Dissecting the role of bank1 in systemic lupus erythematosusidentification of new binding partners

Resumen

Systemic lupus erythematosus (SLE) is an autoimmune disorder characterized by the presence of pathogenic autoantibodies causing inflammation and tissue damage. It is a heterogeneous relapsing and remitting disease principally showing involvement of the skin, joints, kidneys, and serosal membranes. SLE is a complex disease affecting genetically predisposed individuals who have experienced certain environmental exposures. Both innate and adaptive immune responses become altered in lupus, resulting in loss of tolerance to ubiquitous self-antigens. Given the limited understanding of the disease pathogenesis, most treatments are based on drugs inducing unspecific immunosuppression, and thus are responsible of a plethora of adverse effects. During the last decade, the development of genome-wide association studies (GWAS) has greatly contributed to the identification of lupus susceptibility genes, providing evidence about the altered functional pathways contributing to the disease. Most of these genes encode proteins participating in the immune system. BANK1 has been consistently described as a SLE susceptibility gene. It encodes an adaptor protein specifically expressed in B cells that is tyrosine phosphorylated upon B cell receptor (BCR) stimulation. Two spliced variants have been described: a 785 aminoacid full-length isoform and a short isoform lacking exon 2 (¿2 variant). The function of BANK1 in BCR signalling is not well understood. It is known that Src-kinases LYN and BLK, also encoded by lupus risk genes, are able to phosphorylate BANK1. In order to better understand the function of BANK1, a yeast-two hybrid assay for the identification of protein-protein interactions was performed. Several non-previously described interacting partners of BANK1 were found. One of the most confident interactions was obtained with Phospholipase C Gamma 2 (PLCG2), an important effector in BCR signalling that catalyses the production of the second messengers IP3 and DAG. The recovered clones for PLCG2 contained SH2 and SH3 domains, suggesting their implication in binding to BANK1. The interaction between BANK1 and PLCG2, which was validated by co-immunoprecipitation in B cell lines, is transient and highly dependent on BCR stimulation. In HEK293 cells expressing the recombinant proteins, the interaction required the phosphorylation of BANK1 by Src-kinases BLK or LYN. Microscopy analysis of ectopically expressed BANK1 and PLCG2 showed that both proteins highly co-localized in the cytoplasm. Silencing of the BLK kinase in a B cell line reduced the interaction between BANK1 and PLCG2. Mutational analysis of BANK1 demonstrated that proline rich regions and specific tyrosines in BANK1 contribute to PLCG2 binding. The effect of BANK1 on PLCG2 was further studied. No difference in PLCG2 degradation was observed, but an enhanced activating Src-kinase dependent phosphorylation of PLCG2 was apparent, indicating that BANK1 contributes to activation of PLCG2. Differential binding between BANK1 and Src-kinases LYN and BLK was also studied. It is shown that BANK1 sequence codified by exon 2 is required for binding to BLK, whereas it is dispensable for the interaction with LYN. The 40C variant (rs35978636) located in exon 2 enhances the interaction with BLK but not LYN. Together, these results support the function of BANK1 as a modulator of PLCG2 function. Genetic variants contributing to an enhanced interaction between BANK1 and BLK or leading to an increased expression of BANK1 could promote the activating phosphorylation of PLCG2. Thus, an augmented PLCG2 activation could contribute to the B cell autoreactivity observed in SLE patients. BANK1, BLK and PLCG2 participate in a B cell functional pathway. The additional effect of genetic variants within these genes probably contributes to lower thresholds in immune signalling. By understanding this functional pathway, we will be able to identify new putative therapeutic targets for disease management.