RESUMO
Arkadia (RNF111) is a positive regulator of the TGF-ß 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.
RESUMO
Arkadia is a positive regulator of the TGFß-SMAD2/3 pathway, acting through its C-terminal RING-H2 domain and targeting for degradation of its negative regulators. Here we explore the role of regions outside the RING domain (non-RING elements) of Arkadia on the E2-E3 interaction. The contribution of the non-RING elements was addressed using Arkadia RING 68 aa and Arkadia 119 aa polypeptides. The highly conserved NRGA (asparagine-arginine-glycine-alanine) and TIER (threonine-isoleucine-glutamine-arginine) motifs within the 119 aa Arkadia polypeptide, have been shown to be required for pSMAD2/3 substrate recognition and ubiquitination in vivo. However, the role of the NRGA and TIER motifs in the enzymatic activity of Arkadia has not been addressed. Here, nuclear magnetic resonance interaction studies with the E2 enzyme, UBCH5B, C85S UBCH5B-Ub oxyester hydrolysis, and auto-ubiquitination assays were used to address the role of the non-RING elements in E2-E3 interaction and in the enzymatic activity of the RING. The results support that the non-RING elements including the NRGA and TIER motifs are required for E2-E3 recognition and interaction and for efficient auto-ubiquitination. Furthermore, while Arkadia isoform-2 and its close homologue Arkadia 2C are known to interact with free ubiquitin, the results here showed that Arkadia isoform-1 does not interact with free ubiquitin.
