Distinct Pathways Regulate Syk Protein Activation Downstream of Immune Tyrosine Activation Motif (ITAM) and hemITAM Receptors in Platelets

Tyrosine kinase pathways are known to play an important role in the activation of platelets. In particular, the GPVI and CLEC-2 receptors are known to activate Syk upon tyrosine phosphorylation of an immune tyrosine activation motif (ITAM) and hemITAM, respectively. However, unlike GPVI, the CLEC-2 receptor contains only one tyrosine motif in the intracellular domain. The mechanisms by which this receptor activates Syk are not completely understood. In this study, we identified a novel signaling mechanism in CLEC-2-mediated Syk activation. CLEC-2-mediated, but not GPVI-mediated, platelet activation and Syk phosphorylation were abolished by inhibition of PI3K, which demonstrates that PI3K regulates Syk downstream of CLEC-2. Ibrutinib, a Tec family kinase inhibitor, also completely abolished CLEC-2-mediated aggregation and Syk phosphorylation in human and murine platelets. Furthermore, embryos lacking both Btk and Tec exhibited cutaneous edema associated with blood-filled vessels in a typical lymphatic pattern similar to CLEC-2 or Syk-deficient embryos. Thus, our data show, for the first time, that PI3K and Tec family kinases play a crucial role in the regulation of platelet activation and Syk phosphorylation downstream of the CLEC-2 receptor.     

Syk and Src Family Kinases Regulate C-type Lectin Receptor 2 (CLEC-2)-mediated Clustering of Podoplanin and Platelet Adhesion to Lymphatic Endothelial Cells

The interaction of C-type lectin receptor 2 (CLEC-2) on platelets with Podoplanin on lymphatic endothelial cells initiates platelet signaling events that are necessary for prevention of blood-lymph mixing during development. In the present study, we show that CLEC-2 signaling via Src family and Syk tyrosine kinases promotes platelet adhesion to primary mouse lymphatic endothelial cells at low shear. Using supported lipid bilayers containing mobile Podoplanin, we further show that activation of Src and Syk in platelets promotes clustering of CLEC-2 and Podoplanin. Clusters of CLEC-2-bound Podoplanin migrate rapidly to the center of the platelet to form a single structure. Fluorescence lifetime imaging demonstrates that molecules within these clusters are within 10 nm of one another and that the clusters are disrupted by inhibition of Src and Syk family kinases. CLEC-2 clusters are also seen in platelets adhered to immobilized Podoplanin using direct stochastic optical reconstruction microscopy. These findings provide mechanistic insight by which CLEC-2 signaling promotes adhesion to Podoplanin and regulation of Podoplanin signaling, thereby contributing to lymphatic vasculature development.     

Phosphorylation on Syk Y342 is important for both ITAM and hemITAM signaling in platelets

Immune cells express receptors bearing an immune tyrosine activation motif (ITAM) containing two YXXL motifs or hemITAMs containing only one YXXL motif. Phosphorylation of the ITAM/hemITAM is mediated by Src family kinases allowing for the binding and activation of spleen tyrosine kinase (Syk). It is believed that Syk must be phosphorylated on tyrosine residues for activation, and Tyr342, а conserved tyrosine in the interdomain B region, has been shown to be critical for regulating Syk in FcεR1-activated mast cells. Syk is a key mediator of signaling pathways downstream of several platelet pathways including the ITAM bearing glycoprotein VI (GPVI)/Fc receptor gamma chain collagen receptor and the hemITAM containing C-type lectin-like receptor-2 (CLEC-2). Since platelet activation is a crucial step in both hemostasis and thrombosis, we evaluated the importance of Syk Y342 in these processes by producing an Syk Y342F knock-in mouse. When using a CLEC-2 antibody as an agonist, reduced aggregation and secretion were observed in Syk Y342F mouse platelets when compared with control mouse platelets. Platelet reactivity was also reduced in response to the GPVI agonist collagen-related peptide. Signaling initiated by either GPVI or CLEC-2 was also greatly inhibited, including Syk Y519/520 phosphorylation. Hemostasis, as measured by tail bleeding time, was not altered in Syk Y342F mice, but thrombus formation in response to FeCl₃ injury was prolonged in Syk Y342F mice. These data demonstrate that phosphorylation of Y342 on Syk following stimulation of either GPVI or CLEC-2 receptors is important for the ability of Syk to transduce a signal.     

Critical Role for an Acidic Amino Acid Region in Platelet Signaling by the HemITAM (Hemi-immunoreceptor Tyrosine-based Activation Motif) Containing Receptor CLEC-2 (C-type Lectin Receptor-2)

CLEC-2 is a member of new family of C-type lectin receptors characterized by a cytosolic YXXL downstream of three acidic amino acids in a sequence known as a hemITAM (hemi-immunoreceptor tyrosine-based activation motif). Dimerization of two phosphorylated CLEC-2 molecules leads to recruitment of the tyrosine kinase Syk via its tandem SH2 domains and initiation of a downstream signaling cascade. Using Syk-deficient and Zap-70-deficient cell lines we show that hemITAM signaling is restricted to Syk and that the upstream triacidic amino acid sequence is required for signaling. Using surface plasmon resonance and phosphorylation studies, we demonstrate that the triacidic amino acids are required for phosphorylation of the YXXL. These results further emphasize the distinct nature of the proximal events in signaling by hemITAM relative to ITAM receptors.     

Tyrosine Phosphorylation on Spleen Tyrosine Kinase (Syk) Is Differentially Regulated in Human and Murine Platelets by Protein Kinase C Isoforms

Protein kinase C (PKC) isoforms differentially regulate platelet functional responses downstream of glycoprotein VI (GPVI) signaling, but the role of PKCs regulating upstream effectors such as Syk is not known. We investigated the role of PKC on Syk tyrosine phosphorylation using the pan-PKC inhibitor GF109203X (GFX). GPVI-mediated phosphorylation on Syk Tyr-323, Tyr-352, and Tyr-525/526 was rapidly dephosphorylated, but GFX treatment inhibited this dephosphorylation on Tyr-525/526 in human platelets but not in wild type murine platelets. GFX treatment did not affect tyrosine phosphorylation on FcRγ chain or Src family kinases. Phosphorylation of Lat Tyr-191 and PLCγ2 Tyr-759 was also increased upon treatment with GFX. We evaluated whether secreted ADP is required for such dephosphorylation. Exogenous addition of ADP to GFX-treated platelets did not affect tyrosine phosphorylation on Syk. FcγRIIA- or CLEC-2-mediated Syk tyrosine phosphorylation was also potentiated with GFX in human platelets. Because potentiation of Syk phosphorylation is not observed in murine platelets, PKC-deficient mice cannot be used to identify the PKC isoform regulating Syk phosphorylation. We therefore used selective inhibitors of PKC isoforms. Only PKCβ inhibition resulted in Syk hyperphosphorylation similar to that in platelets treated with GFX. This result indicates that PKCβ is the isoform responsible for Syk negative regulation in human platelets. In conclusion, we have elucidated a novel pathway of Syk regulation by PKCβ in human platelets.     

Rhodocytin induces platelet aggregation by interacting with glycoprotein Ia/IIa (GPIa/IIa, Integrin alpha 2beta 1). Involvement of GPIa/IIa-associated src and protein tyrosine phosphorylation

Although glycoprotein Ia/IIa (GPIa/IIa, integrin alpha(2)beta(1)) has established its role as a collagen receptor, it remains unclear whether GPIa/IIa mediates activation signals. In this study, we show that rhodocytin, purified from the Calloselasma rhodostoma venom, induces platelet aggregation, which can be blocked by anti-GPIa monoclonal antibodies. Studies with rhodocytin-coupled beads and liposomes loaded with recombinant GPIa/IIa demonstrated that rhodocytin directly binds to GPIa/IIa independently of divalent cations. In vitro kinase assays and Western blotting of GPIa immunoprecipitates revealed that Src and Lyn constitutively associate with GPIa/IIa and that Src activity increases transiently after rhodocytin stimulation. Src specifically associates with p130 Crk-associated substrate (Cas) in a manner dependent upon Cas phosphorylation, suggesting that Src is responsible for Cas tyrosine phosphorylation. While all these phenomena occur early after rhodocytin stimulation in a cAMP-resistant manner, tyrosine phosphorylation of Syk and phospholipase Cgamma2, intracellular Ca(2+) mobilization, and platelet aggregation occur later in a cAMP-sensitive manner. Cytochalasin D, which interferes with actin polymerization and blocks receptor clustering, inhibits all the rhodocytin-mediated signals we examined in this study. We suggest that rhodocytin, by clustering GPIa/IIa, activates GPIa/IIa-associated Src, which then mediates downstream activation signals.     

Fucoidan Is a Novel Platelet Agonist for the C-type Lectin-like Receptor 2 (CLEC-2)

Fucoidan, a sulfated polysaccharide from Fucus vesiculosus, decreases bleeding time and clotting time in hemophilia, possibly through inhibition of tissue factor pathway inhibitor. However, its effect on platelets and the receptor by which fucoidan induces cellular processes has not been elucidated. In this study, we demonstrate that fucoidan induces platelet activation in a concentration-dependent manner. Fucoidan-induced platelet activation was completely abolished by the pan-Src family kinase (SFK) inhibitor, PP2, or when Syk is inhibited. PP2 abolished phosphorylations of Syk and Phospholipase C-γ2. Fucoidan-induced platelet activation had a lag phase, which is reminiscent of platelet activation by collagen and CLEC-2 receptor agonists. Platelet activation by fucoidan was only slightly inhibited in FcRγ-chain null mice, indicating that fucoidan was not acting primarily through GPVI receptor. On the other hand, fucoidan-induced platelet activation was inhibited in platelet-specific CLEC-2 knock-out murine platelets revealing CLEC-2 as a physiological target of fucoidan. Thus, our data show fucoidan as a novel CLEC-2 receptor agonist that activates platelets through a SFK-dependent signaling pathway. Furthermore, the efficacy of fucoidan in hemophilia raises the possibility that decreased bleeding times could be achieved through activation of platelets.     

Essential in vivo roles of the platelet activation receptor CLEC-2 in tumour metastasis, lymphangiogenesis and thrombus formation

We have recently identified C-type lectin-like receptor 2 (CLEC-2) as a receptor for the platelet activating snake venom rhodocytin. CLEC-2 elicits powerful platelet activation signals in conjunction with single YxxL motif in its cytoplasmic tail, Src, Syk kinases, and phospholipase Cγ2. An endogenous ligand of CLEC-2 has been identified as podoplanin, which is a membrane protein of tumour cells and facilitates tumour metastasis by inducing platelet activation. Studies of CLEC-2-deficient mice have revealed several physiological roles of CLEC-2. Podoplanin is also expressed in lymphatic endothelial cells. In the developmental stages, when the primary lymph sac is derived from the cardinal vein, podoplanin activates platelets in lymphatic endothelial cells, which facilitates blood/lymphatic vessel separation. Moreover, CLEC-2 is involved in thrombus stabilization under flow conditions in part through homophilic interactions. The absence of CLEC-2 does not significantly increase bleeding tendency, implying that CLEC-2 may be a good target protein for anti-platelet drugs in addition to anti-metastatic drugs.     

Activation of Lyn Tyrosine Kinase through Decreased Membrane Cholesterol Levels during a Change in Its Membrane Distribution upon Cell Detachment

Cellular membranes, which can serve as scaffolds for signal transduction, dynamically change their characteristics upon cell detachment. Src family kinases undergo post-translational lipid modification and are involved in a wide range of signaling events at the plasma membrane, such as cell proliferation, cell adhesion, and survival. Previously, we showed the differential membrane distributions among the members of Src family kinases by sucrose density gradient fractionation. However, little is known about the regulation of the membrane distribution of Src family kinases upon cell detachment. Here, we show that cell detachment shifts the main peak of the membrane distribution of Lyn, a member of Src family kinase, from the low density to the high density membrane fractions and enhances the kinase activity of Lyn. The change in Lyn distribution upon cell detachment involves both dynamin activity and a decrease in membrane cholesterol. Cell detachment activates Lyn through decreased membrane cholesterol levels during a change in its membrane distribution. Furthermore, cholesterol incorporation decreases Lyn activity and reduces the viability of suspension cells. These results suggest that cell detachment-induced Lyn activation through the change in the membrane distribution of Lyn plays an important role in survival of suspension cells.     

Functional analysis of Csk in signal transduction through the B-cell antigen receptor.

In B cells, two classes of protein tyrosine kinases (PTKs), the Src family of PTKs (Lyn, Fyn, Lck, and Blk) and non-Src family of PTKs (Syk), are known to be involved in signal transduction induced by the stimulation of the B-cell antigen receptor (BCR). Previous studies using Lyn-negative chicken B-cell clones revealed that Lyn is necessary for transduction of signals through the BCR. The kinase activity of the Src family of PTKs is negatively regulated by phosphorylation at the C-terminal tyrosine residue, and the PTK Csk has been demonstrated to phosphorylate this C-terminal residue of the Src family of PTKs. To investigate the role of Csk in BCR signaling, Csk-negative chicken B-cell clones were generated. In these Csk-negative cells, Lyn became constitutively active and highly phosphorylated at the autophosphorylation site, indicating that Csk is necessary to sustain Lyn in an inactive state. Since the C-terminal tyrosine phosphorylation of Lyn is barely detectable in the unstimulated, wild-type B cells, our data suggest that the activities of Csk and a certain protein tyrosine phosphatase(s) are balanced to maintain Lyn at a hypophosphorylated and inactive state. Moreover, we show that the kinase activity of Syk was also constitutively activated in Csk-negative cells. The degree of activation of both the Lyn and Syk kinases in Csk-negative cells was comparable to that observed in wild-type cells after BCR stimulation. However, BCR stimulation was still necessary in Csk-negative cells to elicit tyrosine phosphorylation of cellular proteins, as well as calcium mobilization and inositol 1,4,5-trisphosphate generation. These results suggest that not only activation of the Lyn and Syk kinases but also additional signals induced by the cross-linking of the BCR are required for full transduction of BCR signaling.     

Structure-Function Analysis of Lyn Kinase Association with Lipid Rafts and Initiation of Early Signaling Events after Fcɛ Receptor I Aggregation

The first step in immunoreceptor signaling is represented by ligand-dependent receptor aggregation, followed by receptor phosphorylation mediated by tyrosine kinases of the Src family. Recently, sphingolipid- and cholesterol-rich plasma membrane microdomains, called lipid rafts, have been identified and proposed to function as platforms where signal transduction molecules may interact with the aggregated immunoreceptors. Here we show that aggregation of the receptors with high affinity for immunoglobulin E (FcepsilonRI) in mast cells is accompanied by a co-redistribution of the Src family kinase Lyn. The co-redistribution requires Lyn dual fatty acylation, Src homology 2 (SH2) and/or SH3 domains, and Lyn kinase activity, in cis or in trans. Palmitoylation site-mutated Lyn, which is anchored to the plasma membrane but exhibits reduced sublocalization into lipid rafts, initiates the tyrosine phosphorylation of FcepsilonRI subunits, Syk protein tyrosine kinase, and the linker for activation of T cells, along with an increase in the concentration of intracellular Ca(2+). However, Lyn mutated in both the palmitoylation and myristoylation sites does not anchor to the plasma membrane and is incapable of initiating FcepsilonRI phosphorylation and early signaling events. These data, together with our finding that a constitutively tyrosine-phosphorylated FcepsilonRI does not exhibit an increased association with lipid rafts, suggest that FcepsilonRI phosphorylation and early activation events can be initiated outside of lipid rafts.     

Tyrosine phosphorylation of SLP-76 is downstream of Syk following stimulation of the collagen receptor in platelets

Collagen-related peptide (CRP), a collagen homologue, induces platelet activation through a tyrosine kinase-dependent pathway, leading to sequential tyrosine phosphorylation of Fc receptor (FcR) gamma-chain, Syk, and phospholipase C-gamma2. Here we report that CRP and the platelet low affinity immune receptor FcgammaRIIA stimulate tyrosine phosphorylation of the T cell adapter SLP-76, whereas the G protein-coupled receptor agonist thrombin induces only minor tyrosine phosphorylation. This suggests that SLP-76 has a specific role downstream of receptors that signal via an immunoreceptor tyrosine-based activation motif. Immunoprecipitation studies demonstrate association of SLP-76 with SLAP-130, Vav, Fyn, Lyn, and the FcR gamma-chain in CRP-stimulated platelets. Several of these proteins, including SLP-76, undergo tyrosine phosphorylation in in vitro kinase assays performed on SLP-76 immunoprecipitates. Tyrosine phosphorylation of all of these proteins in the in vitro kinase assay was abrogated by the Src family kinase inhibitor PP1, suggesting that it is mediated by either Fyn or Lyn. The physiological significance of this is uncertain, however, since tyrosine phosphorylation of SLP-76 in vivo is not altered in either Fyn- or Lyn-deficient platelets. CRP stimulation of Syk-deficient platelets demonstrated that in vivo tyrosine phosphorylation of SLP-76 is downstream of Syk. The absence of Syk in the SLP-76 immunoprecipitates raises the possibility that another protein is responsible for bringing SLP-76 to Syk. Candidates for this include those proteins that co-immunoprecipitate with SLP-76, including the FcR gamma-chain. Tyrosine phosphorylation of PLC-gamma2 and Ca2+ mobilization is markedly attenuated in SLP-76-deficient platelets following CRP stimulation, suggesting that the adapter plays a critical role in the regulation of the phospholipase. The increase in tyrosine phosphorylation of SLAP-130 in response to CRP is also inhibited in SLP-76-deficient platelets, placing it downstream of SLP-76. This work identifies SLP-76 as an important adapter molecule that is regulated by Syk and lies upstream of SLAP-130 and PLC-gamma2 in CRP-stimulated platelets.     

Striatal-enriched protein-tyrosine phosphatase (STEP) regulates Pyk2 kinase activity

Proline-rich tyrosine kinase 2 (Pyk2) is a member of the focal adhesion kinase family and is highly expressed in brain and hematopoietic cells. Pyk2 plays diverse functions in cells, including the regulation of cell adhesion, migration, and cytoskeletal reorganization. In the brain, it is involved in the induction of long term potentiation through regulation of N-methyl-d-aspartate receptor trafficking. This occurs through the phosphorylation and activation of Src family tyrosine kinase members, such as Fyn, that phosphorylate GluN2B at Tyr(1472). Phosphorylation at this site leads to exocytosis of GluN1-GluN2B receptors to synaptic membranes. Pyk2 activity is modulated by phosphorylation at several critical tyrosine sites, including Tyr(402). In this study, we report that Pyk2 is a substrate of striatal-enriched protein-tyrosine phosphatase (STEP). STEP binds to and dephosphorylates Pyk2 at Tyr(402). STEP KO mice showed enhanced phosphorylation of Pyk2 at Tyr(402) and of the Pyk2 substrates paxillin and ASAP1. Functional studies indicated that STEP opposes Pyk2 activation after KCl depolarization of cortical slices and blocks Pyk2 translocation to postsynaptic densities, a key step required for Pyk2 activation and function. This is the first study to identify Pyk2 as a substrate for STEP.     

Syk regulates multiple signaling pathways leading to CX3CL1 chemotaxis in macrophages

Several studies have clearly established the importance of the interaction between macrophages and CX3CL1 in the progression of disease. A previous study demonstrated that Syk was required for CX3CL1-mediated actin polymerization and chemotaxis. Here, we delineated the signaling cascade of Syk-mediated cell migration in response to CX3CL1. Inhibition of Syk in bone marrow-derived macrophages or reduction of Syk expression using siRNA in RAW/LR5 cells indicated that Syk was required for the activation of PI3K, Cdc42, and Rac1. Also, reduction in WASP or WAVE2 levels, common downstream effectors of Cdc42 or Rac1, resulted in impaired cell migration to CX3CL1. Syk indirectly regulated WASP tyrosine phosphorylation through Cdc42 activation. Altogether, our data identify that Syk mediated chemotaxis toward CX3CL1 by regulating both Rac1/WAVE2 and Cdc42/WASP pathways, whereas Src family kinases were required for proper WASP tyrosine phosphorylation.     

Differential involvement of Src family kinases in Fc gamma receptor-mediated phagocytosis

The tyrosine phosphorylation cascade originated from Fc gamma receptors (Fc gamma Rs) is essential for macrophage functions including phagocytosis. Although the initial step is ascribed to Src family tyrosine kinases, the role of individual kinases in phagocytosis signaling is still to be determined. In reconstitution experiments, we first showed that expression in the RAW 264.7 cell line of C-terminal Src kinase (Csk) inhibited and that of a membrane-anchored, gain-of-function Csk abolished the Fc gamma R-mediated signaling that leads to phagocytosis in a kinase-dependent manner. We next tested reconstruction of the signaling in the membrane-anchored, gain-of-function Csk-expressing cells by introducing Src family kinases the C-terminal negative regulatory sequence of which was replaced with a c-myc epitope. Those constructs derived from Lyn and Hck (a-Lyn and a-Hck) that associated with detergent-resistant membranes successfully reconstructed Fc gamma R-mediated Syk activation, filamentous actin rearrangement, and phagocytosis. In contrast, c-Src-derived construct (a-Src), that was excluded from detergent-resistant membranes, could not restore the series of phagocytosis signaling. Tyrosine phosphorylation of Vav and c-Cbl was restored in common by a-Lyn, a-Hck, and a-Src, but Fc gamma RIIB tyrosine phosphorylation, which is implicated in negative signaling, was reconstituted solely by a-Lyn and a-Hck. These findings suggest that Src family kinases are differentially involved in Fc gamma R-signaling and that selective kinases including Lyn and Hck are able to fully transduce phagocytotic signaling.     

Phosphorylation of Cyclin-dependent Kinase 5 (Cdk5) at Tyr-15 Is Inhibited by Cdk5 Activators and Does Not Contribute to the Activation of Cdk5

Cdk5 is a member of the cyclin-dependent kinase (Cdk) family. In contrast to other Cdks that promote cell proliferation, Cdk5 plays a role in regulating various neuronal functions, including neuronal migration, synaptic activity, and neuron death. Cdks responsible for cell proliferation need phosphorylation in the activation loop for activation in addition to binding a regulatory subunit cyclin. Cdk5, however, is activated only by binding to its activator, p35 or p39. Furthermore, in contrast to Cdk1 and Cdk2, which are inhibited by phosphorylation at Tyr-15, the kinase activity of Cdk5 is reported to be stimulated when phosphorylated at Tyr-15 by Src family kinases or receptor-type tyrosine kinases. We investigated the activation mechanism of Cdk5 by phosphorylation at Tyr-15. Unexpectedly, however, it was found that Tyr-15 phosphorylation occurred only on monomeric Cdk5, and the coexpression of activators, p35/p25, p39, or Cyclin I, inhibited the phosphorylation. In neuron cultures, too, the activation of Fyn tyrosine kinase did not increase Tyr-15 phosphorylation of Cdk5. Further, phospho-Cdk5 at Tyr-15 was not detected in the p35-bound Cdk5. In contrast, expression of active Fyn increased p35 in neurons. These results indicate that phosphorylation at Tyr-15 is not an activation mechanism of Cdk5 but, rather, indicate that tyrosine kinases could activate Cdk5 by increasing the protein amount of p35. These results call for reinvestigation of how Cdk5 is regulated downstream of Src family kinases or receptor tyrosine kinases in neurons, which is an important signaling cascade in a variety of neuronal activities.