Karyopherin alpha 1 is a putative substrate of the RAG1 ubiquitin ligase

The RAG1 recombinase, which participates in DNA manipulation during rearrangement of antigen receptor genes in developing immune cells, possesses ubiquitin ligase activity. 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. While RAG1 auto-ubiquitylation is dependent on the ubiquitin conjugating enzyme CDC34, ubiquitylation of KPNA1 was best supported by UbcH2/Rad6 and UbcH5a. 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. This is the first putative substrate identified for the RAG1 ubiquitin ligase, and to our knowledge it is the first reported case of ubiquitylation of KPNA1.