BOTH PHOSPHATIDYLINOSITOL 3-KINASE AND PHOSPHATIDYLINOSITOL 4-KINASE PRODUCTS ARE INCREASED BY ANTIGEN RECEPTOR SIGNALING IN B-CELLS

The effects of Ag binding on B cell development and activation are mediated by intracellular signals initiated by the B cell AgR. In this report, we show that the B cell AgR regulates the production of inositol phospholipids involved in two different signal transduction pathways, the phosphatidylinositol 3-kinase (PtdIns 3-kinase) pathway and the phospholipase C (PLC) pathway. Phosphatidylinositol 3-phosphate (PtdIns3P), phosphatidylinositol 3,4-bisphosphate [PtdIns(3,4)P2], and phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P3] are produced by PtdIns 3-kinase, an enzyme that appears to be an essential component of tyrosine kinase-mediated signaling. Both PtdIns(3,4)P2 and PtdIns(3,4,5)P3 are likely to function as second messengers in vivo because they can activate the zeta isoform of protein kinase C (PKC) in vitro. We show that cross-linking of the B cell AgR with anti-Ig antibodies caused a five- to sixfold increase in the levels of PtdIns(3,4)P2 in both the mature B cell line BAL 17 and the immature B cell line WEHI-231. PtdIns(3,4)P2 levels increased within 15 s of anti-Ig addition and remained elevated for at least 5 min. AgR cross-linking also caused a slower increase in PtdIns3P levels (approximately 50% over control) and a small, transient increase in PtdIns(3,4,5)P3 levels. Thus, the B cell AgR activates the PtdIns 3-kinase pathway. The other inositol phospholipid signaling pathway involves PLC, which cleaves phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2], yielding second messengers that increase intracellular calcium and activate other isoforms of PKC. We analyzed the effects of AgR signaling on PtdIns(4,5)P2 and its precursor, phosphatidylinositol 4-phosphate (PtdIns4P). Consistent with its ability to activate PLC, AgR ligation decreased the levels of PtdIns(4,5)P2. In contrast, AgR cross-linking increased the levels of PtdIns4P. Increased synthesis of PtdIns4P followed by phosphorylation at the D-5 position may prevent depletion of PtdIns(4,5)P2. Thus, signaling by the B cell AgR increases the levels of PtdIns 4-kinase products and PtdIns 3-kinase products. The simplest interpretation of our results is that the B cell AgR activates both PtdIns 3-kinase and PtdIns 4-kinase.