Merlin tumor suppressor function is regulated by PIP2-mediated dimerization

PLOS ONE RESEARCH ARTICLE Merlin tumor suppressor function is regulated by PIP2-mediated dimerization Robert F. Hennigan ID*, Craig S. Thomson¤, Kye Stachowski ID, Nicolas Nassar, Nancy Ratner Division of Experimental Hematology and Cancer Biology, Cincinnati Children’s Hospital Medical Center, Department of Pediatrics, University of Cincinnati, Cincinnati, OH, United States of America ¤ Current address: Medpace, Cincinnati, Ohio, United States of America * a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 OPEN ACCESS Citation: Hennigan RF, Thomson CS, Stachowski K, Nassar N, Ratner N (2023) Merlin tumor suppressor function is regulated by PIP2-mediated dimerization. PLoS ONE 18(2): e0281876. https:// doi.org/10.1371/journal.pone.0281876 Editor: Patrick van der Wel, Rijksuniversiteit Groningen, NETHERLANDS Received: October 24, 2022 Accepted: January 30, 2023 Published: February 21, 2023 Copyright: © 2023 Hennigan et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the manuscript and its Supporting Information files. Funding: This work was funded by a Neurofibromatosis Research Program Investigator-Initiated Research Award NF190083 from the Department of Defense, Office of Congressionally Directed Medical Research Programs (https://cdmrp.health.mil/nfrp/) to RFH and NIH Grants R37 NS083580 and 1 R01 NS120892 to NR. KS is supported by T32CA236764-01A0, KS and NN were supported Abstract Neurofibromatosis Type 2 is an inherited disease characterized by Schwann cell tumors of cranial and peripheral nerves. The NF2 gene encodes Merlin, a member of the ERM family consisting of an N-terminal FERM domain, a central α-helical region, and a C-terminal domain. Changes in the intermolecular FERM-CTD interaction allow Merlin to transition between an open, FERM accessible conformation and a closed, FERM-inaccessible conformation, modulating Merlin activity. Merlin has been shown to dimerize, but the regulation and function Merlin dimerization is not clear. We used a nanobody based binding assay to show that Merlin dimerizes via a FERM-FERM interaction, orientated with each C-terminus close to each other. Patient derived and structural mutants show that dimerization controls interactions with specific binding partners, including HIPPO pathway components, and correlates with tumor suppressor activity. Gel filtration experiments showed that dimerization occurs after a PIP2 mediated transition from closed to open conformation monomers. This process requires the first 18 amino acids of the FERM domain and is inhibited by phosphorylation at serine 518. The discovery that active, open conformation Merlin is a dimer represents a new paradigm for Merlin function with implications for the development of therapies designed to compensate for Merlin loss. Introduction Neurofibromatosis Type 2 is an inherited disease characterized by benign Schwann cell tumors of cranial and peripheral nerves known as schwannomas. Schwannomas that arise in the general population also contain NF2 mutations and represent 8% of all intracranial tumors [1, 2]. Targeted deletion of the Nf2 gene in mouse Schwann cells leads to schwannoma [3, 4] and Nf2-null cells have impaired contact inhibition of growth in vitro [5, 6]. The NF2 gene encodes Merlin, a 70-kDa member of the Ezrin-Radixin-Moesin (ERM) branch of the band 4.1 superfamily [7, 8]. Merlin is predominately localized to the inner face of the plasma membrane [9, 10] where it associates with lipid rafts, which are membrane microdomains enriched with a variety of signaling molecules [11]. Merlin is also a component of cell junctional complexes PLOS ONE | https://doi.org/10.1371/journal.pone.0281876 February 21, 2023 1 / 21 PLOS ONE by R01 CA237016-01 (https://www.nih.gov). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. Merlin dimerization including adherens junctions and focal adhesions [12, 13]. This is consistent with reports indicating that Merlin is a component of the HIPPO pathway, a growth suppressive signaling system which acts downstream of cell junctions and is responsible for contact inhibition of growth [14–16]. Merlin loss also activates other oncogenic signaling networks, including RasERK, Rac, src, β-catenin, and the mTOR protein kinase complex [6, 17–26]. However, the mechanism by which these systems are activated is not fully defined. Merlin has a conserved secondary structure consisting of an N-terminal FERM domain followed by a central α-helical region that folds over itself to position the C-terminal domain (CTD) for an intramolecular interaction with the FERM domain [5, 27–29]. Merlin also has a unique N-terminal 20 amino acid sequence, absent in other ERM proteins, that is necessary for its tumor suppressor activity [30]. The intramolecular interaction between the Merlin CTD and FERM domains mask a large portion of the FERM domain surface area, resulting in a closed, FERM-inaccessible conformation [31]. Upon the release of the CTD from the FERM domain, Merlin transitions to an open conformation that renders the FERM domain accessible to critical interacting proteins [32]. A Merlin mutant designed to stabilize the FERM-CTD interaction results in a more closed conformation that has impaired tumor suppressor activity [32]. Conversely, a more open, FERM-accessible conformation mutant retains activity [32], demonstrating that Merlin’s tumor suppressor function is facilitated by the open conformation. Merlin is phosphorylated at serine 518 by PAK2 [33] and PKA [34]. This phosphorylation promotes a closed conformation with reduced Merlin tumor suppressor activity [32, 35–37]. Merlin is also regulated by binding the lipid signaling molecule phosphatidylinositol 4,5 bisphosphate (PIP2) [38]. This interaction promotes Merlin localization to the plasma membrane and is necessary for tumor-suppressive activity [38]. PIP2 binding causes Merlin to assume an open, FERM accessible conformation [39] that is mediated by a structural change in the N-terminal portion of the central α-helical domain that forces the CTD and FERM domains apart [40]. These reports show that Merlin activity is determined by conformational changes that are regulated by both phosphorylation and lipid based signaling systems. However, the specific mechanism by which open conformation Merlin interacts with binding partners to facilitate tumor suppression is not known. The idea that Merlin forms dimers has long been established in the literature, initially by analogy to other ERM proteins and supported by the observation that its central α-helical domain may arrang (...truncated)