Stimulation of B‐cell antigen receptor (BCR) induces a rapid increase in cytoplasmic free calcium due to its release from intracellular stores and influx from the extracellular environment. Inositol 1,4,5‐trisphosphate receptors (IP3Rs) are ligand‐gated channels that release intracellular calcium stores in response to the second messenger, inositol 1,4,5‐trisphosphate. Most hematopoietic cells, including B cells, express at least two of the three different types of IP3R. We demonstrate here that B cells in which a single type of IP3R has been deleted still mobilize calcium in response to BCR stimulation, whereas this calcium mobilization is abrogated in B cells lacking all three types of IP3R. Calcium mobilization by a transfected G protein‐coupled receptor (muscarinic M1 receptor) was also abolished in only triple‐deficient cells. Capacitative Ca2+ entry, stimulated by thapsigargin, remains unaffected by loss of all three types of IP3R. These data establish that IP3Rs are essential and functionally redundant mediators for both BCR‐ and muscarinic receptor‐induced calcium mobilization, but not for thapsigargin‐induced Ca2+ influx. We further show that the BCR‐induced apoptosis is significantly inhibited by loss of all three types of IP3R, suggesting an important role for Ca2+ in the process of apoptosis.

Signaling through the B cell receptor (BCR) is essential for B cell function and development. Despite the key role of Syk in BCR signaling, little is known about the mechanism by which Syk transmits downstream effectors. BLNK (B cell LiNKer protein), a substrate for Syk, is now shown to be essential in activating phospholipase C (PLC)gamma 2 and JNK. The BCR-induced PLC gamma 2 activation, but not the JNK activation, was restored by introduction of PLC gamma 2 membrane-associated form into BLNK-deficient B cells. As JNK activation requires both Rac1 and PLC gamma 2, our results suggest that BLNK regulates the Rac1-JNK pathway, in addition to modulating PLC gamma 2 localization.

SummaryMitogen-activated protein (MAP) kinase family members, including extracellular signal-regulated kinase (ERK), c-Jun NH 2 -terminal kinase ( JNK), and p38 MAP kinase, have been implicated in coupling the B cell antigen receptor (BCR) to transcriptional responses. However, the mechanisms that lead to the activation of these MAP kinase family members have been poorly elucidated. Here we demonstrate that the BCR-induced ERK activation is reduced by loss of Grb2 or expression of a dominant-negative form of Ras, RasN17, whereas this response is not affected by loss of Shc. The inhibition of the ERK response was also observed in phospholipase C (PLC)-␥ 2-deficient DT40 B cells, and expression of RasN17 in the PLC-␥ 2-deficient cells completely abrogated the ERK activation. The PLC-␥ 2 dependency of ERK activation was most likely due to protein kinase C (PKC) activation rather than calcium mobilization, since loss of inositol 1,4,5-trisphosphate receptors did not affect ERK activation. Similar to cooperation of Ras with PKC activation in ERK response, both PLC-␥ 2-dependent signal and GTPase are required for BCR-induced JNK and p38 responses. JNK response is dependent on Rac1 and calcium mobilization, whereas p38 response requires Rac1 and PKC activation.