Paus D, Phan TG, Chan TD, Gardam S, Basten A, Brink R. period of antigen relationships and respond accordingly. In this study, we asked if rate of metabolism of the second messenger diacylglycerol (DAG) by diacylglycerol kinase enzymes (DGKs) played a role in modulating the magnitude of signaling by this second messenger downstream of the BCR. In the absence of DGK, the threshold for BCR signaling through the Ras-ERK MAP kinase pathway was markedly reduced in mature follicular B cells, resulting in exaggerated reactions to antigen in vitro and in vivo. Inhibition of DAG signaling by DGK was especially important for limiting the number of antibody-secreting cells generated early in response to both T-independent type 2 antigens and T cell-dependent antigens. Furthermore, deficiency in DGK closely resembled the effects of increasing antigen affinity for the BCR during the T cell-dependent antibody response, strongly indicating that the magnitude of DAG signaling, likely through the degree of ERK activation, is definitely important for translating the affinity of the BCR for antigen into the amount of antibody produced during early stages of an immune response. Intro Engagement of the B cell antigen receptor (BCR) by specific antigen induces a complex cascade of Tanaproget intracellular signaling events that play essential tasks in B cell development, activation, survival, and proliferation (1). Early signaling from the BCR entails the activation of Src and Syk family protein tyrosine kinases, which activate a number of downstream signaling events, including activation of phospholipase C-2 (PLC-2) to generate the second messengers inositol trisphosphate (IP3) and diacylglycerol (DAG) (2-4). Whereas IP3 is required for calcium mobilization and activation of Tanaproget the NFAT family of transcription factors, DAG signals through PKC and the Ras guanine exchange element, RasGRP, leading to activation of the NF-B and Ras-MEK-ERK mitogen-activated protein kinase (MAPK) pathways respectively (5-10). Activation of these signaling pathways downstream of the BCR results in rapid transmission of signals to the nucleus and alterations in gene manifestation necessary for subsequent B cell practical reactions. The ERK MAPK signaling cascade is critical for a number of aspects of B cell function and fate decisions (11). During early B cell development, ERK signaling is required for proliferative growth induced by signaling through the pre-BCR, as well as for differentiation of immature transitional B cells to the mature follicular stage in the spleen (12, 13). In adult B cells, pharmacological inhibition of MEK, or genetic deficiency in the key signaling intermediates for Ras activation, RasGRP1 and RasGRP3, seriously impairs survival and proliferation in response to BCR activation (5, 14). Antigen activation of adult B cells in vivo induces antibody production through the quick formation of extrafollicular plasma cells, as well as a slower germinal center response, which gives rise to plasma cells that secrete higher affinity antibodies. ERK signaling in germinal center B cells is required for terminal differentiation Tanaproget to antibody-secreting plasma cells through induction of the key transcription element Blimp1 (15), however its part in early formation of plasmablasts has not been examined. Previous work has shown that B cell maturation from your immature transitional stage to the adult follicular stage in the spleen is definitely accompanied by an attenuation in BCR-induced ERK activation (16), suggesting the possibility that ERK is definitely differentially controlled inside a pathway-specific manner during B cell maturation. One possible mechanism of such rules is definitely by the action of diacylglycerol kinase (DGK) family members, which phosphorylate DAG and convert it to phosphatidic acid, therefore limiting signaling by this second messenger (17). Interesting in this regard, previous studies in T cells found that the degree of ERK activation is definitely controlled at the level of DAG rate of metabolism through the actions of the and isoforms of DGK (18-21). Here we report evidence for an important part for DGK-dependent rules of DAG signaling in mature B cells. We observed that inhibition of DGK enzymatic activity enhanced BCR-mediated activation of ERK selectively in adult follicular B cells, and this correlated with increased mRNA manifestation Eno2 of DGK and DGK during B cell maturation in the spleen. Interestingly, while adult follicular B cells from mice deficient in.