Amino acid resolution mapping of interactions of NF2

Loss of NF2 tumor suppressor protein function contributes to a variety of human cancers. Functional NF2 suppresses growth in response to cell-cell contacts through modulation of essential cellular signal transduction pathways. NF2 tumor suppressor function is isoform specific and depends on its subcellular localization, the interaction with other proteins as well as on its post-translational modification status. These features are inextricably linked with NF2 protein conformation; however the actual relationship between these features and the tumor suppressor function of NF2 is not fully understood. Which conformation, which modification state and which NF2 interaction partners determine tumor suppressor function remain open questions. We will investigate novel posttranslational-modification- and isoform- dependent protein interactions involving NF2 as well as NF2 intra- and inter- interactions at amino acid resolution. Our approach will allow to systematically map NF2 interaction surfaces and will reveal single amino acid mutant NF2 versions that selectively perturb specific NF2 interactions and NF2 conformation states. This protein interaction approach to disrupt NF2 functions will help to better define NF2 structure-function relationship. These mutants will genetically define diverse NF2 functional states that will then be characterized for growth phenotypes related to cancer in cell culture. It will clarify iso-form dependent and modification dependent effects in NF2 cellular signaling, and reveal the role of conditional interacting partners in tumor suppressor activity. The project is methodologically driven by a newly developed stringent reverse yeast two-hybrid (Y2H) system that is coupled to a second generation sequencing readout. We will apply a recently reported microarray-programmed oligonucleotide synthesis approach to create comprehensive sets of single point mutations exchanging every amino acid in NF2 against several other amino acids. With the reverse- Y2H-seq system we select for those protein variants that interfere with a protein interaction. As such we assess every amino acid for its requirement in NF2 intra-, inter- and PTM-dependent interaction with other protein interaction partners. The discovery of new somatic mutations in health and disease, in particular in cancer, has vastly outpaced our ability to assess their functional roles. The field is poised to address the large numbers of genotypic variations mechanistically. In this project we exemplify a proteomics interaction approach that can serve as a universal strategy to systematically assess potential functional categories of protein variation.

Protein Neurofibromine 2 (NF2) is an important tumor suppressor and contact inhibitor. Homozygous mutation or deletion of the NF2 gene frequently results in tumor formation. NF2 shares high sequence homology and specific intramolecular interactions with the mechanistically characterized Ezrin-Radixin-Moesin (ERM) family of membrane linker proteins, which has led to inferences of NF2 protein regulation. Function of ERM proteins is controlled by conformational opening or closing driven by intramolecular interaction. However, how the different NF2 conformational states relate to tumor suppressor function of the protein remains elusive. Deep mutational scanning methods, where every amino acid of a protein is replaced by another, offers the possibility to identify genetic variants with functional impact on the protein in a comprehensive large-scale approach. Pairing the system with a yeast based protein interaction readout with conformation-dependent binding partners, highlights NF2 mutations directly influencing specific protein interactions and protein regions sensitive for conformational changes. With the approach, we were able to enrich protein interaction perturbing mutations of NF2, some of which were found in structural proximity in defined protein domains. A subset of 53 NF2 mutations was generated in each of two NF2 isoforms and further tested individually in protein protein interaction assays. 15 interaction changing single amino acid mutations that influence protein function in the context of expression, cellular localization, and proliferation in mammalian cell lines were characterized. The 3H region of NF2 appears critical in NF2-NF2 interactions. The N terminal FERM domain emerged as a key regulatory entity of the tumor suppressor protein. Inducing conformational changes, mutations in the FERM F3 subdomain imbalanced protein interactions and protein function. F2 / F3 subdomain mutants modulated cell proliferation and one variant severely impacted cellular localization of NF2. Our functional characterization revealed the high impact of conformational changes within the FERM F3 subdomain on NF2 tumor suppressor activity.