Role of RACK1 in RAG1 degradation and B cell development

点击翻译按钮获取中文摘要

PROJECT SUMMARY B and T lymphocytes form the foundation of our adaptive immune system, which is based on specific recognition of foreign molecules by structurally diverse surface antigen receptors. Structural diversity in these receptors originates through site-specific rearrangement of the antigen receptor genes during lymphocyte development. This rearrangement process, called V(D)J recombination, is initiated when the RAG1/2 proteins introduce DNA double-strand breaks (DSBs) at antigen receptor gene segments, and is completed when the DSBs are sensed and repaired by non-homologous end-joining. This process is subjected to many layers of regulation, but an elementary means to constrain V(D)J recombination is to control the level of the RAG proteins themselves. Work in this laboratory suggests that RAG1 levels are controlled by a RAG1 interacting protein we identified called Vpr binding protein (VprBP; also called DCAF1), which exerts its control through two different mechanisms: first, by promoting timely proteasome-dependent degradation of RAG1 through VprBP’s association with a Cul4-DDB1 E3 ubiquitin ligase complex; and second, by regulating Rag transcriptional induction under conditions that stimulate V(D)J recombination. The molecular details underlying these mechanisms remain unclear. We have recently performed more detailed interactome studies, confirming VprBP and DDB1 association with full-length RAG1 and identifying Receptor of Activated C Kinase 1 (RACK1) as a promising co-factor in mediating RAG1 degradation based on previous work establishing it as an adaptor for other modular cullin E3 ubiquitin ligases. These findings lead us to hypothesize that RACK1 regulates RAG1 degradation and V(D)J recombination. To test this hypothesis, we will (i) establish the role of RACK1 in regulating full-length RAG1 degradation in vitro and in cell models and, based on previous studies, its potential for competitive inhibition by HSP90 and modulation by signaling pathways involved in activating Protein Kinase C; and (ii) establish how loss of RACK1 in B cells affects B cell development, RAG1 protein levels, and V(D)J recombination. Establishing the molecular basis for RACK1 interactions with full-length RAG1 and the Cul4- DDB1 ubiquitin ligase complex, the role of RACK1 (and potentially HSP90 and receptor signaling pathways) in regulating RAG1 degradation, and the consequence of losing RACK1 expression in B cells on B cell development, RAG1 protein levels, and V(D)J recombination, would define a new physiological role for RACK1 (and HSP90) in V(D)J recombination, and provide new insights into sources for altered immune repertoire and genomic instability caused by dysregulated RAG1 expression. This work would also extend the paradigm for RACK1 as a multi-functional adaptor to E3 ubiquitin ligases and potentially reveal new avenues to therapeutically regulate RACK1-dependent protein turnover.