(alpha)v(beta)3 integrins and Pyk2 mediate insulin-like growth factor I activation of Src and mitogen-activated protein kinase in 3T3-L1 cells

IGF-I stimulates cell growth through interaction of the IGF receptor with multiprotein signaling complexes. However, the mechanisms of IGF-I receptor-mediated signaling are not completely understood. We have previously shown that IGF-I-stimulated 3T3-L1 cell proliferation is dependent on Src activation of the ERK-1/2 MAPK pathway. We hypothesized that IGF-I activation of the MAPK pathway is mediated through integrin activation of Src-containing signaling complexes. The disintegrin echistatin decreased IGF-I phosphorylation of Src and MAPK, and blocking antibodies to (alpha)v and beta3 integrin subunits inhibited IGF-I activation of MAPK, suggesting that (alpha)v(beta)3 integrins mediate IGF-I mitogenic signaling. IGF-I increased ligand binding to (alpha)v(beta)3 as detected by immunofluorescent staining of ligand-induced binding site antibody and stimulated phosphorylation of the beta3 subunit, consistent with inside-out activation of (alpha)v(beta)3 integrins. IGF-I increased tyrosine phosphorylation of the focal adhesion kinase (FAK) Pyk2 (calcium-dependent proline-rich tyrosine kinase-2) to a much greater extent than FAK, and increased association of Src with Pyk2 but not FAK. The intracellular calcium chelator BAPTA prevented IGF-I phosphorylation of Pyk2, Src, and MAPK, suggesting that IGF-I activation of Pyk2 is calcium dependent. Transient transfection with a dominant-negative Pyk2, which lacks the autophosphorylation and Src binding site, decreased IGF-I activation of MAPK, but no inhibition was seen with transfected wild-type Pyk2. These results indicate that IGF-I signaling to MAPK is dependent on inside-out activation of (alpha)v(beta)3 integrins and integrin-facilitated multiprotein complex formation involving Pyk2 activation and association with Src.     

Novel Role of Src in Priming Pyk2 Phosphorylation

Proline-rich tyrosine kinase 2 (Pyk2) is a member of the focal adhesion kinase (FAK) family of non-receptor tyrosine kinases and plays an important role in diverse cellular events downstream of the integrin-family of receptors, including cell migration, proliferation and survival. Here, we have identified a novel role for Src kinase in priming Pyk2 phosphorylation and subsequent activation upon cell attachment on the integrin-ligand fibronectin. By using complementary methods, we show that Src activity is indispensable for the initial Pyk2 phosphorylation on the Y402 site observed in response to cell attachment. In contrast, the initial fibronectin-induced autophosphorylation of FAK in the homologous Y397 site occurs in a Src-independent manner. We demonstrate that the SH2-domain of Src is required for Src binding to Pyk2 and for Pyk2 phosphorylation at sites Y402 and Y579. Moreover, Y402 phosphorylation is a prerequisite for the subsequent Y579 phosphorylation. While this initial phosphorylation of Pyk2 by Src is independent of Pyk2 kinase activity, subsequent autophosphorylation of Pyk2 in trans is required for full Pyk2 phosphorylation and activation. Collectively, our studies reveal a novel function of Src in priming Pyk2 (but not FAK) phosphorylation and subsequent activation downstream of integrins, and shed light on the signaling events that regulate the function of Pyk2.     

Targeting Pyk2 to beta 1-integrin-containing focal contacts rescues fibronectin-stimulated signaling and haptotactic motility defects of focal adhesion kinase-null cells

Focal adhesion kinase-null (FAK(-/-) fibroblasts exhibit morphological and motility defects that are reversed by focal adhesion kinase (FAK) reexpression. The FAK-related kinase, proline-rich tyrosine kinase 2 (Pyk2), is expressed in FAK(-/-) cells, yet it exhibits a perinuclear distribution and does not functionally substitute for FAK. Chimeric Pyk2/FAK proteins were created and expressed in FAK(-/-) cells to determine the impact of Pyk2 localization to focal contacts. Whereas an FAK/Pyk2 COOH-terminal (CT) domain chimera was perinuclear distributed, stable expression of a Pyk2 chimera with the FAK-CT domain (Pyk2/FAK-CT) localized to focal contact sites and enhanced fibronectin (FN)-stimulated haptotactic cell migration equal to FAK-reconstituted cells. Disruption of paxillin binding to the FAK-CT domain (S-1034) inhibited Pyk2/FAK-CT localization to focal contacts and its capacity to promote cell motility. Paxillin binding to the FAK-CT was necessary but not sufficient to mediate the indirect association of FAK or Pyk2/FAK-CT with a beta 1-integrin-containing complex. Both FAK and Pyk2/FAK-CT but not Pyk2/FAK-CT S-1034 reconstituted FAK(-/-) cells, exhibit elevated FN-stimulated extracellular signal-regulated kinase 2 (ERK2) and c-Jun NH(2)-terminal kinase (JNK) kinase activation. FN-stimulated FAK or Pyk2/FAK-CT activation enhanced both the extent and duration of FN-stimulated ERK2 activity which was necessary for cell motility. Transient overexpression of the FAK-CT but not FAK-CT S-1034 domain inhibited both FN-stimulated ERK2 and JNK activation as well as FN-stimulated motility of Pyk2/FAK-CT reconstituted cells. These gain-of-function studies show that the NH(2)-terminal and kinase domains of Pyk2 can functionally substitute for FAK in promoting FN-stimulated signaling and motility events when localized to beta-integrin-containing focal contact sites via interactions mediated by the FAK-CT domain.     

The Pyk2 FERM regulates Pyk2 complex formation and phosphorylation

The focal adhesion kinase Pyk2 integrates signals from cell adhesion receptors, growth factor receptors, and G-protein-coupled receptors leading to the activation of intracellular signaling pathways that regulate cellular phenotypes. The intrinsic mechanism for the activation of Pyk2 activity remains to be fully defined. Previously, we reported that mutations in the N-terminal FERM domain result in loss of Pyk2 activity and expression of the FERM domain as an autonomous fragment inhibits Pyk2 activity. In the present study, we sought to determine the mechanism that underlies these effects. Utilizing differentially epitope-tagged Pyk2 constructs, we observed that Pyk2 forms oligomeric complexes in cells and that complex formation correlates positively with tyrosine phosphorylation. Similarly, when expressed as an autonomous fragment, the Pyk2 FERM domain formed a complex with other Pyk2 FERM domains but not the FAK FERM domain. When co-expressed with full-length Pyk2, the autonomously expressed Pyk2 FERM domain formed a complex with full-length Pyk2 preventing the formation of Pyk2 oligomers and resulting in reduced Pyk2 phosphorylation. Deletion of the FERM domain from Pyk2 enhanced Pyk2 complex formation and phosphorylation. Together, these data indicate that the Pyk2 FERM domain is involved in the regulation of Pyk2 activity by acting to regulate the formation of Pyk2 oligomers that are critical for Pyk2 activity.     

Small molecule inhibitors of the Pyk2 and FAK kinases modulate chemoattractant-induced migration, adhesion and Akt activation in follicular and marginal zone B cells

B-lymphocytes produce protective antibodies but also contribute to autoimmunity. In particular, marginal zone (MZ) B cells recognize both microbial components and self-antigens. B cell trafficking is critical for B cell activation and is controlled by chemoattactants such as CXCL13 and sphingosine 1-phosphate (S1P). The related tyrosine kinases focal adhesion kinase (FAK) and proline-rich tyrosine kinase (Pyk2) regulate cell migration and adhesion but their roles in B cells are not fully understood. Using a novel Pyk2-selective inhibitor described herein (PF-719), as well as a FAK-selective inhibitor, we show that both Pyk2 and FAK are important for CXCL13- and S1P-induced migration of B-2 cells and MZ B cells. In contrast, LFA-1-mediated adhesion required only Pyk2 whereas activation of the Akt pro-survival kinase required FAK but not Pyk2. Thus Pyk2 and FAK mediate critical processes in B cells and these inhibitors can be used to further elucidate their functions in B cells.     

Integrin-linked kinase (ILK): a regulator of integrin and growth-factor signalling.

Interaction of cells with the extracellular matrix (ECM) results in the regulation of cell growth, differentiation and migration by coordinated signal transduction through integrins and growth-factor receptors. Integrins achieve signalling by interacting with intracellular effectors that couple integrins and growth-factor receptors to downstream components. One well-studied effector is focal-adhesion kinase (FAK), but recently another protein kinase, integrin-linked kinase (ILK), has been identified as a receptor-proximal effector of integrin and growth-factor signalling. ILK appears to interact with and be influenced by a number of different signalling pathways, and this provides new routes for integrin-mediated signalling. This article discusses ILK structure and function and recent genetic and biochemical evidence about the role of ILK in signal transduction.     

Pyk2 and Src-family protein-tyrosine kinases compensate for the loss of FAK in fibronectin-stimulated signaling events but Pyk2 does not fully function to enhance FAK- cell migration.

The focal adhesion kinase (FAK) protein-tyrosine kinase (PTK) links transmembrane integrin receptors to intracellular signaling pathways. We show that expression of the FAK-related PTK, Pyk2, is elevated in fibroblasts isolated from murine fak-/- embryos (FAK-) compared with cells from fak+/+ embryos (FAK+). Pyk2 was localized to perinuclear regions in both FAK+ and FAK- cells. Pyk2 tyrosine phosphorylation was enhanced by fibronectin (FN) stimulation of FAK- but not FAK+ cells. Increased Pyk2 tyrosine phosphorylation paralleled the time-course of Grb2 binding to Shc and activation of ERK2 in FAK- cells. Pyk2 in vitro autophosphorylation activity was not enhanced by FN plating of FAK- cells. However, Pyk2 associated with active Src-family PTKs after FN but not poly-L-lysine replating of the FAK- cells. Overexpression of both wild-type (WT) and kinase-inactive (Ala457), but not the autophosphorylation site mutant (Phe402) Pyk2, enhanced endogenous FN-stimulated c-Src in vitro kinase activity in FAK- cells, but only WT Pyk2 overexpression enhanced FN-stimulated activation of co-transfected ERK2. Interestingly, Pyk2 overexpression only weakly augmented FAK- cell migration to FN whereas transient FAK expression promoted FAK- cell migration to FN efficiently compared with FAK+ cells. Significantly, repression of endogenous Src-family PTK activity by p50(csk) overexpression inhibited FN-stimulated cell spreading, Pyk2 tyrosine phosphorylation, Grb2 binding to Shc, and ERK2 activation in the FAK- but not in FAK+ cells. These studies show that Pyk2 and Src-family PTKs combine to promote FN-stimulated signaling events to ERK2 in the absence of FAK, but that these signaling events are not sufficient to overcome the FAK- cell migration defects.     

Signaling through focal adhesion kinase

Integrin receptor binding to extracellular matrix proteins generates intracellular signals via enhanced tyrosine phosphorylation events that are important for cell growth, survival, and migration. This review will focus on the functions of the focal adhesion kinase (FAK) protein-tyrosine kinase (PTK) and its role in linking integrin receptors to intracellular signaling pathways. FAK associates with several different signaling proteins such as Src-family PTKs, p130^Cas, Shc, Grb2, PI 3-kinase, and paxillin. This enables FAK to function within a network of integrin-stimulated signaling pathways leading to the activation of targets such as the ERK and JNK/mitogen-activated protein kinase pathways. Focus will be placed on the structural domains and sites of FAK tyrosine phosphorylation important for FAK-mediated signaling events and how these sites are conserved in the FAK-related PTK, Pyk2. We will review what is known about FAK activation by integrin receptor-mediated events and also non-integrin stimuli. In addition, we discuss the emergence of a consensus FAK substrate phosphorylation sequence. Emphasis will also be placed on the role of FAK in generating cell survival signals and the cleavage of FAK during caspase-mediated apoptosis. An in-depth discussion will be presented of integrin-stimulated signaling events occurring in the FAK knockout fibroblasts (FAK⁻) and how these cells exhibit deficits in cell migration. FAK re-expression in the FAK⁻ cells confirms the role of this PTK in the regulation of cell morphology and in promoting cell migration events. In addition, these results reinforce the potential role for FAK in promoting an invasive phenotype in human tumors.     

Activation of NPFF2 receptor stimulates neurite outgrowth in Neuro 2A cells through activation of ERK signaling pathway

Neurite outgrowth is an important process in neural regeneration and plasticity, especially after neural injury, and recent evidence indicates that several G_αi/o protein-coupled receptors play an important role in neurite outgrowth. The neuropeptide (NP)FF system contains two G_αi/o protein-coupled receptors, NPFF₁ and NPFF₂ receptors, which are mainly distributed in the central nervous system. The aim of the present study was to determine whether the NPFF system is involved in neurite outgrowth in Neuro 2A cells. We showed that Neuro 2A cells endogenously expressed NPFF₂ receptor, and the NPFF₂ receptor agonist dNPA inhibited cyclic adenosine monophosphate (cAMP) production stimulated by forskolin in Neuro 2A cells. We also demonstrated that NPFF and dNPA dose-dependently induced neurite outgrowth in Neuro 2A cells, which was completely abolished by the NPFF receptor antagonist RF9. Pretreatment with mitogen-activated protein kinase inhibitors PD98059 and U0126 decreased dNPA-induced neurite outgrowth. In addition, dNPA increased phosphorylation of extracellular signal-regulated kinase (ERK) in Neuro 2A cells, which was completely antagonized by pretreatment with U0126. Our results suggest that activation of NPFF₂ receptor stimulates neurite outgrowth in Neuro 2A cells through activation of the ERK signaling pathway. Moreover, NPFF₂ receptor may be a potential therapeutic target for neural injury and degeneration in the future.     

The influence of Pyk2 on the mechanical properties in fibroblasts

The cell surface receptor integrin is involved in signaling mechanical stresses via the focal adhesion complex (FAC) into the cell. Within FAC, the focal adhesion kinase (FAK) and Pyk2 are believed to act as important scaffolding proteins. Based on the knowledge that many signal transducing molecules are transiently immobilized within FAC connecting the cytoskeleton with integrins, we applied magnetic tweezer and atomic force microscopic measurements to determine the influence of FAK and Pyk2 in cells mechanically. Using mouse embryonic fibroblasts (MEF; FAK^+/+, FAK^−/−, and siRNA-Pyk2 treated FAK^−/− cells) provided a unique opportunity to describe the function of FAK and Pyk2 in more detail and to define their influence on FAC and actin distribution.     

A role for the SHP-2 tyrosine phosphatase in nerve growth-induced PC12 cell differentiation.

SHP-1 and SHP-2 are intracellular protein tyrosine phosphatases containing two adjacent src homology 2 domains that target these phosphatases to cell surface receptor signaling complexes and play a role in receptor signal transduction. In this report the PC12 cell system was used to investigate the potential roles of SHP-1 and SHP-2 in the induction of neuronal differentiation by nerve growth factor (NGF). By using neurite outgrowth as a marker for differentiation, the effects of transfected constructs of SHP-1 and SHP-2 were assessed. Overexpression of a catalytically inactive SHP-2, but not a catalytically inactive SHP-1, blocked NGF-stimulated neurite outgrowth. The mitogen-activated protein kinase (MAPK) signaling cascade is important for the morphological differentiation in PC12 cells, and both SHP-1 and SHP-2 have been implicated to act upstream of MAPK in other receptor signaling systems. A positive role for SHP-2 but not SHP-1 in the activation of MAPK by NGF was demonstrated by introduction of the SHP-2 phosphatase mutants along with hemagglutinin-tagged MAPK. Coexpression studies with the SHP-2 mutant along with mutant forms of MAPK kinase suggested that SHP-2 functions upstream of MAPK kinase and MAPK in NGF-induced neurite outgrowth.     

Non-overlapping functions for Pyk2 and FAK in osteoblasts during fluid shear stress-induced mechanotransduction

Mechanotransduction, the process by which cells convert external mechanical stimuli such as fluid shear stress (FSS) into biochemical changes, plays a critical role in maintenance of the skeleton. We have proposed that mechanical stimulation by FSS across the surfaces of bone cells results in formation of unique signaling complexes called mechanosomes that are launched from sites of adhesion with the extracellular matrix and with other bone cells [1]. Deformation of adhesion complexes at the cell membrane ultimately results in alteration of target gene expression. Recently, we reported that focal adhesion kinase (FAK) functions as a part of a mechanosome complex that is required for FSS-induced mechanotransduction in bone cells. This study extends this work to examine the role of a second member of the FAK family of non-receptor protein tyrosine kinases, proline-rich tyrosine kinase 2 (Pyk2), and determine its role during osteoblast mechanotransduction. We use osteoblasts harvested from mice as our model system in this study and compared the contributions of Pyk2 and FAK during FSS induced mechanotransduction in osteoblasts. We exposed Pyk2(+/+) and Pyk2(-/-) primary calvarial osteoblasts to short period of oscillatory fluid flow and analyzed downstream activation of ERK1/2, and expression of c-fos, cyclooxygenase-2 and osteopontin. Unlike FAK, Pyk2 was not required for fluid flow-induced mechanotransduction as there was no significant difference in the response of Pyk2(+/+) and Pyk2(-/-) osteoblasts to short periods of fluid flow (FF). In contrast, and as predicted, FAK(-/-) osteoblasts were unable to respond to FF. These data indicate that FAK and Pyk2 have distinct, non-redundant functions in launching mechanical signals during osteoblast mechanotransduction. Additionally, we compared two methods of generating FF in both cell types, oscillatory pump method and another orbital platform method. We determined that both methods of generating FF induced similar responses in both primary calvarial osteoblasts and immortalized calvarial osteoblasts.     

Phosphotyrosine signaling networks in epidermal growth factor receptor overexpressing squamous carcinoma cells

Overexpression and enhanced activation of the epidermal growth factor (EGF) receptor are frequent events in human cancers that correlate with poor prognosis. Anti-phosphotyrosine and anti-EGFr affinity chromatography, isotope-coded muLC-MS/MS, and immunoblot methods were combined to describe and measure signaling networks associated with EGF receptor activation and pharmacological inhibition. The squamous carcinoma cell line HN5, which overexpresses EGF receptor and displays sustained receptor kinase activation, was used as a model system, where pharmacological inhibition of EGF receptor kinase by erlotinib markedly reduced auto and substrate phosphorylation, Src family phosphorylation at EGFR Y845, while increasing total EGF receptor protein. Diverse sets of known and poorly described functional protein classes were unequivocally identified by affinity selection, comprising either proteins tyrosine phosphorylated or complexed therewith, predominantly through EGF receptor and Src family kinases, principally 1) immediate EGF receptor signaling complexes (18%); 2) complexes involved in adhesion and cell-cell contacts (34%); and 3) receptor internalization and degradation signals. Novel and known phosphorylation sites could be located despite the complexity of the peptide mixtures. In addition to interactions with multiple signaling adaptors Grb2, SHC, SCK, and NSP2, EGF receptors in HN5 cells were shown to form direct or indirect physical interactions with additional kinases including ACK1, focal adhesion kinase (FAK), Pyk2, Yes, EphA2, and EphB4. Pharmacological inhibition of EGF receptor kinase activity by erlotinib resulted in reduced phosphorylation of downstream signaling, for example through Cbl/Cbl-B, phospholipase Cgamma (PLCgamma), Erk1/2, PI-3 kinase, and STAT3/5. Focal adhesion proteins, FAK, Pyk2, paxillin, ARF/GIT1, and plakophillin were down-regulated by transient EGF stimulation suggesting a complex balance between growth factor induced kinase and phosphatase activities in the control of cell adhesion complexes. The functional interactions between IGF-1 receptor, lysophosphatidic acid (LPA) signaling, and EGF receptor were observed, both direct and/or indirectly on phospho-Akt, phospho-Erk1/2, and phospho-ribosomal S6.     

Depolarization-induced differentiation of PC12 cells is mediated by phospholipase D2 through the transcription factor CREB pathway

The present study examined the role of phospholipase D2 (PLD2) in the regulation of depolarization-induced neurite outgrowth and the expression of growth-associated protein-43 (GAP-43) and synapsin I in rat pheochromocytoma (PC12) cells. Depolarization of PC12 cells with 50 mmol/L KCl increased neurite outgrowth and elevated mRNA and protein expression of GAP-43 and synapsin I. These increases were suppressed by inhibition of Ca²⁺-calmodulin-dependent protein kinase II (CaMKII), PLD, or mitogen-activated protein kinase kinase (MEK). Knockdown of PLD2 by small interfering RNA (siRNA) suppressed the depolarization-induced neurite outgrowth, and the increase in GAP-43 and synapsin I expression. Depolarization evoked a Ca²⁺ rise that activated various signaling enzymes and the cAMP response element-binding protein (CREB). Silencing CaMKIIδ by siRNA blocked KCl-induced phosphorylation of proline-rich protein tyrosine kinase 2 (Pyk2), Src kinase, and extracellular signal-regulated kinase (ERK). Inhibition of Src or MEK abolished phosphorylation of ERK and CREB. Furthermore, phosphorylation of Pyk2, ERK, and CREB was suppressed by the PLD inhibitor, 1-butanol and transfection of PLD2 siRNA, whereas it was enhanced by over-expression of wild-type PLD2. Depolarization-induced PLD2 activation was suppressed by CaMKII and Src inhibitors, but not by MEK or protein kinase A inhibitors. These results suggest that the signaling pathway of depolarization-induced PLD2 activation was downstream of CaMKIIδ and Src, and upstream of Pyk2(Y881) and ERK/CREB, but independent of the protein kinase A. This is the first demonstration that PLD2 activation is involved in GAP-43 and synapsin I expression during depolarization-induced neuronal differentiation in PC12 cells.     

Beta1,4-N-Acetylglucosaminyltransferase III down-regulates neurite outgrowth induced by costimulation of epidermal growth factor and integrins through the Ras/ERK signaling pathway in PC12 cells

A rat pheochromocytoma cell line (PC12), when transfected with beta1,4-N-acetylglucosaminyltransferase III (GnT-III), which catalyzes the formation of a bisecting GlcNAc structure in N-glycans, resulted in the suppression of neurite outgrowth induced by costimulation of epidermal growth factor (EGF) and integrins. The neurite outgrowth was restored by the overexpression of a constitutively activated mitogen- or extracellular signal-regulated kinase kinase-1 (MEK-1). Consistent with this, the EGF receptor (EGFR)-mediated ERK activation was blocked in GnT-III transfectants. Conversely, the overexpression of dominant negative MEK-1 or treatment with PD98059, a specific inhibitor of MEK-1, inhibited neurite outgrowth in controls transfected with mock. Furthermore GnT-III activity is required for these inhibitions, because the overexpression of a dominant negative GnT-III mutant (D321A) failed to reduce neurite outgrowth and EGFR-mediated ERK activation. Lectin blot analysis confirmed that EGFR from wild-type GnT-III transfectants had been modified by bisecting GlcNAc in its N-glycan structures. This modification led to a significant decrease in EGF binding and EGFR autophosphorylation. Collectively, the results constitute a comprehensive body of evidence to show clearly that the overexpression of GnT-III prevents neurite outgrowth induced by costimulation of EGF and integrins through the Ras/MAPK activation pathway and indicates that GnT-III may be an important regulator for cell differentiation in neural tissues.     

RAFTK/Pyk2-mediated cellular signalling

Intracellular signal transduction following extracellular ligation by a wide variety of surface molecules involves the activation and tyrosine phosphorylation of protein tyrosine kinases (PTKs). Tyrosine phosphorylation, controlled by the coordinated actions of protein tyrosine phosphatases (PTPs) and tyrosine kinases, is a critical regulatory mechanism for various physiological processes, including cell growth, differentiation, metabolism, cell cycle regulation and cytoskeleton function. The focal adhesion PTK family consists of the focal adhesion kinase (FAK) and the RAFTK/Pyk2 kinase (also known as CAK-beta and CADTK). RAFTK/Pyk2 can be activated by a variety of extracellular signals that elevate intracellular calcium concentration, and by stress signals. RAFTK/Pyk2 is expressed mainly in the central nervous system and in cells derived from hematopoietic lineages, while FAK is widely expressed in various tissues and links transmembrane integrin receptors to intracellular pathways. This review describes the role of RAFTK/Pyk2 in various signalling cascades and details the differential signalling by FAK and RAFTK/Pyk2.     

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

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 Rap-dependent 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 antigen-presenting 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 antigen-engaged BCRs are surrounded by a ring of ligand-bound integrins. Formation of this immune synapse reduces the amount of antigen that is required for B cell activation (12, 13).Recent work has shown that B cells in lymphoid organs may contact soluble antigens by extending membrane processes into a highly organized network of lymph-filled conduits (14). These conduits are created by fibroblastic reticular cells that partially ensheathe collagen fibrils. In addition to being rich in collagen, fibronectin, and other extracellular matrix (ECM) components, the fibroblastic reticular cells that form these conduits express high levels of intercellular adhesion molecule-1, the ligand for the α_Lβ₂ integrin (lymphocyte function-associated antigen-1 (LFA-1)) on B cells (10). Thus B cells interacting with these conduits are likely to be in contact with integrin ligands, and integrin-dependent spreading may enhance the ability of B cells to extend membrane processes into the fibroblastic reticular cell conduit.In addition to promoting cell spreading, integrins can act as co-stimulatory receptors that enhance signaling by many receptors including the T cell receptor and the BCR (15–17). Thus signaling proteins that regulate B cell spreading and that are also targets of BCR/integrin co-stimulation may play a key role in the activation of B cells by membrane-bound antigens as well as soluble antigens that are delivered to lymphoid organs by fibroblastic reticular cell conduits.Proline-rich tyrosine kinase (Pyk2) and focal adhesion kinase (FAK) are related non-receptor protein-tyrosine kinases that integrate signals from multiple receptors and play an important role in regulating cell adhesion, cell morphology, and cell migration in many cell types (18–20). Integrins, receptor tyrosine kinases, antigen receptors, and G protein-coupled chemokine receptors all stimulate tyrosine phosphorylation of Pyk2 and FAK, a modification that increases the enzymatic activity of these kinases and allows them to bind SH2 domain-containing signaling proteins (21). FAK, which is expressed in almost all tissues (21), is a focal adhesion component that mediates integrin-dependent cell migration (22), cell spreading, and cell adhesion (18) in adherent cells as well as co-clustering of LFA-1 with the T cell receptor in lymphocytes (23). Pyk2 is expressed mainly in hematopoietic cells, osteoclasts, and the central nervous system (24) and is critical for chemokine-induced migration of B cells, macrophages, and natural killer cells (20, 25, 26) as well as the spreading of osteoclasts on vitronectin (27). FAK and Pyk2 are thought to mediate overlapping but distinct functions because Pyk2 expression only partially reverses the cell adhesion and migration defects in FAK-deficient fibroblasts (28).In B cells, clustering of the BCR, β₁ integrins, or β₇ integrins induces tyrosine phosphorylation of both Pyk2 and FAK (29–33). FAK is involved in the chemokine-induced adhesion of B cell progenitors (34), and Pyk2 is required for chemokine-induced migration of mature B cells (25). However, the role of these kinases in BCR- and integrin-induced B cell spreading has not been investigated, and the signaling pathways that link the BCR and integrins to tyrosine phosphorylation of Pyk2 and FAK have not been elucidated.We have shown previously that the ability of the BCR to induce integrin activation, B cell spreading, and immune synapse formation requires activation of the Rap GTPases (6, 17). In addition to binding effector proteins such as RapL and Rap1-interacting adaptor molecule (RIAM) that promote integrin activation (35–37), the active GTP-bound forms of Rap1 and Rap2 bind multiple proteins that control actin dynamics and cell morphology (38). Moreover we showed that BCR/integrin-induced phosphorylation of Pyk2 in B cells is dependent on Rap activation (17). However, this previous study did not address how Rap-GTP links the BCR and integrins to Pyk2 phosphorylation, whether Rap activation is important for FAK phosphorylation in B cells, or whether B cell spreading is regulated by Pyk2 or FAK. We now show that Pyk2 and FAK are differentially expressed and localized in B cells, that Pyk2 and FAK are important for B cell spreading, and that integrin engagement enhances BCR-induced phosphorylation of Pyk2 and FAK, a process that depends on both Rap activation and actin dynamics.