Convergence of Signaling Pathways on the Activation of ERK in B Cells

Abstract

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...

B Cell Receptor (BCR) Cross-Talk: IL-4 Creates an Alternate Pathway for BCR-Induced ERK Activation That Is Phosphatidylinositol 3-Kinase Independent

“…Further, inhibition of PI3K with LY294002 before addition of anti-Ig eliminated BCR-induced ERK phosphorylation, as reported (17). However, the situation was very different for B cells previously treated with IL-4.…”

“…Thus, Bam32 and Carma1 appear to channel BCR-induced intracellular signaling toward distinct pathways (11). However, interconnections between these pathways certainly exist, and it has been shown that ERK activation is dependent on molecules such as PI3K and/or PKC that are key mediators of BCR-triggered NF-B induction (12)(13)(14)(15)(16)(17).…”

Phosphatidylinositol 3-Kinase-Dependent Mitogen-Activated Protein/Extracellular Signal-Regulated Kinase Kinase 1/2 and NF-κB Signaling Pathways Are Required for B Cell Antigen Receptor-Mediated Cyclin D2 Induction in Mature B Cells

“…This result contrasts with our observations in normal splenic B cells treated with wortmannin in which a requirement for PI-3K is clearly established in BCR-initiated MEK1/2-p42/44ERK phosphorylation. We also observed that LY294004 blocks p42/44ERK phosphorylation following BCR cross-linking, in agreement with a recent report by Jacob et al (57). The molecular basis underlying this discrepancy is currently unknown; however, because all mammalian class I, II, and III PI-3K members show sensitivity to wortmannin and LY294004, it is plausible that BCR-dependent MEK1/ 2-p42/44ERK activation requires PI-3K members other than class I A (58).…”

B Cell Receptor Signaling Down-Regulates Forkhead Box Transcription Factor Class O 1 mRNA Expression via Phosphatidylinositol 3-Kinase and Bruton’s Tyrosine Kinase

“…PI3K has also been shown to mediate BCR-induced activation of Erk (43). To determine whether this pathway contributes to the down-regulation of FOXO1 mRNA expression, B cells were treated with the Erk inhibitor U0126 before incubation with antiIgM.…”