E2 Partner Tunes the Ubiquitylation Specificity of Arkadia E3 Ubiquitin Ligase

Simple Summary Ubiquitin-mediated proteasomal degradation is a fundamental and tightly coordinated process that controls the cellular concentration of proteins. E3 ubiquitin ligases are responsible for substrate recognition, and their deregulation is associated with various diseases, most notably, cancer. The E3 ligase Arkadia (RNF111) targets for proteasomal degradation negative regulators of the TGF-beta SMAD2/3 signaling pathway and poly-SUMOylated proteins, e.g., promyelocytic leukemia protein. Herein, we provide valuable insights into the interaction and function of Arkadia with its physiological E2 partners, UbcH5 Beta and UbcH13, as well as UbcH7. Specifically, we identify key amino acid residues on these E2 enzymes and Arkadia that are essential for their pairing and control of the enzymatic activity of Arkadia in ubiquitylation machinery. Our findings enrich the current knowledge on the pivotal components of the ubiquitylation system and can be the basis for future drug development. Arkadia (RNF111) is a positive regulator of the TGF-beta signaling that mediates the proteasome-dependent degradation of negative factors of the pathway. It is classified as an E3 ubiquitin ligase and a SUMO-targeted ubiquitin ligase (STUBL), implicated in various pathological conditions including cancer and fibrosis. The enzymatic (ligase) activity of Arkadia is located at its C-terminus and involves the RING domain. Notably, E3 ligases require E2 enzymes to perform ubiquitylation. However, little is known about the cooperation of Arkadia with various E2 enzymes and the type of ubiquitylation that they mediate. In the present work, we study the interaction of Arkadia with the E2 partners UbcH5B and UbcH13, as well as UbcH7. Through NMR spectroscopy, we found that the E2-Arkadia interaction surface is similar in all pairs examined. Nonetheless, the requirements and factors that determine an enzymatically active E2-Arkadia complex differ in each case. Furthermore, we revealed that the cooperation of Arkadia with different E2s results in either monoubiquitylation or polyubiquitin chain formation via K63, K48, or K11 linkages, which can determine the fate of the substrate and lead to distinct biological outcomes.