RACK1 is required for normal B cell development and signaling but not RAG1 degradation.

Abstract

V(D)J recombination is constrained by timely degradation of the RAG1 and RAG2 proteins through distinct mechanisms. Previously, we showed that full-length RAG1 stability is regulated by viral protein R binding protein (VprBP) through its association with an amino-terminal region in RAG1, but the mechanism remains unclear. As an unbiased approach to uncover potential cofactors involved in the process, we compared protein interactomes between RAG1/RAG2 complexes formed when the amino-terminal third of RAG1 was present or absent. These experiments identified RACK1 as preferentially associating with full-length RAG1. Because RACK1 is implicated in mediating protein degradation in other contexts, we evaluated how loss of RACK1 in B cells affects B cell development and V(D)J recombination. We find that conditional disruption of Rack1 expression in the B lineage in mice blocks B cell development at the pro-B cell stage and impairs V(D)J recombination after Igh DH-JH rearrangement. In this background, enforced Bcl2 expression does not significantly rescue B cell development but does enable the V(D)J recombination defect to be bypassed. However, the phenotype of these mice does not show the excessive Igk rearrangement, skewing toward Igλ+ B cells, or increased RAG1 protein levels observed when VprBP expression is similarly disrupted in B cells, arguing against RACK1 serving as a cofactor in RAG1 degradation. Further studies provide evidence that loss of RACK1 in primary B cells dysregulates cell cycle progression, apoptosis, proliferation, and signaling through MAPK and NF-κB pathways.