The SLE variant Ala71Thr of BLK severely decreases protein abundance and binding to BANK1 through impairment of the SH3 domain function

Abstract

The B-lymphocyte kinase (BLK) gene is associated genetically with several human autoimmune diseases including systemic lupus erythematosus. We recently described that the genetic risk is given by two haplotypes: one covering several strongly linked single-nucleotide polymorphisms within the promoter of the gene that correlated with low transcript levels, and a second haplotype that includes a rare nonsynonymous variant (Ala71Thr). Here we show that this variant, located within the BLK SH3 domain, is a major determinant of protein levels. In vitro analyses show that the 71Thr isoform is hyperphosphorylated and promotes kinase activation. As a consequence, BLK is ubiquitinated, its proteasomal degradation enhanced and the average life of the protein is reduced by half. Altogether, these findings suggest that an intrinsic autoregulatory mechanism previously unappreciated in BLK is disrupted by the 71Thr substitution. Because the SH3 domain is also involved in protein interactions, we sought for differences between the two isoforms in trafficking and binding to protein partners. We found that binding of the 71Thr variant to the adaptor protein BANK1 is severely reduced. Our study provides new insights on the intrinsic regulation of BLK activation and highlights the dominant role of its SH3 domain in BANK1 binding.

Functional rare and low frequency variants in BLK and BANK1 contribute to human lupus

“…The tyrosine at 350, mutated in family J, resides within the kinase domain and we hypothesized would restrict BLK catalytic activity. BLK A71T is known to reduce protein stability and thus minimize available protein 32 . Therefore, we postulated that the identified SNVs would alter kinase activity of BLK by impairing availability of protein or protein function.…”

Immunogenetics in systemic lupus erythematosus: Transitioning from genetic associations to cellular effects

“…In addition to the non‐coding SLE risk variants, a low‐frequency mutation (Ala71Thr) resulting in decreased protein levels of BLK through enhanced ubiquitin‐mediated proteasomal degradation is also associated with SLE 74,75 . Moreover, several rare BLK missense variants (minor allele frequency (MAF) <0.5%) with a reduced capacity to phosphorylate IRF5 were recently identified 76 .…”

Review: Abnormal B Cell Development in Systemic Lupus Erythematosus: What the Genetics Tell Us

“…The disease‐associated SNPs for Blk (rs922483 and rs1382568) both result in the down‐regulation of Blk expression and potential failure of adequate pre‐BCR signaling (Table ). A third and rarer coding variant of Blk associated with SLE that results in an alanine‐for‐threonine substitution in its SH3 domain leads to hypophosphorylation, inactivation, and rapid degradation of the protein . This variant also demonstrates impaired binding to B cell scaffold protein with ankyrin repeats 1 (BANK‐1) scaffolding, which is important in signal transduction .…”

“…Two polymorphisms in BANK1 have been identified as associated with risk (Table ). Both impair BCR and CD40 signaling and are associated with an expansion of memory B cells in vivo (step 4 in Figure 2).…”