The negative regulatory role of the Src homology 2 domain-containing inositol 5-phosphatase (SHIP) has been invoked in a variety of receptor-mediated signaling pathways. In B lymphocytes, co-clustering of antigen receptor surface immunoglobulin with Fc␥RIIb promotes the negative effects of SHIP, but how SHIP activity is regulated is unknown. To explore this issue, we investigated the effect of SHIP phosphorylation, receptor tyrosine engagement by its Src homology 2 domain, and membrane recruitment of SHIP on its enzymatic activity. We examined two SHIP phosphorylation kinase candidates, Lyn and Syk, and observed that the Src protein-tyrosine kinase, Lyn is far superior to Syk in its ability to phosphorylate SHIP both in vitro and in vivo. However, we found a minimal effect of phosphorylation or receptor tyrosine engagement of SHIP on its enzymatic activity, whereas membrane localization of SHIP significantly reduced cellular phosphatidylinositol 3,4,5-triphosphate levels. Based on our results, we propose that a membrane localization of SHIP is the crucial event in the induction of its phosphatase effects.Clustering of the B cell surface immunoglobulin (sIg) 1 antigen receptor by binding of foreign antigen initiates a set of biochemical events termed positive signaling which activate B lymphocytes to proliferate and secrete soluble antigen specific Ig (reviewed in Refs. 1-3). The most proximal signaling event is the stimulation of the Src family of protein-tyrosine kinases (PTKs), which phosphorylates tyrosine residues within conserved immunoreceptor tyrosine-based activation motifs (ITAMs), found in receptor-associated proteins (4). Once the tyrosines in the ITAM are phosphorylated, they serve as docking sites for numerous proteins and enzymes containing Src homology 2 (SH2) domains including the PTK Syk and the p85 adapter subunit of phosphatidylinositol 3-kinase (PtdIns 3-kinase; reviewed in Refs. 5 and 6). These activation signals are then propagated through additional tyrosine phosphorylation and protein-protein interactions and result in changes in B cell biology.PtdIns 3-kinase is comprised of a p85 adapter subunit and a p110 catalytic subunit. By catalyzing the phosphorylation of the D-3 position of the inositol ring (7), PtdIns 3-kinase generates phosphatidylinositol 3,4,5-trisphosphate (PtdIns-(3,4,5)P 3 ), which acts as an intracellular mediator for several enzymes. PtdIns-(3,4,5)P 3 binds to pleckstrin homology domains of enzymes (8) such as Akt (9) and Btk (10 -12), thereby promoting re-localization to the membrane and providing enzyme access to new lipid substrates or regulatory kinases (13).In contrast to activating signals generated upon sIg clustering, co-clustering of sIg with the B cell Fc receptor for IgG (Fc␥RIIb) aborts B cell activation. It has been proposed (14, 15) that co-clustering of sIg and Fc␥RIIb occurs late in the humoral immune response to block continued Ig production. We have termed sIg-Fc␥RIIb co-clustering "negative signaling," to contrast with positive signaling initiated by ...

The B cell receptor (BCR) initiates three major signaling pathways: the Ras pathway, which leads to extracellular signal-regulated kinase (ERK) activation; the phospholipase C-␥ pathway, which causes calcium mobilization; and the phosphoinositide 3-kinase (PI 3-kinase) pathway. These combine to induce different biological responses depending on the context of the BCR signal. Both the Ras and PI 3-kinase pathways are important for B cell development and activation. Several model systems show evidence of cross-regulation between these pathways. Here we demonstrate through the use of PI 3-kinase inhibitors and a dominant-negative PI 3-kinase construct that the BCR-induced phosphorylation and activation of ERK is dependent on PI 3-kinase. PI 3-kinase feeds into the Ras signaling cascade at multiple points, both upstream and downstream of Ras. We also show that ERK activation is dependent on phospholipase C-␥, in keeping with its dependence on calcium mobilization. Last, the activation of PI 3-kinase itself is completely dependent on Ras. We conclude that the PI 3-kinase and Ras signaling cascades are intimately connected in B cells and that the activation of ERK is a signal integration point, since it requires simultaneous input from all three major signaling pathways. Triggering the B cell antigen receptor (BCR)1 by antigen or polyclonal activators initiates signaling cascades that lead to various cellular responses (1). Receptor ligation is followed by the phosphorylation of the immunoreceptor tyrosine-based activation motifs (2-4), which recruit cytosolic proteins and enzymes. Receptor recruitment brings enzymes into proximity of their substrates or kinases, which permits the sequential reactions that form the basis of signaling cascades. Three such cascades stimulated by immunoreceptors involve Ras, phosphoinositide 3-kinase (PI 3-kinase), and phospholipase C-␥ (PLC-␥) (reviewed in Ref. 1).The activation of PI 3-kinases results in the rapid accumulation of D-3 phosphorylated inositol phospholipids in the cell membrane (5). PI 3-kinase products bind with high specificity to pleckstrin homology domains of signaling proteins, to recruit pleckstrin homology domain-containing enzymes to the plasma membrane and promote their subsequent activation (6, 7). Class IA PI 3-kinases, the major class induced by tyrosine kinase-dependent receptors (6) consist of an SH2 domain-containing adapter p85 subunit that is constitutively bound to a catalytic p110 subunit. Animals lacking the p85 isoform p85␣ show a defect in B cell development, and mature B cells from these animals are impaired in their response to anti-IgM (8, 9). The activation of Ras requires the membrane localization of a guanine nucleotide exchange factor (GEF) such as Sos (10). In growth factor-stimulated cells, Sos translocation is mediated by the adapter proteins, Grb2 and Shc (11). BCR triggering causes the association of Sos with the adaptors . The ternary complex of Shc, Grb2, and Sos is associated with the plasma membrane after BCR triggering, consistent wi...

TNF receptor superfamily members, such as CD40 and the Toll-like receptors (TLRs), regulate many aspects of B cell differentiation and activation. TRAF6 is an intracellular signaling adaptor molecule for these receptors, but its role in B cells has not been clarified by previous genetic approaches, as the systemic deletion of the TRAF6 gene results in perinatal lethality. Here we show that B cell-specific TRAF6 deficiency results in a reduced number of mature B cells in the bone marrow and spleen. Optimal T cell-dependent (TD) antigen responses, as characterized by isotype switching and long-lived plasma cell generation, are also impaired in B cell-specific TRAF6-deficient mice. B cell-specific TRAF6-deficient mice also exhibit lower levels of serum IgM and IgG2b and defective antigen-specific IgM production in response to T cell-independent (TI) antigens. Unexpectedly, TRAF6-deficient B cell progenitors are unable to generate CD5+ B-1 cells. These results reveal critical roles for TRAF6 in TD and TI humoral immune responses and in inductive fate decisions necessary to generate the B-1 B cell compartment.

The importance of ligand deprotonation to the on rates and the fact that large ligands bind readily indicate that the heme pocket is open and apolar. Ligand basicity strongly influences the strength of binding. The destabilization of inhibitory ligands by the presence of the kinase domain is consistent with a 'load' imposed by coupling to the inactivating mechanism.

We examined activation of the serine/threonine kinase Akt in the murine B cell line A20. Akt is activated in a phosphoinositide 3-kinase (PtdIns 3-kinase)-dependent manner upon stimulation of the antigen receptor, surface immunoglobulin (sIg). In contrast, Akt induction is reduced upon co-clustering of sIg with the B cell IgG receptor, Fc␥RIIb. Co-clustering of sIg-Fc␥RIIb transmits a dominant negative signal and is associated with reduced accumulation of the PtdIns 3-kinase product phosphatidylinositol 3,4,5-trisphosphate (PtdIns 3,4,5-P 3 ), known to be a potent activator of Akt. PtdIns 3-kinase is activated to the same extent with and without Fc␥RIIb co-ligation, indicating conditions supporting the generation of PtdIns 3,4,5-P 3 . We hypothesized that the decreased Akt activity arises from the consumption of PtdIns 3,4,5-P 3 by the inositol-5-phosphatase Src homology 2-containing inositol 5-phosphatase (SHIP), which has been shown by us to be tyrosine-phosphorylated and associated with Fc␥RIIb when the latter is co-ligated. In direct support of this hypothesis, we report here that Akt induction is greatly reduced in fibroblasts expressing catalytically active but not inactive SHIP. Likewise, the reduction in Akt activity upon sIgFc␥RIIb co-clustering is absent from avian B cells lacking expression of SHIP. These findings indicate that SHIP acts as a negative regulator of Akt activation.Cellular survival in many systems is regulated by the serine/ threonine kinase Akt, also known as protein kinase B (reviewed in Ref. 1). Akt prevents apoptosis in growth factorresponsive cells (1, 2) by phosphorylation of the protein Bad (3, 4), which causes the latter to dissociate from Bcl-2 and Bcl-x L (5, 6). Akt activation in most systems studied to date is dependent on the activity of the lipid kinase phosphoinositide 3-kinase (PtdIns 3-kinase), 1 which generates 3-phosphorylated inositol phospholipids. The PtdIns 3-kinase class IA enzymes, which are regulated by tyrosine kinases, appear to be responsible for Akt activation (7). This class of PtdIns 3-kinase contains two distinct and constitutively associated subunits, an 85-kDa regulatory protein, which contains two Src homology 2 (SH2) domains, and a catalytic 110-kDa subunit. Which PtdIns 3-kinase products are most important for Akt activity is currently a matter of debate. Both phosphatidylinositol 3,4-bisphosphate (PtdIns 3,4-P 2 ) and phosphatidylinositol 3,4,5-trisphosphate (PtdIns 3,4,5-P 3 ) bind to the Akt pleckstrin homology domain, but with different affinities, so that PtdIns-3,4,5-P 3 binding is considerably more avid (8). Both 3-phosphoinositide lipids were reported to promote Akt activation, but whether this is a direct effect is unclear (9 -12). Besides regulation by 3-phosphoinositides, Akt must also be phosphorylated at conserved serine and threonine residues for maximal activation (reviewed in Ref. 13). At least one kinase responsible for phosphorylating and activating Akt has been isolated (9, 10). The activity of this kinase, PtdIns 3,4,5-P 3 -depen...