Rac-mediated stimulation of phospholipase Cγ₂ amplifies B cell receptor-induced calcium signaling

The Rho GTPase Rac is crucially involved in controlling multiple B cell functions, including those regulated by the B cell receptor (BCR) through increased cytosolic Ca²⁺. The underlying molecular mechanisms and their relevance to the functions of intact B cells have thus far remained unknown. We have previously shown that the activity of phospholipase Cγ₂ (PLCγ₂), a key constituent of the BCR signalosome, is stimulated by activated Rac through direct protein-protein interaction. Here, we use a Rac-resistant mutant of PLCγ₂ to functionally reconstitute cultured PLCγ₂-deficient DT40 B cells and to examine the effects of the Rac-PLCγ₂ interaction on BCR-mediated changes of intracellular Ca²⁺ and regulation of Ca²⁺-regulated and nuclear-factor-of-activated-T-cell-regulated gene transcription at the level of single, intact B cells. The results show that the functional Rac-PLCγ₂ interaction causes marked increases in the following: (i) sensitivity of B cells to BCR ligation; (ii) BCR-mediated Ca²⁺ release from intracellular stores; (iii) Ca²⁺ entry from the extracellular compartment; and (iv) nuclear translocation of the Ca²⁺-regulated nuclear factor of activated T cells. Hence, Rac-mediated stimulation of PLCγ₂ activity serves to amplify B cell receptor-induced Ca²⁺ signaling.