction partners may exist in adherens junctions, tight junctions, and focal adhesions. With mutant forms of Merlin that cannot bind to phosphatidylinositol 4,5-bisphosphate (PIP₂) or that constitutively adopt a closed conformation, we confirmed a critical role for PIP₂ binding in Merlin function and identified a large cohort of proteins that specifically interacted with Merlin in the closed conformation. Among these proteins, we identified a previously unreported Merlin-binding protein, apoptosis-stimulated p53 protein 2 (ASPP2, also called Tp53bp2), that bound to closed-conformation Merlin predominately through the FERM domain. Our results demonstrate that Merlin is a component of cell junctional mechanosensing complexes and defines a specific set of proteins through which it acts.

Fig. 1.. Proximity biotinylation constructs.

(A) A schematic diagram depicting the six BirA^R118G fusion constructs expressed from Dox-inducible plasmids in immortalized NF2^–/– mouse Schwann cells. Constructs include a negative control (BirA^R118G–A-fos), two wild-type Merlin constructs with BirA fused to either the N or C terminus (BirA^R118G-Merlin and Merlin-BirA^R118G), a PIP₂ binding–deficient mutant (Merlin-6N–BirA^R118G), a C-terminal deletion mutant intended to mimic the open, FERM-accessible conformation (Merlin-FH–BirA^R118G), and a mutant intended to mimic a closed, FERM-inaccessible conformation (Merlin-AR–BirA^R118G). (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-BirA^R118G.

(A) Venn diagram showing the 52 proteins from cells expressing Merlin-BirA^R118G (pink) and the 12 proteins from cells expressing BirA^R118G-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–BirA^R118G, 81 proteins from cells expressing Merlin-AR-BirA^R118G, and 27 proteins from cells expressing Merlin-6N–BirA^R118G 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-BirA^R118G dataset in (A).

Fig. 3.

Merlin-BirA^R118Gbiotinylates 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–BirA^R118G and Paxillin-BirA^R118G (55)], tight junctions [ZO-1–BirA^R118G and BirA^R118G–ZO-1 (57)], adherens junctions [E-cadherin– BirA^R118G (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 AN18. 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.