Btk regulation in human and mouse B cells via protein kinase C phosphorylation of IBtkγ

The inhibitor of Bruton tyrosine kinase gamma (IBtk gamma) is a negative regulator of the Bruton tyrosine kinase (Btk), which plays a major role in B-cell differentiation; however, the mechanisms of IBtk gamma-mediated regulation of Btk are unknown. Here we report that B-cell receptor (BCR) triggering caused serine-phosphorylation of IBtk gamma at protein kinase C consensus sites and dissociation from Btk. By liquid chromatography and mass-mass spectrometry and functional analysis, we identified IBtk gamma-S87 and -S90 as the critical amino acid residues that regulate the IBtk(gamma) binding affinity to Btk. Consistently, the mutants IBtk gamma carrying S87A and S90A mutations bound constitutively to Btk and down-regulated Ca2+ fluxes and NF-kappa B activation on BCR triggering. Accordingly, spleen B cells from Ibtk gamma(-/-) mice showed an increased activation of Btk, as evaluated by Y551-phosphorylation and sustained Ca2+ mobilization on BCR engagement. These findings identify a novel pathway of Btk regulation via protein kinase C phosphorylation of IBtk gamma. (Blood. 2011;117(24):6520-6531)