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.
Subject(s)
Adaptor Proteins, Signal Transducing/genetics , Amino Acid Substitution , Lupus Erythematosus, Systemic/genetics , Membrane Proteins/genetics , Mutation , src-Family Kinases/genetics , Adaptor Proteins, Signal Transducing/immunology , Amino Acid Sequence , B-Lymphocytes/immunology , B-Lymphocytes/pathology , Binding Sites , Cell Line, Tumor , Gene Expression , Half-Life , Haplotypes , Humans , Lupus Erythematosus, Systemic/immunology , Lupus Erythematosus, Systemic/pathology , Membrane Proteins/immunology , Models, Molecular , Molecular Sequence Data , Promoter Regions, Genetic , Proteasome Endopeptidase Complex/metabolism , Protein Binding , Protein Isoforms/genetics , Protein Isoforms/immunology , Proteolysis , Sequence Alignment , Ubiquitination , src-Family Kinases/immunologyABSTRACT
31 patients with confirmed angina pectoris and selected to form an homogenous group entered this double-blind study. Following a period of observation during which the patients were on a placebo and only took nitroglycerin to relieve pain, a randomized treatment of either dipyramidole (DPM) 75 mg or pentaerythritol tetranitrate (PETN) 80 mg 8-hourly was given during 4 weeks. At the end of this period the anti-anginal effects of the drugs were assessed, using the following criteria: incidence of anginal attacks, nitroglycerin consumption, number of attacks per days, changes in exercise capacity and changes in repolarization on ECG. PETN proved distinctly more effective than DPM. The number of attacks per week decreased from 11.5 to 1.5 under PETN and from 12.5 to 9 under DPM. Similarly, nitroglycerin weekly consumption fell from 8 to 1.5 tablets under PETN and from 9 to 7 tablets under DPM.
