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.immortalized mouse 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.

Proximity Dependent Biotinylation: Key Enzymes and Adaptation to Proteomics Approaches

“…This study was followed by many others, including an investigation of the Hippo signaling pathway in which phosphorylation-specific interactions could be detected in the absence of sustained signal, revealing a propensity for signal amplification in BioID experiments (19,55). Since its initial development, BioID has been widely used to explore proximal associations in both large and small-scale experimentations in various experimental models (reviewed in (20)) that importantly now include the characterization of the proteome composition and organization of membraneless organelles, including focal adhesions (56) and cell junctions (57)(58)(59)(60), the centrosome (61, 62), P-bodies and stress granules (63), and the generation of a draft proximity map of a human cell (64). As we discuss in the later sections, continued development of this technology has enabled the identification of more active enzymes and permitted the implementation of new assay designs.…”

The mitochondrial protein ERAL1 suppresses RNA virus infection by facilitating RIG-I-like receptor signaling

“…ERAL1 positively regulates RNA virus-triggered innate immune signaling MAVS plays a crucial role in innate immune responses to RNA virus infection. Proximity-dependent biotin identification is an effec-tive method for identifying protein-protein interactions in living cells (Hennigan et al, 2019). To identify potential regulatory mechanisms of MAVS activation, we cloned MAVS into a mammalian expression vector with a TurboID tag and performed proximitybased labeling screening (Liu et al, 2018).…”

ASPP2/PP1 complexes maintain the integrity of pseudostratified epithelia undergoing remodelling during morphogenesis

“…Because ASPP2 does not possess any known F-actin binding domain, we looked if previously identified ASPP2 binding partners could provide this link. Interestingly, ASPP2 has been found to interact with Afadin in a number of proteomic studies 29,30 . Afadin is an F-actin-binding protein that has previously been shown to not only be enriched at tricellular junctions but to also regulate their architecture 31 .…”