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Activation of the NLRP3 inflammasome is crucial for immune defense, but improper and excessive activation causes inflammatory diseases. We previously reported that Pyk2 is essential for NLRP3 inflammasome activation. Here we show that the Src-family kinases (SFKs)-Cbl axis plays a pivotal role in suppressing NLRP3 inflammasome activation in response to stimulation by nigericin or ATP, as assessed using gene knockout and gene knockdown cells, dominant active/negative mutants, and pharmacological inhibition. We reveal that the phosphorylation of Cbl is regulated by SFKs, and that phosphorylation of Cbl at Tyr371 suppresses NLRP3 inflammasome activation. Mechanistically, Cbl decreases the level of phosphorylated Pyk2 (p-Pyk2) through ubiquitination-mediated proteasomal degradation and reduces mitochondrial ROS (mtROS) production by contributing to the maintenance of mitochondrial size. The lower levels of p-Pyk2 and mtROS dampen NLRP3 inflammasome activation. In vivo, inhibition of Cbl with an analgesic drug, hydrocotarnine, increases inflammasome-mediated IL-18 secretion in the colon, and protects mice from dextran sulphate sodium-induced colitis. Together, our novel findings provide new insights into the role of the SFK-Cbl axis in suppressing NLRP3 inflammasome activation and identify a novel clinical utility of hydrocortanine for disease treatment.

CD4 T cells play a central role as helper cells in adaptive immunity. Presentation of exogenous antigens in MHC class II by professional antigen-presenting cells is a crucial step in induction of specific CD4 T cells in adaptive immune responses. For efficient induction of immunity against intracellular threats such as viruses or malignant transformations, antigens from HLA class II-negative infected or transformed cells need to be transferred to surrounding antigen-presenting cells to allow efficient priming of naive CD4 T cells. Here we show indirect antigen presentation for a subset of natural HLA class II ligands that are created by genetic variants and demonstrated that (neo)antigens can be transferred between cells by extracellular vesicles. Intercellular transfer by extracellular vesicles was not dependent on the T-cell epitope, but rather on characteristics of the full-length protein. This mechanism of (neo)antigen transfer from HLA class II-negative cells to surrounding antigen-presenting cells may play a crucial role in induction of anti-tumor immunity., (© 2018 The Authors. European Journal of Immunology published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Monocytes are short-lived myeloid cells that perform functions essential for tissue homeostasis and disease resolution. However, the cellular mechanisms controlling the maintenance and turnover of monocyte populations are largely undefined. Proline-rich tyrosine kinase 2 (Pyk2) is a nonreceptor tyrosine kinase that regulates numerous immune cell functions, but its role in monocytes is currently unknown. In this study, we sought to characterize the expression and function of Pyk2 in lineage-committed monocyte populations. Here, we report that Pyk2 protein expression is increased in the Ly6C - monocyte population. Using a Pyk2 knockout mouse model (Pyk2 -/- ), we show that Pyk2 regulates the relative proportion of monocyte subsets normally represented in the bone marrow (BM) at steady state. In support of this conclusion, a similar phenotype was observed in the peripheral blood and spleen. Data from reciprocal BM chimera experiments indicate that the alterations in monocyte populations exhibited by Pyk2 -/- mice are due to factors intrinsic to the monocytes. Lineage-tracing of monocyte populations suggests that Pyk2 promotes apoptosis in BM monocytes, thereby acting as an important homeostatic regulator of turnover in these short-lived, innate immune cells., (© Society for Leukocyte Biology.)

The inflammasome adaptor protein, ASC, contributes to both innate immune responses and inflammatory diseases via self-oligomerization, which leads to the activation of the protease, caspase-1. Here, we report that the cytosolic tyrosine kinases, FAK and Pyk2, are differentially involved in NLRP3 and AIM2 inflammasome activation. The inhibition of FAK and Pyk2 with RNA interference or chemical inhibitors dramatically abolished ASC oligomerization, caspase-1 activation, and IL-1β secretion in response to NLRP3 or AIM2 stimulation. Pyk2 is phosphorylated by the kinase Syk and relocalizes to the ASC specks upon NLRP3 inflammasome activation. Pyk2, but not FAK, could directly phosphorylate ASC at Tyr146, and only the phosphorylated ASC could participate in speck formation and trigger IL-1β secretion. Moreover, the clinical-trial-tested Pyk2/FAK dual inhibitor PF-562271 reduced monosodium urate-mediated peritonitis, a disease model used for studying the consequences of NLRP3 activation. Our results suggest that although Pyk2 and FAK are involved in inflammasome activation, only Pyk2 directly phosphorylates ASC and brings ASC into an oligomerization-competent state by allowing Tyr146 phosphorylation to participate ASC speck formation and subsequent NLRP3 inflammation.

Protein tyrosine kinase 2 (Pyk2) is required for T cell adhesion to ICAM-1; however, the mechanism by which it regulates adhesion remains unexplored. Pyk2 function in murine CTL clones and activated ex vivo CD8(+) T cells was disrupted by pharmacological inhibition, knockdown of expression with small interfering RNA, or expression of the dominant-negative C-terminal domain. We found that Pyk2 is not absolutely required for adhesion of CTL to ICAM-1, but rather delays the initial adhesion. Disruption of Pyk2 function caused cells to display an unusual elongated appearance after 1 h on ICAM-1, consistent with abnormally strong adhesion. Furthermore, the random mobility of CTL on ICAM-1 was severely compromised using all three methods of disrupting Pyk2 function. Live-cell imaging studies revealed that the decreased migration is the result of a defect in the detachment from ICAM-1 at the trailing edge when Pyk2 function is inhibited. Examination of Pyk2 tyrosine phosphorylation in normal polarized cells demonstrated that Pyk2 phosphorylated at Y579 and Y580 preferentially localizes to the leading edge, whereas Y881-phosphorylated Pyk2 is enriched at the trailing edge, suggesting that the tyrosine phosphorylation of Pyk2 is spatially regulated in migrating CTL. Additionally, inhibition of Pyk2 caused cells to form multiple LFA-1-rich tails at the trailing edge, most likely resulting from a defect in LFA-1 release required for forward movement. Our results show that Pyk2 contributes to CTL migration by regulating detachment of CTL at the trailing edge, which could explain why Pyk2 is important for chemotactic and migratory responses., (Copyright © 2016 by The American Association of Immunologists, Inc.)

The immunological synapse (IS) formed at the interface between T cells and antigen-presenting cells represents a hallmark of initiation of acquired immunity. T cell activation is initiated at T cell receptor (TCR) microclusters (MCs), in which TCRs and signaling molecules assemble at the interface before IS formation. We found that each TCR-MC was transiently bordered by a ring structure made of integrin and focal adhesion molecules in the early phase of activation, which is similar in structure to the IS in microscale. The micro-adhesion ring is composed of LFA-1, focal adhesion molecules paxillin and Pyk2, and myosin II (MyoII) and is supported by F-actin core and MyoII activity through LFA-1 outside-in signals. The formation of the micro-adhesion ring was transient but especially sustained upon weak TCR stimulation to recruit linker for activation of T cells (LAT) and SLP76. Perturbation of the micro-adhesion ring induced impairment of TCR-MC development and resulted in impaired cellular signaling and cell functions. Thus, the synapse-like structure composed of the core TCR-MC and surrounding micro-adhesion ring is a critical structure for initial T cell activation through integrin outside-in signals., (© 2016 Hashimoto-Tane et al.)

During inflammation, dendritic cells emigrate from inflamed tissue across the lymphatic endothelium into the lymphatic vasculature and travel to regional lymph nodes to initiate immune responses. However, the processes that regulate dendritic cell tissue egress and migration across the lymphatic endothelium are not well defined. The mammalian lectin galectin-1 is highly expressed by vascular endothelial cells in inflamed tissue and has been shown to regulate immune cell tissue entry into inflamed tissue. Here, we show that galectin-1 is also highly expressed by human lymphatic endothelial cells, and deposition of galectin-1 in extracellular matrix selectively regulates migration of specific human dendritic cell subsets. The presence of galectin-1 inhibits migration of immunogenic dendritic cells through the extracellular matrix and across lymphatic endothelial cells, but it has no effect on migration of tolerogenic dendritic cells. The major galectin-1 counter-receptor on both dendritic cell populations is the cell surface mucin CD43; differential core 2 O-glycosylation of CD43 between immunogenic dendritic cells and tolerogenic dendritic cells appears to contribute to the differential effect of galectin-1 on migration. Binding of galectin-1 to immunogenic dendritic cells reduces phosphorylation and activity of the protein-tyrosine kinase Pyk2, an effect that may also contribute to reduced migration of this subset. In a murine lymphedema model, galectin-1(-/-) animals had increased numbers of migratory dendritic cells in draining lymph nodes, specifically dendritic cells with an immunogenic phenotype. These findings define a novel role for galectin-1 in inhibiting tissue emigration of immunogenic, but not tolerogenic, dendritic cells, providing an additional mechanism by which galectin-1 can dampen immune responses., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

TCR-induced signaling controls T cell activation that drives adaptive immunity against infections, but it can also induce dysfunctional T cell responses that promote pathologic disease. The PI3K pathway regulates many downstream effector responses after TCR stimulation. However, the molecular mechanisms that induce PI3K function downstream of the TCR are not fully understood. We have previously shown that Pyk2 is activated downstream of the TCR in a PI3K-independent manner. Although Pyk2 controls adhesion, proliferation, and cytokine production in T cells, the mechanisms by which it controls these processes are not known. In this study, we generated Pyk2-deficient human T cells to elucidate further the role that this kinase plays in TCR-induced effector functions and signaling. We observed that Pyk2 localized with the p85 regulatory subunit of PI3K at the LAT complex and that PI3K-dependent signaling was impaired in Pyk2-deficient T cells. Likewise, functions downstream of PI3K, including IFN-γ production and proliferation, were also suppressed in human T cells deficient in Pyk2. Collectively, these data demonstrate that Pyk2 is a critical regulator of PI3K function downstream of the TCR., (© Society for Leukocyte Biology.)

T cells control the focus and extent of adaptive immunity in infectious and pathological diseases. The activation of T cells occurs when the T cell antigen receptor (TCR) and costimulatory and/or adhesion receptors are engaged by their ligands. This process drives signaling that promotes cytoskeletal rearrangement and transcription factor activation, both of which regulate the quality and magnitude of the T cell response. However, it is not fully understood how different receptor-induced signals combine to alter T cell activation. The related non-receptor tyrosine kinases focal adhesion kinase (FAK) and proline-rich tyrosine kinase 2 (Pyk2) are phosphorylated downstream of the TCR and several costimulatory and adhesion receptors. FAK family proteins integrate receptor-mediated signals that influence actin cytoskeletal rearrangement and effector T cell responses. In this review, we summarize the receptor-specific roles that FAK and Pyk2 control to influence T cell development and activation.

Dendritic cells (DCs) capture and process antigens in peripheral tissues, migrate to lymphoid tissues, and present the antigens to T cells. PTPN12, also known as PTP-PEST, is an intracellular protein tyrosine phosphatase (PTP) involved in cell-cell and cell-substratum interactions. Herein, we examined the role of PTPN12 in DCs, using a genetically engineered mouse lacking PTPN12 in DCs. Our data indicated that PTPN12 was not necessary for DC differentiation, DC maturation, or cytokine production in response to inflammatory stimuli. However, it was needed for full induction of T cell-dependent immune responses in vivo. This function largely correlated with the need of PTPN12 for DC migration from peripheral sites to secondary lymphoid tissues. Loss of PTPN12 in DCs resulted in hyperphosphorylation of the protein tyrosine kinase Pyk2 and its substrate, the adaptor paxillin. Pharmacological inhibition of Pyk2 or downregulation of Pyk2 expression also compromised DC migration, suggesting that Pyk2 deregulation played a pivotal role in the migration defect caused by PTPN12 deficiency. Together, these findings identified PTPN12 as a key regulator in the ability of DCs to induce antigen-induced T cell responses. This is due primarily to the role of PTPN12 in DC migration from peripheral sites to secondary lymphoid organs through regulation of Pyk2.

It is well known that allo-reactive T cells play a crucial role in graft-versus-leukemia and graft-versus-host disease after allogeneic hematopoietic stem cell transplantation (alloSCT). Allo-reactive CD4(+) T cells can mediate direct cytolysis, but may also stimulate production of IgG antibodies as helper cells. Immune complexes may subsequently be processed and presented by professional antigen presenting cells and stimulate induction of specific CD8(+) T cells. As such, proteins targeted in coordinated T- and B-cell responses may represent a class of immunodominant antigens in clinical responses after alloSCT. We previously identified LB-PTK2B-1T as HLA class II restricted polymorphic antigen in a patient treated with donor lymphocyte infusion for relapsed chronic myeloid leukemia after HLA-matched alloSCT. Since PTK2B has also been described as antibody target, we here investigated whether a coordinated T- and B-cell response against PTK2B was induced. Patient serum before and after alloSCT and donor lymphocyte infusion (DLI) was screened for antibodies, and we indeed observed development of a humoral immune response against PTK2B. Antibodies against PTK2B were only found after DLI and, in contrast to the CD4(+) T cells, recognized a monomorphic region of the protein. To our knowledge, this is the first description of a coordinated allo-reactive CD4(+) T-cell and auto-reactive antibody response against an autosomal antigen.

The role of tyrosine phosphatase Src homology region 2 domain-containing phosphatase (SHP)-1 in LPS-activated cytokine production and inflammation was investigated by determining TNF-α and IL-10 production in splenic macrophages employing SHP-1-null (me/me) mouse model. LPS-stimulated me/me splenic macrophages secreted significantly less IL-10 with concomitantly elevated levels of TNF-α compared with wild-type (WT) macrophages irrespective of LPS dose and duration of stimulation. IL-10 significantly inhibited LPS-induced TNF-α production in both me/me and WT macrophages. The critical requirement for SHP-1 in regulating LPS-induced IL-10 and TNF-α production was confirmed by interfering with SHP-1 expression in WT macrophages and by reconstituting me/me macrophages with the SHP-1 gene. To delineate the role of SHP-1 in positive regulation of LPS-induced IL-10 production, signaling proteins representing SHP-1 targets were examined. The results reveal that tyrosine kinases Src and proline-rich tyrosine kinase 2 (Pyk2) regulate SHP-1-dependent LPS-induced IL-10 production and infer that optimal LPS-induced IL-10 production requires an assembly of a protein complex consisting of SHP-1-Pyk2-Src proteins. Moreover, LPS-induced IL-10 production also requires activation of the p38 MAPK independent of SHP-1 function. Overall, to our knowledge our results show for the first time that SHP-1 acts as a positive regulator of LPS-induced IL-10 production in splenic macrophages through two distinct and independent SHP-1-Pyk2-Src and p38 MAPK pathways.

Nucleotide-binding domain leucine-rich repeat (NLR) protein complexes sense infections and trigger robust immune responses in plants and humans. Activation of plant NLR resistance (R) proteins by pathogen effectors launches convergent immune responses, including programmed cell death (PCD), reactive oxygen species (ROS) production and transcriptional reprogramming with elusive mechanisms. Functional genomic and biochemical genetic screens identified six closely related Arabidopsis Ca²⁺-dependent protein kinases (CPKs) in mediating bifurcate immune responses activated by NLR proteins, RPS2 and RPM1. The dynamics of differential CPK1/2 activation by pathogen effectors controls the onset of cell death. Sustained CPK4/5/6/11 activation directly phosphorylates a specific subgroup of WRKY transcription factors, WRKY8/28/48, to synergistically regulate transcriptional reprogramming crucial for NLR-dependent restriction of pathogen growth, whereas CPK1/2/4/11 phosphorylate plasma membrane-resident NADPH oxidases for ROS production. Our studies delineate bifurcation of complex signaling mechanisms downstream of NLR immune sensors mediated by the myriad action of CPKs with distinct substrate specificity and subcellular dynamics.

Mycobacterium avium subsp. paratuberculosis is the causative agent of Johne's disease in cattle. The complex, multifaceted interaction of M. avium subsp. paratuberculosis with its host includes dampening the ability of infected cells to respond to stimuli that promote M. avium subsp. paratuberculosis clearance. By disrupting host defenses, M. avium subsp. paratuberculosis creates an intracellular environment that favors the establishment and maintenance of infection. Toll-like receptors (TLRs) are important sensors that initiate innate immune responses to microbial challenge and are also immunotherapeutic targets. For example, TLR9 contributes to host defense against M. avium subsp. paratuberculosis, and its agonists (CpG oligodeoxynucleotides [ODNs]) are under investigation for treatment of Johne's disease and other infections. Here we demonstrate that M. avium subsp. paratuberculosis infection changes the responsiveness of bovine monocytes to TLR9 stimulation. M. avium subsp. paratuberculosis inhibits classical TLR9-mediated responses despite a 10-fold increase in TLR9 expression and maintained uptake of CpG ODNs. Other TLR9-mediated responses, such as oxidative burst, which occur through noncanonical signaling, remain functional. Kinome analysis verifies that classic TLR9 signaling is blocked by M. avium subsp. paratuberculosis infection and that signaling instead proceeds through a Pyk2-mediated mechanism. Pyk2-mediated signaling does not hinder infection, as CpG ODNs fail to promote M. avium subsp. paratuberculosis clearance. Indeed, Pyk2 signaling appears to be an important aspect of M. avium subsp. paratuberculosis infection, as Pyk2 inhibitors significantly reduce the number of intracellular M. avium subsp. paratuberculosis bacteria. The actions of M. avium subsp. paratuberculosis on TLR9 signaling may represent a strategy to generate a host environment which is better suited for infection, revealing potential new targets for therapeutic intervention.

The adaptors DOCK8 and MyD88 have been linked to serological memory. Here we report that DOCK8-deficient patients had impaired antibody responses and considerably fewer CD27(+) memory B cells. B cell proliferation and immunoglobulin production driven by Toll-like receptor 9 (TLR9) were considerably lower in DOCK8-deficient B cells, but those driven by the costimulatory molecule CD40 were not. In contrast, TLR9-driven expression of AICDA (which encodes the cytidine deaminase AID), the immunoglobulin receptor CD23 and the costimulatory molecule CD86 and activation of the transcription factor NF-κB, the kinase p38 and the GTPase Rac1 were intact. DOCK8 associated constitutively with MyD88 and the tyrosine kinase Pyk2 in normal B cells. After ligation of TLR9, DOCK8 became tyrosine-phosphorylated by Pyk2, bound the Src-family kinase Lyn and linked TLR9 to a Src-kinase Syk-transcription factor STAT3 cascade essential for TLR9-driven B cell proliferation and differentiation. Thus, DOCK8 functions as an adaptor in a TLR9-MyD88 signaling pathway in B cells.

Cell-mediated transmission and dissemination of sexually-acquired human immunodeficiency virus 1 (HIV-1) in the host involves the migration of immature dendritic cells (iDCs). iDCs migrate in response to the HIV-1 envelope protein, gp120, and inhibiting such migration may limit the mucosal transmission of HIV-1. In this study, we elucidated the mechanism of HIV-1-gp120-induced transendothelial migration of iDCs. We found that gp120 enhanced the binding of Wiskott-Aldrich Syndrome protein (WASp) and the Actin-Related Protein 2/3 (Arp2/3) complex with β-actin, an interaction essential for the proper formation of podosomes, specialized adhesion structures required for the migration of iDCs through different tissues. We further identified Leukocyte-Specific Protein 1 (LSP1) as a novel component of the WASp-Arp2/3-β-actin complex. Pretreating iDCs with an active fragment of the secretory glycoprotein Slit2 (Slit2N) inhibited HIV-1-gp120-mediated migration and podosome formation, by inducing the cognate receptor Roundabout 1 (Robo1) to bind to and sequester WASp and LSP1 from β-actin. Slit2N treatment also inhibited Src signaling and the activation of several downstream molecules, including Rac1, Pyk2, paxillin, and CDC42, a major regulator of podosome formation. Taken together, our results support a novel mechanism by which Slit2/Robo1 may inhibit the HIV-1-gp120-induced migration of iDCs, thereby restricting dissemination of HIV-1 from mucosal surfaces in the host.

Pyk2 is a non-receptor tyrosine kinase that regulates cellular adhesion. We generated antibodies to a peptide corresponding to the N-terminus (NT) of Pyk2 and another to a portion of the C-terminal (CT) domain. Only the CT antiserum recovered paxillin-associated Pyk2. These antibodies recognized overlapping but biochemically distinct molecular species of Pyk2 since the CT antiserum recovered Pyk2 after NT antibody immunodepletion. Furthermore, the CT antibody could not immunoblot NT antibody-captured Pyk2. Phosphorylation partially accounts for the differential binding of these antibodies as dephosphorylation of Pyk2 recovered with the NT antibodies allows for recognition by the CT antibody. Additionally, Pyk2 recovered with the NT antibody displays increased serine/threonine phosphorylation. We suggest that the NT epitope is inaccessible to the antibody because Pyk2 is in a closed confirmation in association with paxillin. Upon induction of serine and/or threonine phosphorylation of Pyk2, it opens to a confirmation that allows for antibody binding to the NT epitope but at the same time no longer binds paxillin or the CT antiserum. These antibodies also display differential staining of Pyk2 in both T cells and macrophages. Pyk2 recognized by the CT antibody, but not the NT antibody, colocalized with paxillin at the microtubule-organizing center (MTOC). The MTOC-bound Pyk2 was not tyrosine phosphorylated upon T cell activation. We hypothesize that a reservoir of primarily inactive Pyk2 associates with paxillin at the MTOC, which may allow for rapid delivery of Pyk2 to specific sites of adhesion., (Copyright © 2010 Elsevier Inc. All rights reserved.)

T-cell interactions with antigen-presenting cells are important for CD8 T-cell effector or memory fate determination. The integrin leukocyte function-associated antigen-1 (LFA-1) mediates T-cell adhesion but the contribution of LFA-1-induced signaling pathways to T-cell responses is poorly understood. Here we demonstrate that proline-rich tyrosine kinase-2 (PYK2) deficiency impairs CD8 T-cell activation by synergistic LFA-1 and T-cell receptor stimulation. Furthermore, PYK2 is essential for LFA-1-mediated CD8 T-cell adhesion and LFA-1 costimulation of CD8 T-cell migration. During lymphocytic choriomeningitis virus infection in vivo, PYK2 deficiency results in a specific loss of short-lived effector CD8 T cells but does not affect memory-precursor CD8 T-cell development. Similarly, lack of LFA-1 primarily impairs the generation of short-lived effector cells. Thus, PYK2 facilitates LFA-1-dependent CD8 T-cell responses and promotes CD8 T-cell short-lived effector fate, suggesting that PYK2 may be an interesting therapeutic target to suppress exacerbated CD8 T-cell responses.

PTP-PEST (encoded by Ptpn12) is an intracellular protein tyrosine phosphatase belonging to the same family as LYP. LYP inhibits secondary T cell responses by suppressing Src family protein tyrosine kinases and is implicated in human autoimmunity. To determine the function of PTP-PEST in T cells, we generated mice with a conditionally deleted allele of Ptpn12. By removing PTP-PEST in T cells, we determined that PTP-PEST was not necessary for T cell development or primary responses. However, PTP-PEST was required for secondary T cell responses, anergy prevention, and autoimmunity induction. PTP-PEST specifically regulated the phosphorylation of Pyk2, a substrate of the Src family kinase Fyn. It also promoted the formation of T cell homoaggregates, which are known to enhance T cell activation. Thus, PTP-PEST controls Pyk2 activity and is a positive regulator of secondary T cell activation. These data illustrate the critical role of protein tyrosine phosphatases in T cell regulation., (Copyright 2010 Elsevier Inc. All rights reserved.)

The tyrosine kinase Pyk2 is vital for integrating receptor-mediated signals controlling adhesion and motility in neuronal, epithelial, and hematopoietic cell types. In T cells, the stimulation of the TCR and costimulatory, chemokine, cytokine, and integrin receptors leads to the phosphorylation of Pyk2 and the induction of its catalytic activity. However, our understanding of the mechanism of the TCR-induced, site-specific phosphorylation of this kinase is incomplete and contradictory. To address this issue, the role of individual signaling pathways in the phosphorylation of Pyk2 tyrosines 402 and 580 upon TCR activation was assessed in human T cells. In contrast to other receptor systems, the TCR-induced phosphorylation of Pyk2 tyrosines 402 and 580 was dependent on the Src family kinases, Fyn or Lck. Interestingly, the TCR-mediated phosphorylation of Pyk2 tyrosines 402 and 580 did not require Ca(2+) influx, ZAP-70 activation, actin cytoskeleton rearrangement, or PI3K function. These observations are different than other receptor systems, which require the induction of one or more of these pathways. Together, these data have defined more fully the mechanism for the TCR-induced phosphorylation of specific sites on Pyk2, suggesting that the TCR has a distinct pathway for the activation of Pyk2 compared with other receptor systems.

The tyrosine kinase Pyk2 plays a unique role in intracellular signal transduction by linking Ca(2+) influx to tyrosine phosphorylation, but the molecular mechanism of Pyk2 activation is unknown. We report that Pyk2 oligomerization by antibodies in vitro or overexpression of PSD-95 in PC6-3 cells induces trans-autophosphorylation of Tyr402, the first step in Pyk2 activation. In neurons, Ca(2+) influx through NMDA-type glutamate receptors causes postsynaptic clustering and autophosphorylation of endogenous Pyk2 via Ca(2+)- and calmodulin-stimulated binding to PSD-95. Accordingly, Ca(2+) influx promotes oligomerization and thereby autoactivation of Pyk2 by stimulating its interaction with PSD-95. We show that this mechanism of Pyk2 activation is critical for long-term potentiation in the hippocampus CA1 region, which is thought to underlie learning and memory.

Potent graft-versus-leukemia (GVL) effects can be mediated by donor-derived T cells recognizing minor histocompatibility antigens (mHags) in patients treated with donor lymphocyte infusion (DLI) for relapsed hematologic malignancies after HLA-matched allogeneic stem cell transplantation (alloSCT). Donor-derived T cells, however, may not only induce GVL, but also mediate detrimental graft-versus-host disease (GVHD). Because HLA-class II is under noninflammatory conditions predominantly expressed on hematopoietic cells, CD4+ T cells administered late after alloSCT may selectively confer GVL without GVHD. Although a broad range of different HLA-class I-restricted mHags have been identified, the first 2 autosomal HLA-class II-restricted mHags have only recently been characterized. By screening a recombinant bacteria cDNA expression library, we identified 4 new HLA-class II-restricted mHags recognized by CD4+ T cells induced in a patient with relapsed chronic myeloid leukemia who achieved long-term complete remission and experienced only mild GVHD of the skin after DLI. All CD4+ T cells were capable of recognizing the mHags presented by HLA-DR surface molecules on primary hematopoietic cells, but not on skin-derived (cytokine-treated) fibroblasts. The selective recognition of hematopoietic cells as well as the balanced population frequencies and common HLA-DR restriction elements make the novel mHags possible targets for development of immunotherapeutic strategies.

MIP-1beta/CCL4 is a principal regulator of macrophage migration and signals through CCR5. Several protein kinases are linked to CCR5 in macrophages including the src kinase Lyn, PI3K, focal adhesion related kinase Pyk2, and members of the MAPK family, but whether and how these kinases regulate macrophage chemotaxis are not known. To define the role of these signaling molecules, we examined the functions and interactions of endogenous proteins in primary human macrophages. Using siRNA gene silencing and pharmacologic inhibition, we show that chemotaxis in response to CCR5 stimulation by MIP-1beta requires activation of Pyk2, PI3K p85, and Lyn, as well as MAPK ERK. MIP-1beta activation of CCR5 triggered translocation of Pyk2 and PI3K p85 from the cytoplasm to colocalize with Lyn at the plasma membrane with formation of a multimolecular complex. We show further that arrestins were recruited into the complex, and arrestin down-regulation impaired complex formation and macrophage chemotaxis toward MIP-1beta. Together, these results identify a novel mechanism of chemokine receptor regulation of chemotaxis and suggest that arrestins may serve as scaffolding proteins linking CCR5 to multiple downstream signaling molecules in a biologically important primary human cell type.

The activity of tyrosine kinases is central to many cellular processes, and accumulating evidence suggests that their role in inflammation is no less profound. Three main tyrosine kinase families, the Src, Tec and Syk kinase families are intimately involved in TLR signalling, the critical first step in cellular recognition of invading pathogens and tissue damage. Their activity results in changes in gene expression in affected cells. Key amongst these genes are the cytokines, which orchestrate both the duration and extent of inflammation. Tyrosine kinases also play important roles in cytokine function, and are implicated in signalling through both pro- and anti-inflammatory cytokines such as TNF, IL-6 and IL-10. Thus, strategies to modulate tyrosine kinase activity have significant therapeutic potential in combating the chronic inflammatory state that is typical of many major health issues that face us today, including Rheumatoid Arthritis, Cardiovascular disease and cancer. Here we review current knowledge of the role of tyrosine kinases in inflammation with particular emphasis on their role in TLR signalling.

Bacterial endotoxin (lipopolysaccharide or LPS) has potent pro-inflammatory properties and acts on many cell types including endothelial cells. Secretion of the CC chemokine, MCP-1 (CCL2) by LPS-activated endothelial cells contributes substantially to the pathogenesis of sepsis. However, the mechanism involved in LPS-induced MCP-1 production in endothelial cells is not well understood. Using human microvascular endothelial cells (HMVEC), we analyzed the involvement of the non-receptor tyrosine kinase, Pyk2, in LPS-mediated MCP-1 production. There was a marked activation of the non-receptor tyrosine kinase, Pyk2, in response to LPS. Inhibition of Pyk2 activity using a pharmacological inhibitor, Tyrphostin A9 significantly attenuated LPS-induced Pyk2 tyrosine phosphorylation, p38 MAP kinase (MAPK) activation, NF-kappaB activation, and MCP-1 expression. Furthermore, specific inactivation of Pyk2 activity by transducing microvascular endothelial cells with catalytically inactive Pyk2 mutant (AAV-Pyk2MT) or Pyk2-specific siRNA significantly blocked LPS-induced MCP-1 production. The supernatants of these LPS-stimulated cells with attenuated Pyk2 activity demonstrated decreased trans-endothelial monocyte migration in comparison to LPS-treated controls, thus confirming the inhibition of functional MCP-1 production. In summary, our data suggest a critical role for the Pyk2 mediated pathway involving p38 MAP kinase and NF-kappaB in LPS-induced MCP-1 production in human microvascular endothelial cells.

Secretion of proinflammatory cytokines by LPS activated endothelial cells contributes substantially to the pathogenesis of sepsis. However, the mechanism involved in this process is not well understood. In the present study, we determined the role of a nonreceptor proline-rich tyrosine kinase, Pyk2, in LPS-induced IL-8 (CXCL8) production in endothelial cells. First, we observed a marked activation of Pyk2 in response to LPS. Furthermore, inhibition of Pyk2 activity in these cells by transduction with the catalytically inactive Pyk2 mutant, transfection with Pyk2-specific small interfering RNA, or treatment with Tyrphostin A9 significantly blocked LPS-induced IL-8 production. The supernatants of LPS-stimulated cells exhibiting attenuated Pyk2 activity blocked transendothelial neutrophil migration in comparison to the supernatants of LPS-treated controls, thus confirming the inhibition of functional IL-8 production. Investigations into the molecular mechanism of this pathway revealed that LPS activates Pyk2 leading to IL-8 production through the TLR4. In addition, we identified the p38 MAPK pathway to be a critical step downstream of Pyk2 during LPS-induced IL-8 production. Taken together, these results demonstrate a novel role for Pyk2 in LPS-induced IL-8 production in endothelial cells.

Src tyrosine kinases (TKs) are signaling proteins involved in cell signaling pathways toward cytoskeletal, membrane and nuclear targets. In the present study, using a selective Src TK inhibitor, PP1, we investigated the roles of Src TKs in the key pulmonary responses, NF-kappaB activation, and integrin signaling during acute lung injury in BALB/C mice intratracheally treated with LPS. LPS resulted in c-Src phosphorylation in lung tissue and the phospho-c-Src was predominantly localized in recruited neutrophils and alveolar macrophages. PP1 inhibited LPS-induced increases in total protein content in bronchoalveolar lavage fluid, neutrophil recruitment, and increases in the production or activity of TNF-alpha and matrix metalloproteinase-9. PP1 also blocked LPS-induced NF-kappaB activation, and phosphorylation and degradation of IkappaB-alpha. The inhibition of NF-kappaB activation by PP1 correlated with a depression of LPS-induced integrin signaling, which included increases in the phosphorylations of integrin beta(3), and of the focal adhesion kinase (FAK) family members, FAK and Pyk2, in lung tissue, and reductions in the fibrinogen-binding activity of alveolar macrophages. Moreover, treatment with anti-alpha(v), anti-beta(3), or Arg-Gly-Asp-Ser (RGDS), inhibited LPS-induced NF-kappaB activation. Taken together, our findings suggest that Src TKs play a critical role in LPS-induced activations of NF-kappaB and integrin (alpha(v)beta(3)) signaling during acute lung injury. Therefore, Src TK inhibition may provide a potential means of ameliorating inflammatory cascade-associated lung injury.

We have previously demonstrated that binding of ephrin-A1 to Eph receptors on human CD4+ T cells stimulates migration. Here, we show that a distinct population of CD8+ T lymphocytes, expressing the chemokine receptor CCR7, also binds ephrin-A1 and is stimulated to migrate after binding. The Eph receptor signaling pathway taking part in the migration event was here investigated. Induced tyrosine phosphorylation of several proteins was seen after ephrin-A1 binding. In particular, induced phosphorylation and kinase activity of the Src kinase family member Lck was observed. An Lck inhibitor inhibited ephrin-A1-induced migration, indicating the involvement of Lck in the migration event. In addition, we observed an induced association of the focal adhesion-like kinase proline-rich tyrosine kinase 2 (Pyk2) and the guanidine exchange factor Vav1 with Lck. PI3K inhibitors also inhibited migration, and studies in transfectants indicate an association of PI3K with EphA1. Further, ephrin-A1-induced migration could be related to the activation of Rho GTPases. This was also observed by using an inhibitor of the Rho-associated kinase ROCK, a downstream effector of Rho. Our results suggest that stimulation of Eph receptors on CD8+CCR7+ T cells leads to migration involving activation of Lck, Pyk2, PI3K, Vav1 and Rho GTPase.

Microfilaments (actin filaments) regulate various dynamic events during meiotic maturation. Relatively, little is known about the regulation of microfilament organization in mammalian oocytes. Proline-rich tyrosine kinase2 (Pyk2), a protein tyrosine kinase related to focal adhesion kinase (FAK) is essential in actin filaments organization. The present study was to examine the expression and localization of Pyk2, and in particular, its function during rat oocyte maturation. For the first time, by using Western blot and confocal laser scanning microscopy, we detected the expression of Pyk2 in rat oocytes and found that Pyk2 and Try402 phospho-Pyk2 were localized uniformly at the cell cortex and surrounded the germinal vesicle (GV) or the condensed chromosomes at the GV stage or after GV breakdown. At the metaphase and the beginning of anaphase, Pyk2 distributed asymmetrically both in the ooplasm and the cortex with a marked staining associated with the chromosomes and the region overlying the meiotic spindle. At telophase, Pyk2 was observed in the cleavage furrows in addition to its cortex and cytoplasm localization. The dynamics of Pyk2 were similar to that of F-actin, and this kinase was found to co-localize with microfilaments in several developmental stages during rat oocyte maturation. Microinjection of Pyk2 antibody demolished the microfilaments assembly and also inhibited the first polar body (PB1) emission. These findings suggest an important role of Pyk2 for rat oocyte maturation by regulating the organization of actin filaments.