Characterizing WW Domain Interactions of Tumor Suppressor WWOX Reveals Its Association with Multiprotein Networks

• Published in: The Journal of biological chemistry 2014-03, Vol.289 (13), p.8865-8880
• Main Authors: Abu-Odeh, Mohammad, Bar-Mag, Tomer, Huang, Haiming, Kim, TaeHyung, Salah, Zaidoun, Abdeen, Suhaib K., Sudol, Marius, Reichmann, Dana, Sidhu, Sachdev, Kim, Philip M., Aqeilan, Rami I.
• Format: Article
• Language: English
• Subjects: Amino Acid Motifs; Cell Biology; E3 Ubiquitin Ligase; HEK293 Cells; Humans; Itch; Mass Spectrometry (MS); Oxidoreductases - chemistry; Oxidoreductases - metabolism; Peptide Library; Protein Binding; Protein Structure, Tertiary; Protein-Protein Interactions; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - metabolism; Tumor Suppressor Gene; Tumor Suppressor Proteins - chemistry; Tumor Suppressor Proteins - metabolism; Ubiquitination; WW Domain; WW Domain-Containing Oxidoreductase; WWOX
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M113.506790

WW domains are small modules present in regulatory and signaling proteins that mediate specific protein-protein interactions. The WW domain-containing oxidoreductase (WWOX) encodes a 46-kDa tumor suppressor that contains two N-terminal WW domains and a central short-chain dehydrogenase/reductase domain. Based on its ligand recognition motifs, the WW domain family is classified into four groups. The largest one, to which WWOX belongs, recognizes ligands with a PPXY motif. To pursue the functional properties of the WW domains of WWOX, we employed mass spectrometry and phage display experiments to identify putative WWOX-interacting partners. Our analysis revealed that the first WW (WW1) domain of WWOX is the main functional interacting domain. Furthermore, our study uncovered well known and new PPXY-WW1-interacting partners and shed light on novel LPXY-WW1-interacting partners of WWOX. Many of these proteins are components of multiprotein complexes involved in molecular processes, including transcription, RNA processing, tight junction, and metabolism. By utilizing GST pull-down and immunoprecipitation assays, we validated that WWOX is a substrate of the E3 ubiquitin ligase ITCH, which contains two LPXY motifs. We found that ITCH mediates Lys-63-linked polyubiquitination of WWOX, leading to its nuclear localization and increased cell death. Our data suggest that the WW1 domain of WWOX provides a versatile platform that links WWOX with individual proteins associated with physiologically important networks. Background: WWOX encodes a 46-kDa tumor suppressor. Results: WW1 domain of WWOX mediates its protein-protein interaction with PY motifs that are involved in molecular processes, including transcription, RNA processing, and metabolism. Conclusion: The WW1 domain of WWOX provides a versatile platform that links WWOX with individual proteins associated with physiologically important networks. Significance: This study provides a better understanding of WWOX biology in normal and disease states.