KPNA1 is a putative substrate of the RAG1 ubiquitin ligase (138.11)

Abstract

Abstract The RAG1 V(D)J recombinase encompasses DNA binding/cleavage and ubiquitin ligase activities. The nuclear transport protein karyopherin alpha 1 (KPNA1) binds to RAG1 upstream of its ubiquitin ligase domain, but this interaction is not required for nuclear localization of RAG1. We found that the isolated ubiquitin ligase domain of RAG1 (amino acids 218-389) promoted ubiquitylation of purified KPNA1 in a reaction supported by the ubiquitin conjugating (E2) enzyme UbcH2/Rad6 or UbcH5a. KPNA1 is the first putative substrate identified for the RAG1 ubiquitin ligase. Ubiquitylation of KPNA1 required the lysine/arginine-rich region spanning RAG1 amino acids 218-263 upstream of the RAG1 ubiquitin ligase domain, but RAG1 was still able to undergo auto-ubiquitylation in this region even in the presence of KPNA1. RAG1 did not promote rapid ubiquitin chain extension following mono-ubiquitylation of substrate, regardless of the E2 used. Substitutions of amino acids surrounding the third, non-canonical Zn coordination site of the RAG1 RING domain abrogated functional interaction with E2 enzymes, and this was significantly correlated with reduction in the ability of full length RAG1 to support recombination of extra-chromosomal substrates. These data suggest that RAG1-dependent mono-ubiquitylation of a substrate, possibly KPNA1, is required for optimal levels of V(D)J recombination.