Insulin treatment stimulates the tyrosine phosphorylation of the alpha-type 85-kDa subunit of phosphatidylinositol 3-kinase in vivo.

After adding insulin to cells overexpressing the insulin receptor, the activity of phosphatidylinositol (PI) 3-kinase in the anti-phosphotyrosine immunoprecipitates was rapidly and greatly increased. This enzyme may therefore be a substrate for the insulin receptor tyrosine kinase and may be one of the mediators of insulin signal transduction. However, it is unclear whether or not activated tyrosine kinase of the insulin receptor directly phosphorylates PI 3-kinase at tyrosine residue(s) and whether insulin stimulates the specific activity of PI 3-kinase. We reported previously that the 85-kDa subunit of purified PI 3-kinase was phosphorylated at tyrosine residue(s) by the insulin receptor in vitro. To examine the tyrosine phosphorylation of PI 3-kinase and change of its activity by insulin treatment in vivo, we used a specific antibody to the 85-kDa subunit of PI 3-kinase. The activity of PI 3-kinase in immunoprecipitates with the antibody against the p85 subunit of PI 3-kinase was increased about 3-fold by insulin treatment of cells overexpressing insulin receptors. Insulin treatment also stimulated the tyrosine, serine, and threonine phosphorylation of the alpha-type 85-kDa subunit of PI 3-kinase in vivo. Phosphatase treatment of the immunoprecipitates abolished the increase in PI 3-kinase activity. The phosphorylation(s) of the kinase itself, tyrosine phosphorylation(s) of associated protein(s), or the complex formation of the phosphorylated PI 3-kinase with associated proteins may increase the activity of PI 3-kinase.     

Nitric oxide inhibits thrombin receptor-activating peptide-induced phosphoinositide 3-kinase activity in human platelets.

Although nitric oxide (NO) has potent antiplatelet actions, the signaling pathways affected by NO in the platelet are poorly understood. Since NO can induce platelet disaggregation and phosphoinositide 3-kinase (PI3-kinase) activation renders aggregation irreversible, we tested the hypothesis that NO exerts its antiplatelet effects at least in part by inhibiting PI3-kinase. The results demonstrate that the NO donor S-nitrosoglutathione (S-NO-glutathione) inhibits the stimulation of PI3-kinase associated with tyrosine-phosphorylated proteins and of p85/PI3-kinase associated with the SRC family kinase member LYN following the exposure of platelets to thrombin receptor-activating peptide. The activation of LYN-associated PI3-kinase was unrelated to changes in the amount of PI3-kinase physically associated with LYN signaling complexes but did require the activation of LYN and other tyrosine kinases. The cyclic GMP-dependent kinase activator 8-bromo-cyclic GMP had similar effects on PI3-kinase activity, consistent with a model in which the cyclic nucleotide mediates the effects of NO. Additional studies showed that wortmannin and S-NO-glutathione have additive inhibitory effects on thrombin receptor-activating peptide-induced platelet aggregation and the surface expression of platelet activation markers. These data provide evidence of a distinct and novel mechanism for the inhibitory effects of NO on platelet function.     

p42 mitogen-activated protein kinase and p90 ribosomal S6 kinase are selectively phosphorylated and activated during thrombin-induced platelet activation and aggregation.

Human platelets provide an excellent model system for the study of phosphorylation events during signal transduction and cell adhesion. Platelets are terminally differentiated cells that exhibit rapid phosphorylation of many proteins upon agonist-induced activation and aggregation. We have sought to identify the kinases as well as the phosphorylated substrates that participate in thrombin-induced signal transduction and platelet aggregation. In this study, we have identified two forms of mitogen-activated protein kinase (MAPK), p42mapk and p44mapk, in platelets. The data demonstrate that p42mapk but not p44mapk becomes phosphorylated on serine, threonine, and tyrosine during platelet activation. Immune complex kinase assays, gel renaturation assays, and a direct assay for MAPK activity in platelet extracts all support the conclusion that p42mapk but not p44mapk shows increased kinase activity during platelet activation. The activation of p42mapk, independently of p44mapk, in platelets is unique since in other systems, both kinases are coactivated by a variety of stimuli. We also show that platelets express p90rsk, a ribosomal S6 kinase that has previously been characterized as a substrate for MAPK. p90rsk is phosphorylated on serine in resting platelets, and this phosphorylation is enhanced upon thrombin-induced platelet activation. Immune complex kinase assays demonstrate that the activity of p90rsk is markedly increased during platelet activation. Another ribosomal S6 protein kinase, p70S6K, is expressed by platelets but shows no change in kinase activity upon platelet activation with thrombin. Finally, we show that the increased phosphorylation and activity of both p42mapk and p90rsk does not require integrin-mediated platelet aggregation. Since platelets are nonproliferative cells, the signal transduction pathways that include p42mapk and p90rsk cannot lead to a mitogenic signal and instead may regulate cytoskeletal or secretory changes during platelet activation.     

Interleukin-2 receptor regulates activation of phosphatidylinositol 3-kinase.

Interleukin-2 (IL-2) stimulates proliferation of T lymphocytes and is involved in the activation of both natural killer and lymphokine-activated killer precursor cells. The intracellular messengers which mediate IL-2-dependent events have not yet been identified. IL-2 receptor is not a protein-tyrosine kinase. Activation of a cellular protein-tyrosine kinase and direct association of a protein-tyrosine kinase activity with the IL-2 receptor occurs within minutes of IL-2 stimulation. We investigated the activation of phosphatidylinositol 3-kinase (PI 3-kinase) in IL-2-mediated signal transduction using the IL-2-dependent murine T-cell line, CTLL-2, and human phytohemagglutinin-stimulated peripheral blood lymphocytes (phytohemagglutinin blasts). Within a minute following stimulation of these cells with IL-2, PI 3-kinase activity could be detected in antiphosphotyrosine (anti-P-Tyr) antibody immunoprecipitates. IL-2 triggered a direct association of PI 3-kinase with the IL-2 receptor as detected in immunoprecipitates using anti-IL-2 receptor beta chain antibody. In vivo labeled CTLL-2 cells have a time-dependent increase in D-3-phosphorylated polyphosphoinositides following stimulation with IL-2. This is the first group of second messengers identified in IL-2-mediated signal transduction.     

Heregulin-dependent activation of phosphoinositide 3-kinase and Akt via the ErbB2/ErbB3 co-receptor

The ErbB2/ErbB3 heregulin co-receptor has been shown to couple to phosphoinositide (PI) 3-kinase in a heregulin-dependent manner. The recruitment and activation of PI 3-kinase by this co-receptor is presumed to occur via its interaction with phosphorylated Tyr-Xaa-Xaa-Met (YXXM) motifs occurring in the ErbB3 C terminus. In this study, mutant ErbB3 receptor proteins expressed in COS7 cells were used to investigate PI 3-kinase-dependent signaling pathways activated by the ErbB2/ErbB3 co-receptor. We observed that a mutant ErbB3 protein with each of its six YXXM motifs containing a Tyr --> Phe substitution was unable to bind either the p85 regulatory or p110 catalytic subunit of PI 3-kinase. However, restoration of a single YXXM motif was sufficient to mediate association with the PI 3-kinase holoenzyme, although at a lower level than wild-type ErbB3. When ErbB3 YXXM motifs were restored in pairs, evidence for cooperativity between two, those incorporating Tyr-1273 and Tyr-1286, was observed. Interestingly, we have shown that an apparent association of PI 3-kinase activity with ErbB2/Neu was due to the residual presence of ErbB3 in ErbB2 immunoprecipitates. The necessity of ErbB3 association with PI 3-kinase for downstream signaling to the effector kinase Akt was also investigated. Here, the heregulin-dependent translocation of Akt to the plasma membrane and its subsequent activation was observed in intact NIH-3T3 fibroblasts. Recruitment of PI 3-kinase to ErbB3 was required for both activities, and it appeared that ErbB2 activation alone was not sufficient to activate PI 3-kinase signaling in these cells.     

Phosphatidylinositol 3'-kinase and tyrosine-phosphatase activation positively modulate Convulxin-induced platelet activation. Comparison with collagen

In this report we have studied the role of phosphatidylinositol 3'-kinase (PI3-K) and tyrosine phosphatase activation on platelet activation by Convulxin (Cvx). Wortmannin, a specific PI3-K inhibitor, and phenylarsine oxide (PAO), a sulfhydryl reagent that inhibits tyrosine phosphatase (PTPase), block Cvx-induced platelet aggregation, granule secretion, inositol phosphate production, and increase in [Ca2+]i. However, PAO does not inhibit Cvx-induced tyrosine phosphorylation of platelet proteins, including Syk and PLCgamma2, but blocked collagen-induced platelet aggregation as well as tyrosine phosphorylation of PLCgamma2. In contrast, Cvx-induced PLCgamma2 tyrosyl phosphorylation was partially inhibited by wortmannin. We conclude that (i) although Cvx and collagen activate platelets by a similar mechanism, different regulatory processes are specific to each agonist; (ii) mechanisms other than tyrosine phosphorylation regulate PLCgamma2 activity; and (iii) besides protein tyrosine kinases, PI3-K (and PTPase) positively modulate platelet activation by both Cvx and collagen, and this enzyme is required for effective transmission of GPVI-Fc receptor gamma chain signal to result in full activation and tyrosine phosphorylation of PLCgamma2 in Cvx-stimulated platelets.     

Aggretin, a C-type lectin protein, induces platelet aggregation via integrin alpha(2)beta(1) and GPIb in a phosphatidylinositol 3-kinase independent pathway.

Aggretin purified from Calloselasma rhodostoma venom was previously identified as alpha(2)beta(1) agonist in triggering platelet aggregation, and exists as a heterodimer sharing a great homologous sequence to GPIb binding proteins. We show here that binding to GPIb is also required in aggregation-inducing activity of aggretin. A2-IIE10, an anti-integrin alpha(2) monoclonal antibody, delayed platelet aggregation while agkistin, a GPIb antagonist, only slightly inhibited platelet aggregation caused by aggretin. However, the aggretin-induced platelet aggregation was completely abolished by a combination of A2-IIE10 and agkistin. Either A2-IIE10 or agkistin significantly inhibited the binding of FITC-aggretin toward fixed platelets. Aggretin and collagen induced a similar signal transduction in platelets involving a time-dependent tyrosine phosphorylation of p125(FAK) and PLCgamma2, but aggretin caused a much-delayed tyrosine-phosphorylation of PI 3-kinase compared with collagen. LY294002, a PI 3-kinase inhibitor, showed a significant inhibitory effect on collagen, but not aggretin-stimulated platelet aggregation. These findings indicate aggretin induces platelet aggregation via binding of alpha(2)beta(1) and GPIb, causing phosphorylation of p125(FAK) and PLCgamma2 leading to platelet activation without the involvement of PI 3-kinase activation.     

Leptin-mediated activation of human platelets: involvement of a leptin receptor and phosphodiesterase 3A-containing cellular signaling complex

An elevated circulating level of the adipocyte-derived satiety hormone leptin is an independent risk factor for cardiovascular disease. Because thrombus formation is a major cause of acute coronary events and leptin was shown previously to facilitate ADP-induced platelet aggregation, we chose to define the signaling events involved in leptin-mediated platelet activation. Using pharmacological, biochemical, and cell biological approaches, we show that leptin-induced platelet activation required activation of a signaling cascade that included the long form of the leptin receptor, three kinases [Janus kinase 2 (JAK2), phosphatidylinositol 3-kinase (PI3K), and protein kinase B (PKB/Akt)], the insulin receptor substrate-1 (IRS-1), and the major human platelet cAMP phosphodiesterase phosphodiesterase 3A (PDE3A). Moreover, we identify a role for an intraplatelet LEPR/JAK2/IRS-1/PI3K/PKB/PDE3A molecular complex that allows for the selective leptin-mediated activation of platelets. Our data demonstrate that leptin promotes platelet activation, provides a mechanistic basis for the prothrombotic effect of this hormone, and identifies a potentially novel therapeutic avenue to limit obesity-associated cardiovascular disease.     

Phosphatidylinositol-3-kinase is a non-tyrosine phosphorylated member of the insulin receptor signalling complex.

In rat HTC cells expressing a large number of human insulin receptors, insulin stimulated phosphatidylinositol-3-kinase (PI-3-kinase) activity. This activity was more effectively immunoprecipitated with anti-phosphotyrosine antibody (alpha-PY) than with anti-insulin receptor antibody (alpha-IR), suggesting that PI-3-kinase was not directly associated with the insulin receptor. alpha-PY immunoprecipitable PI-3 kinase activity, which was regulated by insulin, corresponded to a small pool of the total cellular PI-3-kinase activity. PI-3-kinase was not directly tyrosine phosphorylated by insulin treatment. A comparison of both catalytic activity and content of PI-3-kinase in alpha-PY immunoprecipitates indicated that after insulin treatment PI-3-kinase activity was enhanced by its association with tyrosine phosphorylated proteins. These studies suggest therefore that PI-3-kinase is a non-tyrosine phosphorylated member of the insulin receptor signalling complex.     

A novel pathway for tumor necrosis factor-alpha and ceramide signaling involving sequential activation of tyrosine kinase, p21(ras), and phosphatidylinositol 3-kinase

Treatment of confluent rat2 fibroblasts with C2-ceramide (N-acetylsphingosine), sphingomyelinase, or tumor necrosis factor-alpha (TNFalpha) increased phosphatidylinositol (PI) 3-kinase activity by 3-6-fold after 10 min. This effect of C2-ceramide depended on tyrosine kinase activity and an increase in Ras-GTP levels. Increased PI 3-kinase activity was also accompanied by its translocation to the membrane fraction, increases in tyrosine phosphorylation of the p85 subunit, and physical association with Ras. Activation of PI 3-kinase by TNFalpha, sphingomyelinase, and C2-ceramide was inhibited by tyrosine kinase inhibitors (genistein and PP1). The stimulation of PI 3-kinase by sphingomyelinase and C2-ceramide was not observed in fibroblasts expressing dominant-negative Ras (N17) and the stimulation by TNFalpha was decreased by 70%. PI 3-kinase activation by C2-ceramide was not modified by inhibitors of acidic and neutral ceramidases, and it was not observed with the relatively inactive analog, dihydro-C2-ceramide. It is proposed that activation of Ras and PI 3-kinase by ceramide can contribute to signaling effects of TNFalpha that occur downstream of sphingomyelinase activation and result in increased fibroblasts proliferation.     

Pathological shear stress stimulates the tyrosine phosphorylation of alpha-actinin associated with the glycoprotein Ib-IX complex.

Shear-induced platelet responses are triggered by VWF binding to the platelet GpIb-IX complex, and there is evidence that this ligand-receptor coupling stimulates transmembranous signaling through the cytoplasmic tail of glycoprotein (Gp) Ib alpha. To investigate the mechanism by which signaling is effected, new molecular interactions involving GpIb-IX that develop in response to pathological shearing stress were examined in intact human platelets. Exposure to shear, but not alpha-thrombin, results in the co-immunoprecipitation of the actin cross-linking protein alpha-actinin with the GpIb-IX complex. Blockers of VWF binding to GpIb alpha or actin polymerization inhibit the association of alpha-actinin with the GpIb-IX complex, but the association of alpha-actinin with the GpIb-IX complex is not affected by inhibiting VWF binding to platelet integrin alpha IIb beta 3 (GpIIb-IIIa). alpha-Actinin becomes tyrosine phosphorylated in response to pathological shear stress, and phosphorylated alpha-actinin associates with GpIb-IX. In resting platelets, class IA heterodimeric phosphatidylinositol 3-kinase (PI 3-K) and protein kinase N (PKN) associate with nonphosphorylated alpha-actinin. Shear stress causes PI 3-K to disassociate from alpha-actinin, while it stimulates PKN binding to alpha-actinin. These results demonstrate that shear-induced VWF binding to GpIb alpha causes enhanced binding of cytoskeletal alpha-actinin to GpIb-IX and suggest that alpha-actinin, perhaps through tyrosine phosphorylation, serves as an adapter for a signaling complex that could regulate VWF-induced platelet aggregation.     

Regulation of glycogen synthase kinase 3 in human platelets: a possible role in platelet function?

In this study we show that both glycogen synthase kinase 3 (GSK3) isoforms, GSK3alpha and GSK3beta, are present in human platelets and are phosphorylated on Ser(21) and Ser(9), respectively, in platelets stimulated with collagen, convulxin and thrombin. Phosphorylation of GSK3alpha/beta was dependent on phosphoinositide 3-kinase (PI3K) activity and independent of platelet aggregation, and correlated with a decrease in GSK3 activity that was preserved by pre-incubating platelets with PI3K inhibitor LY294002. Three structurally distinct GSK3 inhibitors, lithium, SB415286 and TDZD-8, were found to inhibit platelet aggregation. This implicates GSK3 as a potential regulator of platelet function.     

Integrin-dependent translocation of phosphoinositide 3-kinase to the cytoskeleton of thrombin-activated platelets involves specific interactions of p85 alpha with actin filaments and focal adhesion kinase

Thrombin-induced accumulation of phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P2) but not of PtdIns(3,4,5,)P3 is strongly correlated with the relocation to the cytoskeleton of 29% of the p85 alpha regulatory subunit of phosphoinositide 3-kinase (PtdIns 3-kinase) and is accompanied by a significant increase in PtdIns 3-kinase activity in this subcellular fraction. Actually, PtdIns(3,4)P2 accumulation and PtdIns 3-kinase, pp60c-src, and p125FAK translocations as well as aggregation were concomitant events occurring with a distinct lag after actin polymerization. The accumulation of PtdIns(3,4)P2 and the relocalization of PtdIns 3-kinase to the cytoskeleton were both dependent on tyrosine phosphorylation, integrin signaling, and aggregation. Furthermore, although p85 alpha was detected in anti- phosphotyrosine immunoprecipitates obtained from the cytoskeleton of thrombin-activated platelets, we failed to demonstrate tyrosine phosphorylation of cytoskeletal p85 alpha. Tyrphostin treatment clearly reduced its presence in this subcellular fraction, suggesting a physical interaction of p85 alpha with a phosphotyrosyl protein. These data led us to investigate the proteins that are able to interact with PtdIns 3-kinase in the cytoskeleton. We found an association of this enzyme with actin filaments: this interaction was spontaneously restored after one cycle of actin depolymerization-repolymerization in vitro. This association with F-actin appeared to be at least partly indirect, since we demonstrated a thrombin-dependent interaction of p85 alpha with a proline-rich sequence of the tyrosine-phosphorylated cytoskeletal focal adhesion kinase, p125FAK. In addition, we show that PtdIns 3-kinase is significantly activated by the p125FAK proline-rich sequence binding to the src homology 3 domain of p85 alpha subunit. This interaction may represent a new mechanism for PtdIns 3-kinase activation at very specific areas of the cell and indicates that the focal contact-like areas linked to the actin filaments play a critical role in signaling events that occur upon ligand engagement of alpha IIb/beta 3 integrin and platelet aggregation evoked by thrombin.     

A role for phosphatidylinositol 3-kinase in platelet aggregation in response to low, but not high, concentrations of PAF or thrombin

In this study we show that platelet activating factor (PAF) activates PI 3-kinase over a rapid time course that correlates closely with the aggregation response. Tyrosine kinases are involved in this response, since there is increased PI 3-kinase activity associated with tyrosine-phosphorylated proteins. PI 3-kinase inhibitors were used to probe the dependence of PAF-induced aggregation on PI 3-kinase. Both wortmannin and LY-294002 inhibited PAF-induced aggregation that correlated with PI 3-kinase inhibition only when using lower concentrations of PAF giving reversible aggregation (primary phase). Similar results were obtained with human platelets using thrombin or thrombin receptor activating peptide. The same pattern of response was observed when activation of GPIIbIIIa was assessed by flow cytometry, i.e., PI 3-kinase inhibitors blocked integrin activation only when lower concentrations of agonist were used. We suggest that PI 3-kinase is important for reversible (primary) aggregation of platelets in response to PAF or thrombin, perhaps by contributing to the 'inside-out' activation of the platelet integrin GPIIbIIIa, only when submaximal concentrations of agonists are used. The lack of effect of PI 3-kinase inhibitors, when high concentrations of agonist are used, suggests that PI 3-kinase-independent pathways contribute to aggregation under these conditions.     

Involvement of the Src kinase Lyn in phospholipase C-gamma 2 phosphorylation and phosphatidylinositol 3-kinase activation in Epo signalling

We examined the role of the Src kinase Lyn in phospholipase C-gamma 2 (PLC-gamma 2) and phosphatidylinositol (PI) 3-kinase activation in erythropoietin (Epo)-stimulated FDC-P1 cells transfected with a wild type (WT) Epo-receptor (Epo-R). We showed that two inhibitors of Src kinases, PP1 and PP2, abolish both PLC-gamma 2 tyrosine phosphorylation and PI 3-kinase activity in WT Epo-R FDC-P1 cells. We also demonstrated that Epo-phosphorylated Lyn is associated with tyrosine phosphorylated PLC-gamma 2 and PI 3-kinase in WT Epo-R FDC-P1-stimulated cells. Moreover Epo-activated Lyn phosphorylates in vitro PLC-gamma 2 immunoprecipitated from unstimulated cells. Our results suggest that the Src kinase Lyn is involved in PLC-gamma 2 phosphorylation and PI 3-kinase activation induced by Epo.     

The protein-tyrosine kinase fer associates with signaling complexes containing insulin receptor substrate-1 and phosphatidylinositol 3-kinase

In a screen for 3T3-F442A adipocyte proteins that bind SH2 domains, we isolated a cDNA encoding Fer, a nonreceptor protein-tyrosine kinase of the Fes/Fps family that contains a functional SH2 domain. A truncated splicing variant, iFer, was also cloned. iFer is devoid of both the tyrosine kinase domain and a functional SH2 domain but displays a unique 42-residue C terminus and retains the ability to form oligomers with Fer. Expression of both Fer and iFer proteins are strikingly increased upon differentiation of 3T3-L1 fibroblasts to adipocytes. Platelet-derived growth factor treatment of the cultured adipocytes caused rapid tyrosine phosphorylation of Fer and its recruitment to complexes containing platelet-derived growth factor receptor and the p85 regulatory subunit of phosphatidylinositol (PI) 3-kinase. Insulin treatment of 3T3-L1 adipocytes stimulated association of Fer with complexes containing tyrosine phosphorylated IRS-1 and PI 3-kinase but did not stimulate tyrosine phosphorylation of Fer. PI 3-kinase activity in anti-Fer immunoprecipitates was also acutely activated by insulin treatment of cultured adipocytes. These data demonstrate the presence of Fer tyrosine kinase in insulin signaling complexes, suggesting a role of Fer in insulin action.     

Integrin-dependent phosphorylation and activation of the protein tyrosine kinase pp125FAK in platelets

We have investigated mechanisms involved in integrin-mediated signal transduction in platelets by examining integrin-dependent phosphorylation and activation of a newly identified protein tyrosine kinase, pp125FAK (FAK, focal adhesion kinase). This kinase was previously shown to be localized in focal adhesions in fibroblasts, and to be phosphorylated on tyrosine in normal and Src-transformed fibroblasts. We show that thrombin and collagen activation of platelets causes an induction of tyrosine phosphorylation of pp125FAK and that pp125FAK molecules isolated from activated platelets display enhanced levels of phosphorylation in immune-complex kinase assays. pp125FAK was not phosphorylated on tyrosine after thrombin or collagen treatment of Glanzmann's thrombasthenic platelets deficient in the fibrinogen receptor GPIIb-IIIa, or of platelets pretreated with an inhibitory monoclonal antibody to GP IIb-IIIa. Fibrinogen binding to GP IIb-IIIa was not sufficient to induce pp125FAK phosphorylation because pp125FAK was not phosphorylated on tyrosine in thrombin-treated platelets that were not allowed to aggregate. These results indicate that tyrosine phosphorylation of pp125FAK is dependent on platelet aggregation mediated by fibrinogen binding to the integrin receptor GP IIb-IIIa. The induction of tyrosine phosphorylation of pp125FAK was inhibited in thrombin- and collagen-treated platelets preincubated with cytochalasin D, which prevents actin polymerization following activation. Under all of these conditions, there was a strong correlation between the induction of tyrosine phosphorylation of pp125FAK in vivo and stimulation of the phosphorylation of pp125FAK in vitro in immune-complex kinase assays. This study provides the first genetic evidence that tyrosine phosphorylation of pp125FAK is dependent on integrin-mediated events, and demonstrates that there is a strong correlation between tyrosine phosphorylation of pp125FAK in platelets, and the activation of pp125FAK-associated phosphorylating activity in vitro.     

GM1-induced activation of phosphatidylinositol 3-kinase: involvement of Trk receptors

The ganglioside GM1 promotes neuronal growth, differentiation, survival, phenotypic expression, and function restoration, by apparently interacting with neurotrophic factors and/or their receptors. In brain, GM1 activates the Trk receptors for neurotrophins and the Raf/MEK/ERK cascade in situ and in vivo . We have expanded these studies and explored whether GM1 recruits the phosphatidylinositol 3 (PI3)-kinase pathway in brain also. Incubating striatal slices with GM1 increased the activity of PI3-kinase in phosphotyrosine immunoprecipitates in a time- and concentration-dependent manner, and the response was blocked by the PI3-kinase inhibitors wortmannin and LY294002. PI3-kinase activation following GM1 was rapid and short lasting with an EC₅₀ of 5 μmol/L. There was a temporally parallel activation of the downstream PI3-kinase target Akt, which was prevented by PI3-kinase inhibition. PI3-kinase activity was found increased in Trk and Gab1 immunoprecipitates, and co-immunoprecipitation studies demonstrated the association of Trk and Gab1 after GM1 treatment. Enhanced PI3-kinase activity associated with Trk or Gab1 immunoprecipitates was blocked by the Trk inhibitor K252a. GM1 did not appear to transactivate Trk and did not alter the efflux of neurotrophins in striatal slices. Our findings suggest that GM1 induces activation of PI3-kinase that is, in part, mediated through Trk and Gab1.     

Wheat germ agglutinin-induced platelet activation via platelet endothelial cell adhesion molecule-1: involvement of rapid phospholipase C gamma 2 activation by Src family kinases.

Platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31) is a 130K transmembrane glycoprotein that belongs to the immunoglobulin gene superfamily and is expressed on the surface of hematological or vascular cells, including platelets and endothelial cells. Although the importance of this adhesion molecule in various cell-cell interactions is established, its function in platelets remains ill-defined. In the process of clarifying the mechanism by which the lectin wheat germ agglutinin (WGA) activates platelets, we unexpectedly discovered that PECAM-1 is involved in signal transduction pathways elicited by this N-acetyl-D-glucosamine (NAGlu)-reactive lectin. WGA, which is a very potent platelet stimulator, elicited a rapid surge in Syk and phospholipase C (PLC)-gamma 2 tyrosine phosphorylation and the resultant intracellular Ca(2+) mobilization; collagen, as reported, induced these responses, but in a much slower and weaker manner. WGA strongly induced tyrosine phosphorylation of a 130-140K protein, which was confirmed to be PECAM-1 by immunoprecipitation and immunodepletion studies. WGA-induced PECAM-1 tyrosine phosphorylation occurred rapidly, strongly and in a manner independent of platelet aggregation or cell-cell contact; these characteristics of PECAM-1 phosphorylation were not mimicked at all by receptor-mediated platelet agonists. In addition, WGA was found to associate with PECAM-1 itself, and anti-PECAM-1 antibody, as well as NAGlu, specifically inhibited WGA-induced platelet aggregation. In PECAM-1 immunoprecipitates, Src family tyrosine kinases existed, and a kinase activity was detected, which increased upon WGA stimulation. Furthermore, the Src family kinase inhibitor PP2 inhibited WGA-induced platelet aggregation, Ca(2+) mobilization, and PLC-gamma 2 tyrosine phosphorylation. Finally, WGA induced PECAM-1 tyrosine phosphorylation and cytoskeletal reorganization in vascular endothelial cells. Our results suggest that (i) PECAM-1 is involved in WGA-induced platelet activation, (ii) PECAM-1 clustering by WGA activates unique and strong platelet signaling pathways, leading to a rapid PLC activation via Src family kinases, and (iii) WGA is a useful tool for elucidating PECAM-1-mediated signaling with wide implications not confined to platelets.