Syk Family Research Articles

Characterization of TREM-1 Signaling in Human Neutrophils By Kinome Array and RNA-Seq

The Triggering Receptor Expressed on Myeloid Cells (TREM)-1 is a member of the Immunoglobulin superfamily and an activating receptor mainly expressed on myeloid cells. TREM-1 ligation leads to immediate activation of polymorphonuclear neutrophils (PMN) resulting in degranulation and release of reactive oxygen species as well as various effector molecules. Beyond its role in acute and chronic inflammatory processes, TREM-1 is also involved in cancer emergence and progression probably by alteration of the tumor associated neutrophils (TAN) and macrophages (TAM). Advanced information about the TREM-1 signaling cascade may reveal novel therapeutic approaches, as various kinases can specifically be targeted with kinase inhibitors. Therefore, we investigated the protein tyrosine kinome (PTK) and serine threonine kinome (STK) of human PMN after TREM-1 activation. To gain further insights beyond the signaling cascade, we performed RNAseq to validate the TREM-1 mediated activation and to reveal the TREM-1 mediated transcriptome. Purified PMN from healthy donors were stimulated with a monoclonal TREM-1 antibody compared to an isotype matched control for 20 minutes (kinome activity) or 60 minutes (RNAseq). For kinomics, PamChip arrays (PamGene) were used to quantify the kinome activity and upstream kinase analysis (UKA) was used to predict the kinase activity. The final data analysis and the UKA was performed by PamGene. A median final score (depending on the significance and the specificity of the kinase activity compared to the control group) > 1.2 was defined as specific kinase activity according to the manufacturer's recommendations. For transcriptomics, Novogene performed library preparation and sequencing, and data analysis was performed with CLC. Differentially expressed genes were defined as FDR adjusted p value ≤ 0.05 and fold change ≥2. Gene set enrichment analysis was performed with Enrichr using the libraries Reactome 2022, WikiPathway 2021 Human, and GO Biological Process 2023 and significance was defined as adjusted p value ≤ 0.05. We identified the activation of 31 PTKs and 38 STKs upon TREM-1 ligation. The kinase statistic revealed changes in the activation state of the kinases present in the TREM-1 treated cells compared to the untreated control group ranging from 2.4 to 5.5 (PTK) and from 1.2 to 1.6 (STK) suggesting more TREM-1 mediated activity alteration of the PTKs. Interestingly, there were no downregulated kinase activities after receptor ligation. Out of the PTK family, we identified ten Src family kinases as well as the Syk family member ZAP70 and other kinases such as BTK, already previously shown to abrogate TREM-1 mediated PMN activation. The RNAseq after TREM-1 ligation revealed 542 differentially expressed genes from which 102 genes were upregulated and 440 genes were downregulated. Gene set enrichment analysis revealed upregulation of genes related to nuclear events (kinase and transcription factor activaton, R-HSA-198725) and cell chemotaxis (GO:0060326) validating the activation of several kinases after TREM-1 ligation. We found increased expressions of genes involved in adipogenesis (WP236) and lung fibrosis (WP3624) suitably to the observed increased activity of Src family kinases that are described to be involved in lung fibrosis. Moreover, some upregulated genes related to NGF-stimulated transcription (R-HSA-9031628) or regulation of transcription in response to stress (GO:0043618) in which the MAPK are known to be involved. However, we did not observe clearly increased MAPK activity. Besides upregulation, TREM-1 ligation led to decreased expression of several genes involved in (positive) regulation of cytokine production involved in inflammatory response (GO:1900017 and GO:1900015) and signaling by interleukins (R-HSA-449147). Taken together, the results contribute to the knowledge about TREM-1 in inflammatory disorders and reveal approaches of its role in cancer. This study revealed various kinases possibly involved in early TREM-1 signaling of neutrophils that may be suitable targets to inhibit TREM-1 mediated cell activation. Moreover, we linked the TREM-1 mediated kinase activity to the resulting gene expression. Further studies are needed to validate the results in vitro and in vivo and and to identify suitable pharmacological inhibitors to modulate TREM-1 mediated activities in inflammatory conditions and cancer.

Spleen tyrosine kinase mediates the γδTCR signaling required for γδT cell commitment and γδT17 differentiation.

The γδT cells that produce IL-17 (γδT17 cells) play a key role in various pathophysiologic processes in host defense and homeostasis. The development of γδT cells in the thymus requires γδT cell receptor (γδTCR) signaling mediated by the spleen tyrosine kinase (Syk) family proteins, Syk and Zap70. Here, we show a critical role of Syk in the early phase of γδT cell development using mice deficient for Syk specifically in lymphoid lineage cells (Syk-conditional knockout (cKO) mice). The development of γδT cells in the Syk-cKO mice was arrested at the precursor stage where the expression of Rag genes and αβT-lineage-associated genes were retained, indicating that Syk is required for γδT-cell lineage commitment. Loss of Syk in γδT cells weakened TCR signal-induced phosphorylation of Erk and Akt, which is mandatory for the thymic development of γδT17 cells. Syk-cKO mice exhibited a loss of γδT17 cells in the thymus as well as throughout the body, and thereby are protected from γδT17-dependent psoriasis-like skin inflammation. Collectively, our results indicate that Syk is a key player in the lineage commitment of γδT cells and the priming of γδT17 cell differentiation.

ArtinM Cytotoxicity in B Cells Derived from Non-Hodgkin's Lymphoma Depends on Syk and Src Family Kinases.

Receptors on the immune cell surface have a variety of glycans that may account for the immunomodulation induced by lectins, which have a carbohydrate recognition domain (CRD) that binds to monosaccharides or oligosaccharides in a specific manner. ArtinM, a D-mannose-binding lectin obtained from Artocarpus heterophyllus, has affinity for the N-glycans core. Immunomodulation by ArtinM toward the Th1 phenotype occurs via its interaction with TLR2/CD14 N-glycans on antigen-presenting cells, as well as recognition of CD3γ N-glycans on murine CD4+ and CD8+ T cells. ArtinM exerts a cytotoxic effect on Jurkat human leukemic T-cell line and human myeloid leukemia cell line (NB4). The current study evaluated the effects of ArtinM on murine and human B cells derived from non-Hodgkin’s lymphoma. We found that murine B cells are recognized by ArtinM via the CRD, and the ArtinM stimulus did not augment the proliferation rate or production of IL-2. However, murine B cell incubation with ArtinM augmented the rate of apoptosis, and this cytotoxic effect of ArtinM was also seen in human B cell-lines sourced from non-Hodgkin’s lymphoma Raji cell line. This cytotoxic effect was inhibited by the phosphatase activity of CD45 on Lck, and the protein kinases of the Src family contribute to cell death triggered by ArtinM.

Spatial requirements for ITAM signaling in an intracellular natural killer cell model membrane

FcγRIIIa-FcεRIγ complexes, upon stimulation by antibodies, cluster to initiate intracellular signaling and activate natural killer (NK) cells. Intracellular signaling involves Lck phosphorylation of ITAMs of each monomer of a FcεRIγ homodimer in a FcγRIIIa-FcεRIγ complex and subsequent binding of two phosphotyrosines (pY) in tandem by a Syk family kinase. However, how FcR clustering triggers ITAM signaling is not resolved. Molecular modeling and dynamics (MD) simulations are applied to generate ensembles of structures of the FcγRIIIa and FcεRIγ homodimeric cytoplasmic tails of FcγRIIIa-FcεRIγ complexes based on the transmembrane helices and cytoplasmic tails spaced 120, 80, and 50 Å apart to model different extents of clustering. Site-identification by ligand competitive saturation method with Monte Carlo sampling (SILCS-MC) is used to model how Lck could phosphorylate a diversity of ITAM conformations. At 80 Å separation between FcγRIIIa-FcεRIγ complexes, Lck can perform multiple phosphorylations on individual and multiple ITAMs across complexes, including potential sequential phosphorylation events. Syk may then potentially bind the two pYs within a single ITAM in tandem in isolated FcγRIIIa-FcεRIγ complexes, as observed in CD3ε and ζ chains of T cell receptors by the Syk family kinase ZAP-70. In addition, at 50 Å separation between complexes, unique to natural killer cells over T cells, Syk could potentially bind in tandem to pYs in different ITAMs across FcγRIIIa-FcεRIγ complexes. Thus, we predict that an ensemble of spatial orientations of the ITAMS of FcγRIIIa-FcεRIγ complexes that occur upon clustering lead to ITAM phosphorylation by Lck and subsequent Syk activity thereby facilitating downstream signaling.

Complexity growth in integrable and chaotic models

We use the SYK family of models with N Majorana fermions to study the complexity of time evolution, formulated as the shortest geodesic length on the unitary group manifold between the identity and the time evolution operator, in free, integrable, and chaotic systems. Initially, the shortest geodesic follows the time evolution trajectory, and hence complexity grows linearly in time. We study how this linear growth is eventually truncated by the appearance and accumulation of conjugate points, which signal the presence of shorter geodesics intersecting the time evolution trajectory. By explicitly locating such “shortcuts” through analytical and numerical methods, we demonstrate that: (a) in the free theory, time evolution encounters conjugate points at a polynomial time; consequently complexity growth truncates at O( sqrt{N} ), and we find an explicit operator which “fast-forwards” the free N-fermion time evolution with this complexity, (b) in a class of interacting integrable theories, the complexity is upper bounded by O(poly(N)), and (c) in chaotic theories, we argue that conjugate points do not occur until exponential times O(eN), after which it becomes possible to find infinitesimally nearby geodesics which approximate the time evolution operator. Finally, we explore the notion of eigenstate complexity in free, integrable, and chaotic models.

Control of Platelet CLEC-2-Mediated Activation by Receptor Clustering and Tyrosine Kinase Signaling

Platelets are blood cells responsible for vascular integrity preservation. The activation of platelet receptor C-type lectin-like receptor II-type (CLEC-2) could partially mediate the latter function. Although this receptor is considered to be of importance for hemostasis, the rate-limiting steps of CLEC-2-induced platelet activation are not clear. Here, we aimed to investigate CLEC-2-induced platelet signal transduction using computational modeling in combination with experimental approaches. We developed a stochastic multicompartmental computational model of CLEC-2 signaling. The model described platelet activation beginning with CLEC-2 receptor clustering, followed by Syk and Src family kinase phosphorylation, determined by the cluster size. Active Syk mediated linker adaptor for T cell protein phosphorylation and membrane signalosome formation, which resulted in the activation of Bruton’s tyrosine kinase, phospholipase and phosphoinositide-3-kinase, calcium, and phosphoinositide signaling. The model parameters were assessed from published experimental data. Flow cytometry, total internal reflection fluorescence and confocal microscopy, and western blotting quantification of the protein phosphorylation were used for the assessment of the experimental dynamics of CLEC-2-induced platelet activation. Analysis of the model revealed that the CLEC-2 receptor clustering leading to the membrane-based signalosome formation is a critical element required for the accurate description of the experimental data. Both receptor clustering and signalosome formation are among the rate-limiting steps of CLEC-2-mediated platelet activation. In agreement with these predictions, the CLEC-2-induced platelet activation, but not activation mediated by G-protein-coupled receptors, was strongly dependent on temperature conditions and cholesterol depletion. Besides, the model predicted that CLEC-2-induced platelet activation results in cytosolic calcium spiking, which was confirmed by single-platelet total internal reflection fluorescence microscopy imaging. Our results suggest a refined picture of the platelet signal transduction network associated with CLEC-2. We show that tyrosine kinase activation is not the only rate-limiting step in CLEC-2-induced activation of platelets. Translocation of receptor-agonist complexes to the signaling region and linker adaptor for T cell signalosome formation in this region are limiting CLEC-2-induced activation as well.

A Phase 2 Study of the Dual SYK/JAK Inhibitor Cerdulatinib Demonstrates Good Tolerability and Clinical Response in Relapsed/Refractory Peripheral T-Cell Lymphoma and Cutaneous T-Cell Lymphoma

A Phase 2 Study of the Dual SYK/JAK Inhibitor Cerdulatinib Demonstrates Good Tolerability and Clinical Response in Relapsed/Refractory Peripheral T-Cell Lymphoma and Cutaneous T-Cell Lymphoma

Tyrosine Kinases in Autoimmune and Inflammatory Skin Diseases.

Tyrosine kinases relay signals from diverse leukocyte antigen receptors, innate immune receptors, and cytokine receptors, and therefore mediate the recruitment and activation of various leukocyte populations. Non-receptor tyrosine kinases of the Jak, Src, Syk, and Btk families play major roles in various immune-mediated disorders, and small-molecule tyrosine kinase inhibitors are emerging novel therapeutics in a number of those diseases. Autoimmune and inflammatory skin diseases represent a broad spectrum of immune-mediated diseases. Genetic and pharmacological studies in humans and mice support the role of tyrosine kinases in several inflammatory skin diseases. Atopic dermatitis and psoriasis are characterized by an inflammatory microenvironment which activates cytokine receptors coupled to the Jak-Stat signaling pathway. Jak kinases are also implicated in alopecia areata and vitiligo, skin disorders mediated by cytotoxic T lymphocytes. Genetic studies indicate a critical role for Src-family kinases and Syk in animal models of autoantibody-mediated blistering skin diseases. Here, we review the various tyrosine kinase signaling pathways and their role in various autoimmune and inflammatory skin diseases. Special emphasis will be placed on identification of potential therapeutic targets, as well as on ongoing preclinical and clinical studies for the treatment of inflammatory skin diseases by small-molecule tyrosine kinase inhibitors.

Genome-wide discovery of somatic coding and noncoding mutations in pediatric endemic and sporadic Burkitt lymphoma

AbstractAlthough generally curable with intensive chemotherapy in resource-rich settings, Burkitt lymphoma (BL) remains a deadly disease in older patients and in sub-Saharan Africa. Epstein-Barr virus (EBV) positivity is a feature in more than 90% of cases in malaria-endemic regions, and up to 30% elsewhere. However, the molecular features of BL have not been comprehensively evaluated when taking into account tumor EBV status or geographic origin. Through an integrative analysis of whole-genome and transcriptome data, we show a striking genome-wide increase in aberrant somatic hypermutation in EBV-positive tumors, supporting a link between EBV and activation-induced cytidine deaminase (AICDA) activity. In addition to identifying novel candidate BL genes such as SIN3A, USP7, and CHD8, we demonstrate that EBV-positive tumors had significantly fewer driver mutations, especially among genes with roles in apoptosis. We also found immunoglobulin variable region genes that were disproportionally used to encode clonal B-cell receptors (BCRs) in the tumors. These include IGHV4-34, known to produce autoreactive antibodies, and IGKV3-20, a feature described in other B-cell malignancies but not yet in BL. Our results suggest that tumor EBV status defines a specific BL phenotype irrespective of geographic origin, with particular molecular properties and distinct pathogenic mechanisms. The novel mutation patterns identified here imply rational use of DNA-damaging chemotherapy in some patients with BL and targeted agents such as the CDK4/6 inhibitor palbociclib in others, whereas the importance of BCR signaling in BL strengthens the potential benefit of inhibitors for PI3K, Syk, and Src family kinases among these patients.

The Novel SYK/JAK Inhibitor Cerdulatinib Demonstrates Good Tolerability and Clinical Response in a Phase 2a Study in Relapsed/Refractory Peripheral T-Cell Lymphoma and Cutaneous T-Cell Lymphoma

The Novel SYK/JAK Inhibitor Cerdulatinib Demonstrates Good Tolerability and Clinical Response in a Phase 2a Study in Relapsed/Refractory Peripheral T-Cell Lymphoma and Cutaneous T-Cell Lymphoma

Mathematical Model of Platelet Intracellular Signaling After Activation by Fucoidan

Blood platelets are the cells responsible for prevention of the blood loss. Fucoidan is a brown algae extract that is known to activate platelets via C-type lectin receptor of the second type. On the other hand, different fucoidans are now considered as perspective immunomodulators. Thus, application of fucoidan as a medicinal drug seems to be contradictory. In this work we studied activation of platelets by fucoidan in silico and in vitro. The computational model describes the behavior of the participants of the fucoidan receptor signaling cascade. The model was validated with available experimental data published earlier. In order to confirm the model predictions, the fucoidan-induced activation of platelets was assessed in flow cytometry and aggregometry experiments. The resultant model describes changes in the activity of tyrosine kinases of Syk and Sarc family and subsequent activation of phospholipase Cγ2. One of the main model prediction is a significant increase in the platelet cytosolic calcium level after the activation by fucoidan. This prediction was confirmed in the experiments. Thus, fucoidan, as a true platelet activator, cannot be applied in therapy.

The protein tyrosine phosphatase PTPN22 negatively regulates presentation of immune complex derived antigens

A C1858T single nucleotide polymorphism within PTPN22 (which encodes PTPN22R620W) is associated with an enhanced susceptibility to multiple autoimmune diseases including type 1 diabetes and rheumatoid arthritis. Many of the associated autoimmune diseases have an autoantibody component to their pathology. Fc receptors (FcRs) recognise autoantibodies when they bind to autoantigens and form immune complexes. After immune complex binding and receptor crosslinking, FcRs signal via Src and Syk family kinases, leading to antigen uptake, presentation and cytokine secretion. Ptpn22 encodes a protein tyrosine phosphatase that negatively regulates Src and Syk family kinases proximal to immunoreceptor signalling cascades. We therefore hypothesised that PTPN22 regulates immune complex stimulated FcR responses in dendritic cells (DCs). Bone marrow derived DCs (BMDCs) from wild type (WT) or Ptpn22−/− mice were pulsed with ovalbumin:anti-ovalbumin immune complexes (ova ICs). Co-culture with WT OT-II T cells revealed that ova IC pulsed Ptpn22−/− BMDCs have an enhanced capability to induce T cell proliferation. This was associated with an increased capability of Ptpn22−/− BMDCs to present immune complex derived antigens and to form ova IC dependent DC-T cell conjugates. These findings highlight PTPN22 as a regulator of FcR mediated responses and provide a link between the association of PTPN22R620W with autoantibody associated autoimmune diseases.

TULA-family proteins: Jacks of many trades and then some.

UBASH3/STS/TULA is a novel two-member family, which exerts several key regulatory effects in multiple cell types. UBASH3B/STS-1/TULA-2 is a highly active protein tyrosine phosphatase; its major target appears to be a specific regulatory site of protein tyrosine kinases of the Syk family, dephosphorylation of which inhibits Syk and Zap-70 kinases and suppresses receptor signaling mediated by these kinases. UBASH3A/STS-2/TULA exhibits substantial homology to UBASH3B/STS-1/TULA-2, but possesses only a small fraction of phosphatase activity of UBASH3B/STS-1/TULA-2, and thus, its regulatory effect may be based also on the phosphatase-independent mechanisms. Critical physiologic effects of these proteins have been demonstrated in T lymphocytes, platelets, stem cells, and other important cell types. These proteins have also been shown to play a key role in such pathologic conditions as autoimmunity, cancer, and thrombosis. The review focuses on the recent studies of this important family of cellular regulators.

Protein tyrosine phosphatase PTPN22 regulates LFA-1 dependent Th1 responses

A missense C1858T single nucleotide polymorphism within PTPN22 is a strong genetic risk factor for the development of multiple autoimmune diseases. PTPN22 encodes a protein tyrosine phosphatase that negatively regulates immuno-receptor proximal Src and Syk family kinases. Notably, PTPN22 negatively regulates kinases downstream of T-cell receptor (TCR) and LFA-1, thereby setting thresholds for T-cell activation. Alterations to the quality of TCR and LFA-1 engagement at the immune synapse and the regulation of downstream signals can have profound effects on the type of effector T-cell response induced. Here we describe how IFNγ+ Th1 responses are potentiated in Ptpn22−/− T-cells and in T-cells from mice expressing Ptpn22R619W (the mouse orthologue of the human genetic variant) as they age, or following repeated immune challenge, and explore the mechanisms contributing to the expansion of Th1 cells. Specifically, we uncover two LFA-1-ICAM dependent mechanisms; one T-cell intrinsic, and one T-cell extrinsic. Firstly, we found that in vitro anti-CD3/LFA-1 induced Th1 responses were enhanced in Ptpn22−/− T-cells compared to WT, whereas anti-CD3/anti-CD28 induced IFNy responses were similar. These data were associated with an enhanced ability of Ptpn22−/− T-cells to engage ICAM-1 at the immune synapse when incubated on planar lipid bilayers, and to form conjugates with dendritic cells. Secondly, we observed a T-cell extrinsic mechanism whereby repeated stimulation of WT OT-II T-cells with LPS and OVA323-339 pulsed Ptpn22−/− bone marrow derived dendritic cells (BMDCs) was sufficient to enhance Th1 cell development compared to WT BMDCs. Furthermore, this response could be reversed by LFA-1 blockade. Our data point to two related but distinct mechanisms by which PTPN22 regulates LFA-1 dependent signals to enhance Th1 development, highlighting how perturbations to PTPN22 function over time to regulate the balance of the immune response.

Multidomain Control Over TEC Kinase Activation State Tunes the T Cell Response.

Signaling through the T cell antigen receptor (TCR) activates a series of tyrosine kinases. Directly associated with the TCR, the SRC family kinase LCK and the SYK family kinase ZAP-70 are essential for all downstream responses to TCR stimulation. In contrast, the TEC family kinase ITK is not an obligate component of the TCR cascade. Instead, ITK functions as a tuning dial, to translate variations in TCR signal strength into differential programs of gene expression. Recent insights into TEC kinase structure have provided a view into the molecular mechanisms that generate different states of kinase activation. In resting lymphocytes, TEC kinases are autoinhibited, and multiple interactions between the regulatory and kinase domains maintain low activity. Following TCR stimulation, newly generated signaling modules compete with the autoinhibited core and shift the conformational ensemble to the fully active kinase. This multidomain control over kinase activation state provides a structural mechanism to account for ITK's ability to tune the TCR signal.

Putting on the Brakes: Regulatory Kinases and Phosphatases Maintaining B Cell Anergy.

B cell antigen receptor (BCR) signaling is a tightly regulated process governed by both positive and negative mediators/regulators to ensure appropriate responses to exogenous and autologous antigens. Upon naïve B cell recognition of antigen CD79 [the immunoreceptor tyrosine-based activation motif (ITAM)-containing signaling subunit of the BCR] is phosphorylated and recruits Src and Syk family kinases that then phosphorylate proximal intermediaries linked to downstream activating signaling circuitry. This plasma membrane localized signalosome activates PI3K leading to generation of PIP3 critical for membrane localization and activation of plecktrin homology domain-containing effectors. Conversely, in anergic B cells, chronic antigen stimulation drives biased monophosphorylation of CD79 ITAMs leading to recruitment of Lyn, but not Syk, which docks only to bi-phosphorylated ITAMS. In this context, Lyn appears to function primarily as a driver of inhibitory signaling pathways promoting the inhibition of the PI3K pathway by inositol phosphatases, SHIP-1 and PTEN, which hydrolyze PIP3 to PIP2. Lyn may also exert negative regulation of signaling through recruitment of SHP-1, a tyrosine phosphatase that dephosphorylates activating signaling molecules. Alleles of genes that encode or regulate expression of components of this axis, including SHIP-1, SHP-1, Csk/PTPn22, and Lyn, have been shown to confer risk of autoimmunity. This review will discuss functional interplay of components of this pathway and the impact of risk alleles on its function.

PTRAPs: Transmembrane adaptors in innate immune signaling.

Transmembrane adaptor proteins (TRAPs) are protein scaffolds and signaling regulators with established roles in signal-induced activation of lymphocytes. A subset of the TRAP family, the palmitoylated TRAPs (pTRAPs), are increasingly emerging with additional roles in innate immune cells. Targeted to lipid rafts, tetraspannin-enriched microdomains, and protein microclusters in membranes, pTRAP scaffolds exert spatiotemporal regulation by recruiting signaling kinases, particularly Src and Syk family members, as well as Csk, and other effectors. In this way, pTRAPs modulate signaling and influence resulting cell responses, including the selective output of inflammatory cytokines and other mediators. Here, we review studies revealing that different pTRAPs work together, often with overlapping or redundant roles, for positive and negative regulation of key innate immune pathways, including Fc receptor and pattern recognition receptor signaling. Recent findings show that pTRAPs can bind directly to innate immune receptors, in addition to other transmembrane binding partners. Thus, pTRAPs are important, multifunctional scaffolds in pathways that are fundamental to diverse innate immune responses.

Protein tyrosine phosphatase PTPN22 regulates IL-1β dependent Th17 responses by modulating dectin-1 signaling in mice.

A single nucleotide polymorphism within the PTPN22 gene is a strong genetic risk factor predisposing to the development of multiple autoimmune diseases. PTPN22 regulates Syk and Src family kinases downstream of immuno‐receptors. Fungal β‐glucan receptor dectin‐1 signals via Syk, and dectin‐1 stimulation induces arthritis in mouse models. We investigated whether PTPN22 regulates dectin‐1 dependent immune responses. Bone marrow derived dendritic cells (BMDCs) generated from C57BL/6 wild type (WT) and Ptpn22−/− mutant mice, were pulsed with OVA323‐339 and the dectin‐1 agonist curdlan and co‐cultured in vitro with OT‐II T‐cells or adoptively transferred into OT‐II mice, and T‐cell responses were determined by immunoassay. Dectin‐1 activated Ptpn22−/− BMDCs enhanced T‐cell secretion of IL‐17 in vitro and in vivo in an IL‐1β dependent manner. Immunoblotting revealed that compared to WT, dectin‐1 activated Ptpn22−/− BMDCs displayed enhanced Syk and Erk phosphorylation. Dectin‐1 activation of BMDCs expressing Ptpn22R619W (the mouse orthologue of human PTPN22R620W) also resulted in increased IL‐1β secretion and T‐cell dependent IL‐17 responses, indicating that in the context of dectin‐1 Ptpn22R619W operates as a loss‐of‐function variant. These findings highlight PTPN22 as a novel regulator of dectin‐1 signals, providing a link between genetically conferred perturbations of innate receptor signaling and the risk of autoimmune disease.

Selective Binders of the Tandem Src Homology 2 Domains in Syk and Zap70 Protein Kinases by DNA-Programmed Spatial Screening.

Members of the Syk family of tyrosine kinases arrange Src homology 2 (SH2) domains in tandem to allow the firm binding of immunoreceptor tyrosine-based interaction motifs (ITAMs). While the advantages provided by the bivalency enhanced interactions are evident, the impact on binding specificity is less-clear. For example, the spleen tyrosine kinase (Syk) and the ζ-chain-associated protein kinase (ZAP-70) recognize the consensus sequence pYXXI/L(X)6-8 pYXXI/L with near-identical nanomolar affinity. The nondiscriminatory recognition, on the one hand, poses a specificity challenge for the design of subtype selective protein binders and, on the other hand, raises the question as to how differential activation of Syk and ZAP-70 is ensured when both kinases are co-expressed. Herein, we identified the criteria for the design of binders that specifically address either the Syk or the Zap-70 tSH2 domain. Our approach is based on DNA-programmed spatial screening. Tyrosine-phosphorylated peptides containing the pYXXI/L motif were attached to oligonucleotides and aligned in tandem on a DNA template by means of nucleic acid hybridization. The distance between the pYXXI/L motifs and the orientation of strands were varied. The exploration exposed remarkably different recognition characteristics. While Syk tSH2 has a rather broad substrate scope, ZAP-70 tSH2 required a proximal arrangement of the phosphotyrosine ligands in defined strand orientation. The spatial screen led to the design of mutually selective, DNA-free binders, which discriminate Zap-70 and Syk tSH2 by 1 order of magnitude in affinity.

The Syk kinases orchestrate cerebellar granule cell tangential migration

The tyrosine kinases of the Syk family are essential components of the well-characterized immunoreceptor ITAM-based signaling pathway. However, ITAM-based signaling typically does not function in isolation. Instead, it is enmeshed in the molecular network controlling cellular adhesion and chemotaxis. Consistent with the increasing number of data involving ITAM-bearing molecules in neuronal functions, we previously depicted a role for Syk kinases in the establishment of neuronal connectivity. In the developing cerebellum, we found that Syk is essentially expressed in the granule cells (GC) and more importantly, phosphorylated on tyrosine residues representative of an active form of the kinase in tangentially migrating GC. In light of these findings, experiments were performed to establish the implication of Syk in this process. We showed that Syk state of phosphorylation is spatiotemporally regulated during GC ontogeny. Moreover, the analysis of external granular layer microexplants treated with a Syk pharmacological inhibitor together with the quantification of ectopic GC in Syk+/−; ZAP-70−/− mutant mice brought evidence of a requirement of Syk in GC tangential migration. Syk phosphorylation was induced by EphB2 engagement and locally turned down by a not yet identified factor that could in part explain the restricted pattern of Syk phosphorylation observed along GC migratory route. Whereas Syk kinase activity appeared not essential for ephrin/Eph-mediated axon extension, it might provide polarization signals required for proper nucleus translocation during GC migration. In conclusion, Syk kinase acts downstream of receptors controlling GC tangential migration.