B Cell Receptor-induced Phosphorylation of Pyk2 and Focal Adhesion Kinase Involves Integrins and the Rap GTPases and Is Required for B Cell Spreading

Abstract

Signaling by the B cell receptor (BCR) promotes integrin-mediated adhesion and cytoskeletal reorganization. This results in B cell spreading, which enhances the ability of B cells to bind antigens and become activated. Proline-rich tyrosine kinase (Pyk2) and focal adhesion kinase (FAK) are related cytoplasmic tyrosine kinases that regulate cell adhesion, cell morphology, and cell migration. In this report we show that BCR signaling and integrin signaling collaborate to induce the phosphorylation of Pyk2 and FAK on key tyrosine residues, a modification that increases the kinase activity of Pyk2 and FAK. Activation of the Rap GTPases is critical for BCR-induced integrin activation as well as for BCR-and integrin-induced reorganization of the actin cytoskeleton. We now show that Rap activation is essential for BCR-induced phosphorylation of Pyk2 and for integrin-induced phosphorylation of Pyk2 and FAK. Moreover Rapdependent phosphorylation of Pyk2 and FAK required an intact actin cytoskeleton as well as actin dynamics, suggesting that Rap regulates Pyk2 and FAK via its effects on the actin cytoskeleton. Importantly B cell spreading induced by BCR/integrin co-stimulation or by integrin engagement was inhibited by short hairpin RNA-mediated knockdown of either Pyk2 or FAK expression and by treatment with PF-431396, a chemical inhibitor that blocks the kinase activities of both Pyk2 and FAK. Thus Pyk2 and FAK are downstream targets of the Rap GTPases that play a key role in regulating B cell morphology. Antibodies (Abs)2 made by B lymphocytes play a critical role in host defense against infection. Antigen-induced signaling by the B cell receptor (BCR) initiates an activation program that leads to B cell proliferation and subsequent differentiation into Ab-producing cells. BCR clustering by antigens or by anti-immunoglobulin (anti-Ig) Abs used as surrogate antigens initiates multiple signaling pathways that control gene expression, cell survival, and proliferation pathways (1-3).BCR signaling also promotes integrin activation (4, 5), localized actin polymerization, reorganization of the actin cytoskeleton, and changes in B cell morphology (6, 7), all of which may facilitate B cell activation. Integrin activation and cell spreading is critical for the activation of B cells by membrane-bound antigens. Macrophages, dendritic cells, and follicular dendritic cells can present arrays of captured antigens to B cells (8, 9), and this may be one of the main ways in which B cells encounter antigens (10). BCR-induced integrin activation prolongs the interaction between the B cell and the antigen-presenting cell and also allows the B cell to spread on the surface of the antigenpresenting cell such that more BCRs can encounter and bind membrane-bound antigens (11). Subsequent contraction of the B cell membrane allows the B cells to gather the BCR-bound antigen into an immune synapse in which clustered antigenengaged BCRs are surrounded by a ring of ligand-bound integrins. Formation of this immune synapse reduces the amount of antig...

Pyk2 Controls Integrin-Dependent CTL Migration through Regulation of De-Adhesion

“…In contrast, Pyk2 positively regulates naive mouse CD8 T cell (17) and NK cell (20) adhesion to ICAM-1, and has no impact on neutrophil (16) or eosinophil (21) adhesion. Knockout or inhibition of Pyk2 enhances macrophage cell spreading (15); decreases cell spreading of naive T cells (17), B cells (22), eosinophils (21), and HL-60 cells (23); and has no effect on neutrophil spreading (16).…”

Rap GTPase-mediated adhesion and migration

“…Moreover the kinase activities of Pyk2 and FAK are required for B cell spreading, a key aspect of cell adhesion and motility. 27 The importance of this Rap/Pyk2 signaling module is supported by the observation that B cells from Pyk2-deficient mice have a severe defect in chemokine-induced migration. 28 Rap effectors also promote the establishment of cell polarity, another key aspect of cell motility.…”

“…27 Rap-dependent actin dynamics is critical for the activation of Pyk2 and FAK in B-lymphoma cells. Moreover the kinase activities of Pyk2 and FAK are required for B cell spreading, a key aspect of cell adhesion and motility.…”

PTK2 expression and immunochemotherapy outcome in chronic lymphocytic leukemia

“…This could potentially explain why inhibition of PTK2 kinase activity blunts R-dependent cell death and the association of increased PTK2 expression the specific benefit provided to R-FC in this study. In addition, similarities between CD20 and BCR signaling have been described previously 19 and these similarities suggest that PTK2 may play a direct role in mediating CD20 signaling. Further studies investigating the mechanism by which PTK2 contributes to R-dependent cell death should be considered.…”

“…PTK2 has typically been described as a key regulator of cell adhesion, proliferation, and migration 18 and has been reported to be regulated by the BCR. 19 Although an association or direct mechanistic connection with R efficacy has not been previously described, the fact that high expression of PTK2 correlates with improved PFS to R-FC, but not FC, in 2 independent data sets suggests that PTK2 might be a contributor to R-dependent cell death. To test this hypothesis, we assessed if inhibition of PTK2 kinase activity would have any consequence on R-dependent cell death in vitro.…”