The p85 subunit of phosphatidylinositol 3-kinase associates with the Fc receptor gamma-chain and linker for activitor of T cells (LAT) in platelets stimulated by collagen and convulxin.

There is extensive evidence to show that phosphatidylinositol 3-kinase plays an important role in signaling by the immune family of receptors, which has recently been extended to include the platelet collagen receptor, glycoprotein VI. In this report we present two potential mechanisms for the regulation of this enzyme on stimulation of platelets by collagen. We show that on stimulation with collagen, the regulatory subunit of phosphatidylinositol 3-kinase associates with the tyrosine-phosphorylated form of the adapter protein linker for activator of T Cells (LAT) and the tyrosine-phosphorylated immunoreceptor tyrosine-based activation motif of the Fc receptor gamma-chain (a component of the collagen receptor complex that includes glycoprotein VI). The associations of the Fc receptor gamma-chain and LAT with p85 are rapid and supported by the Src-homology 2 domains of the regulatory subunit. We did not obtain evidence to support previous observations that the regulatory subunit of phosphatidylinositol 3-kinase is regulated through association with the tyrosine kinase Syk. The present results provide a molecular basis for the regulation of the p85/110 form of phosphatidylinositol 3-kinase by GPVI, the collagen receptor that underlies activation.

Protease-activated receptor 2 sensitizes the capsaicin receptor transient receptor potential vanilloid receptor 1 to induce hyperalgesia

Inflammatory proteases (mast cell tryptase and trypsins) cleave protease-activated receptor 2 (PAR2) on spinal afferent neurons and cause persistent inflammation and hyperalgesia by unknown mechanisms. We determined whether transient receptor potential vanilloid receptor 1 (TRPV1), a cation channel activated by capsaicin, protons, and noxious heat, mediates PAR2-induced hyperalgesia. PAR2 was coexpressed with TRPV1 in small- to medium-diameter neurons of the dorsal root ganglia (DRG), as determined by immunofluorescence. PAR2 agonists increased intracellular [Ca2+] ([Ca2+]i) in these neurons in culture, and PAR2-responsive neurons also responded to the TRPV1 agonist capsaicin, confirming coexpression of PAR2 and TRPV1. PAR2 agonists potentiated capsaicin-induced increases in [Ca2+]i in TRPV1-transfected human embryonic kidney (HEK) cells and DRG neurons and potentiated capsaicin-induced currents in DRG neurons. Inhibitors of phospholipase C and protein kinase C (PKC) suppressed PAR2-induced sensitization of TRPV1-mediated changes in [Ca2+]i and TRPV1 currents. Activation of PAR2 or PKC induced phosphorylation of TRPV1 in HEK cells, suggesting a direct regulation of the channel. Intraplantar injection of a PAR2 agonist caused persistent thermal hyperalgesia that was prevented by antagonism or deletion of TRPV1. Coinjection of nonhyperalgesic doses of PAR2 agonist and capsaicin induced hyperalgesia that was inhibited by deletion of TRPV1 or antagonism of PKC. PAR2 activation also potentiated capsaicin-induced release of substance P and calcitonin gene-related peptide from superfused segments of the dorsal horn of the spinal cord, where they mediate hyperalgesia. We have identified a novel mechanism by which proteases that activate PAR2 sensitize TRPV1 through PKC. Antagonism of PAR2, TRPV1, or PKC may abrogate protease-induced thermal hyperalgesia.

Enhanced hepatitis C virus genome replication and lipid accumulation mediated by inhibition of AMP-activated protein kinase

Hepatitis C virus (HCV) infection is associated with dysregulation of both lipid and glucose metabolism. As well as contributing to viral replication, these perturbations influence the pathogenesis associated with the virus, including steatosis, insulin resistance, and type 2 diabetes. AMP-activated protein kinase (AMPK) plays a key role in regulation of both lipid and glucose metabolism. We show here that, in cells either infected with HCV or harboring an HCV subgenomic replicon, phosphorylation of AMPK at threonine 172 and concomitant AMPK activity are dramatically reduced. We demonstrate that this effect is mediated by activation of the serine/threonine kinase, protein kinase B, which inhibits AMPK by phosphorylating serine 485. The physiological significance of this inhibition is demonstrated by the observation that pharmacological restoration of AMPK activity not only abrogates the lipid accumulation observed in virus-infected and subgenomic replicon-harboring cells but also efficiently inhibits viral replication. These data demonstrate that inhibition of AMPK is required for HCV replication and that the restoration of AMPK activity may present a target for much needed anti-HCV therapies.

MICAL-1 is a negative regulator of MST-NDR kinase signaling and apoptosis.

MICALs (molecules interacting with CasL) are atypical multidomain flavoenzymes with diverse cellular functions. The molecular pathways employed by MICAL proteins to exert their cellular effects remain largely uncharacterized. Via an unbiased proteomics approach, we identify MICAL-1 as a binding partner of NDR (nuclear Dbf2-related) kinases. NDR1/2 kinases are known to mediate apoptosis downstream of the mammalian Ste-20-like kinase MST1, and ablation of NDR1 in mice predisposes the mice to cancer as a result of compromised apoptosis. MST1 phosphorylates NDR1/2 kinases at their hydrophobic motif, thereby facilitating full NDR kinase activity and function. However, if and how this key phosphorylation event is regulated are unknown. Here we show that MICAL-1 interacts with the hydrophobic motif of NDR1/2 and that overexpression or knockdown of MICAL-1 reduces or augments NDR kinase activation or activity, respectively. Surprisingly, MICAL-1 is a phosphoprotein but not an NDR or MST1 substrate. Rather, MICAL-1 competes with MST1 for NDR binding and thereby antagonizes MST1-induced NDR activation. In line with this inhibitory effect, overexpression or knockdown of MICAL-1 inhibits or enhances, respectively, NDR-dependent proapoptotic signaling induced by extrinsic stimuli. Our findings unveil a previously unknown biological role for MICAL-1 in apoptosis and define a novel negative regulatory mechanism of MST-NDR signaling.

Platelet endothelial cell adhesion molecule-1 inhibits platelet response to thrombin and von Willebrand factor by regulating the internalization of glycoprotein Ib via AKT/glycogen synthase kinase-3/dynamin and integrin αIIbβ3

OBJECTIVE: Platelet endothelial cell adhesion molecule-1 (PECAM-1) regulates platelet response to multiple agonists. How this immunoreceptor tyrosine-based inhibitory motif-containing receptor inhibits G protein-coupled receptor-mediated thrombin-induced activation of platelets is unknown. APPROACH AND RESULTS: Here, we show that the activation of PECAM-1 inhibits fibrinogen binding to integrin αIIbβ3 and P-selectin surface expression in response to thrombin (0.1-3 U/mL) but not thrombin receptor-activating peptides SFLLRN (3×10(-7)-1×10(-5) mol/L) and GYPGQV (3×10(-6)-1×10(-4) mol/L). We hypothesized a role for PECAM-1 in reducing the tethering of thrombin to glycoprotein Ibα (GPIbα) on the platelet surface. We show that PECAM-1 signaling regulates the binding of fluorescein isothiocyanate-labeled thrombin to the platelet surface and reduces the levels of cell surface GPIbα by promoting its internalization, while concomitantly reducing the binding of platelets to von Willebrand factor under flow in vitro. PECAM-1-mediated internalization of GPIbα was reduced in the presence of both EGTA and cytochalasin D or latrunculin, but not either individually, and was reduced in mice in which tyrosines 747 and 759 of the cytoplasmic tail of β3 integrin were mutated to phenylalanine. Furthermore, PECAM-1 cross-linking led to a significant reduction in the phosphorylation of glycogen synthase kinase-3β Ser(9), but interestingly an increase in glycogen synthase kinase-3α pSer(21). PECAM-1-mediated internalization of GPIbα was reduced by inhibitors of dynamin (Dynasore) and glycogen synthase kinase-3 (CHIR99021), an effect that was enhanced in the presence of EGTA. CONCLUSIONS: PECAM-1 mediates internalization of GPIbα in platelets through dual AKT/protein kinase B/glycogen synthase kinase-3/dynamin-dependent and αIIbβ3-dependent mechanisms. These findings expand our understanding of how PECAM-1 regulates nonimmunoreceptor signaling pathways and helps to explains how PECAM-1 regulates thrombosis.

GRID and docking analyses reveal a molecular basis for flavonoid inhibition of src-family kinase activity

Flavonoids reduce cardiovascular disease risk through anti-inflammatory, anti-coagulant and anti-platelet actions. One key flavonoid inhibitory mechanism is blocking kinase activity that drives these processes. Flavonoids attenuate activities of kinases including phosphoinositide-3-kinase (PI3K), Fyn, Lyn, Src, Syk, PKC, PIM1/2, ERK, JNK, and PKA. X-ray crystallographic analyses of kinase-flavonoid complexes show that flavonoid ring systems and their hydroxyl substitutions are important structural features for their binding to kinases. A clearer understanding of structural interactions of flavonoids with kinases is necessary to allow construction of more potent and selective counterparts. We examined flavonoid (quercetin, apigenin and catechin) interactions with Src-family kinases (Lyn, Fyn and Hck) applying the Sybyl docking algorithm and GRID. A homology model (Lyn) was used in our analyses to demonstrate that high quality predicted kinase structures are suitable for flavonoid computational studies. Our docking results revealed potential hydrogen bond contacts between flavonoid hydroxyls and kinase catalytic site residues. Identification of plausible contacts indicated that quercetin formed the most energetically stable interactions, apigenin lacked hydroxyl groups necessary for important contacts, and the non-planar structure of catechin could not support predicted hydrogen bonding patterns. GRID analysis using a hydroxyl functional group supported docking results. Based on these findings, we predicted that quercetin would inhibit activities of Src-family kinases with greater potency than apigenin and catechin. We validated this prediction using in vitro kinase assays. We conclude that our study can be used as a basis to construct virtual flavonoid interaction libraries to guide drug discovery using these compounds as molecular templates.

Cardiac protein kinases: the cardiomyocyte kinome and differential kinase expression in human failing hearts

Aims. Protein kinases are potential therapeutic targets for heart failure, but most studies of cardiac protein kinases derive from other systems, an approach that fails to account for specific kinases expressed in the heart and the contractile cardiomyocytes. We aimed to define the cardiomyocyte kinome (i.e. the protein kinases expressed in cardiomyocytes) and identify kinases with altered expression in human failing hearts. Methods and Results. Expression profiling (Affymetrix microarrays) detected >400 protein kinase mRNAs in rat neonatal ventricular myocytes (NVMs) and/or adult ventricular myocytes (AVMs), 32 and 93 of which were significantly upregulated or downregulated (>2-fold), respectively, in AVMs. Data for AGC family members were validated by qPCR. Proteomics analysis identified >180 cardiomyocyte protein kinases, with high relative expression of mitogen-activated protein kinase cascades and other known cardiomyocyte kinases (e.g. CAMKs, cAMP-dependent protein kinase). Other kinases are poorly-investigated (e.g. Slk, Stk24, Oxsr1). Expression of Akt1/2/3, BRaf, ERK1/2, Map2k1, Map3k8, Map4k4, MST1/3, p38-MAPK, PKCδ, Pkn2, Ripk1/2, Tnni3k and Zak was confirmed by immunoblotting. Relative to total protein, Map3k8 and Tnni3k were upregulated in AVMs vs NVMs. Microarray data for human hearts demonstrated variation in kinome expression that may influence responses to kinase inhibitor therapies. Furthermore, some kinases were upregulated (e.g. NRK, JAK2, STK38L) or downregulated (e.g. MAP2K1, IRAK1, STK40) in human failing hearts. Conclusions. This characterization of the spectrum of kinases expressed in cardiomyocytes and the heart (cardiomyocyte and cardiac kinomes) identified novel kinases, some of which are differentially expressed in failing human hearts and could serve as potential therapeutic targets.

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.

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.

CLEC-2 and Syk in the megakaryocytic/platelet lineage are essential for development

The C-type lectin receptor CLEC-2 signals through a pathway that is critically dependent on the tyrosine kinase Syk. We show that homozygous loss of either protein results in defects in brain vascular and lymphatic development, lung inflation, and perinatal lethality. Furthermore, we find that conditional deletion of Syk in the hematopoietic lineage, or conditional deletion of CLEC-2 or Syk in the megakaryocyte/platelet lineage, also causes defects in brain vascular and lymphatic development, although the mice are viable. In contrast, conditional deletion of Syk in other hematopoietic lineages had no effect on viability or brain vasculature and lymphatic development. We show that platelets, but not platelet releasate, modulate the migration and intercellular adhesion of lymphatic endothelial cells through a pathway that depends on CLEC-2 and Syk. These studies found that megakaryocyte/platelet expression of CLEC-2 and Syk is required for normal brain vasculature and lymphatic development and that platelet CLEC-2 and Syk directly modulate lymphatic endothelial cell behavior in vitro.

CLEC-2 is not required for platelet aggregation at arteriolar shear

The C-type lectin receptor CLEC-2 is expressed primarily on the surface of platelets, where it is present as a dimer, and is found at low level on a subpopulation of other hematopoietic cells, including mouse neutrophils [1–4] Clustering of CLEC-2 by the snake venom toxin rhodocytin, specific antibodies or its endogenous ligand, podoplanin, elicits powerful activation of platelets through a pathway that is similar to that used by the collagen receptor glycoprotein VI (GPVI) [4–6]. The cytosolic tail of CLEC-2 contains a conserved YxxL sequence preceded by three upstream acidic amino acid residues, which together form a novel motif known as a hemITAM. Ligand engagement induces tyrosine phosphorylation of the hemITAM sequence providing docking sites for the tandem-SH2 domains of the tyrosine kinase Syk across a CLEC-2 receptor dimer [3]. Tyrosine phosphorylation of Syk by Src family kinases and through autophosphorylation leads to stimulation of a downstream signaling cascade that culminates in activation of phospholipase C γ2 (PLCγ2) [4,6]. Recently, CLEC-2 has been proposed to play a major role in supporting activation of platelets at arteriolar rates of flow [1]. Injection of a CLEC-2 antibody into mice causes a sustained depletion of the C-type lectin receptor from the platelet surface [1]. The CLEC-2-depleted platelets were unresponsive to rhodocytin but underwent normal aggregation and secretion responses after stimulation of other platelet receptors, including GPVI [1]. In contrast, there was a marked decrease in aggregate formation relative to controls when CLEC-2-depleted blood was flowed at arteriolar rates of shear over collagen (1000 s−1 and 1700 s−1) [1]. Furthermore, antibody treatment significantly increased tail bleeding times and mice were unable to occlude their vessels after ferric chloride injury [1]. These data provide evidence for a critical role for CLEC-2 in supporting platelet aggregation at arteriolar rates of flow. The underlying mechanism is unclear as platelets do not express podoplanin, the only known endogenous ligand of CLEC-2. In the present study, we have investigated the role of CLEC-2 in platelet aggregation and thrombus formation using platelets from a novel mutant mouse model that lacks functional CLEC-2.

Syk-dependent phosphorylation of CLEC-2: a novel mechanism of hem-immunoreceptor tyrosine-based activation motif signalling

The C-type lectin-like receptor CLEC-2 signals via phosphorylation of a single cytoplasmic YXXL sequence known as a hem-immunoreceptor tyrosine-based activation motif (hemITAM). In this study, we show that phosphorylation of CLEC-2 by the snake toxin rhodocytin is abolished in the absence of the tyrosine kinase Syk but is not altered in the absence of the major platelet Src family kinases, Fyn, Lyn, and Src, or the tyrosine phosphatase CD148, which regulates the basal activity of Src family kinases. Further, phosphorylation of CLEC-2 by rhodocytin is not altered in the presence of the Src family kinase inhibitor PP2, even though PLCγ2 phosphorylation and platelet activation are abolished. A similar dependence of phosphorylation of CLEC-2 on Syk is also seen in response to stimulation by an IgG mAb to CLEC-2, although interestingly CLEC-2 phosphorylation is also reduced in the absence of Lyn. These results provide the first definitive evidence that Syk mediates phosphorylation of the CLEC-2 hemITAM receptor with Src family kinases playing a critical role further downstream through the regulation of Syk and other effector proteins, providing a new paradigm in signaling by YXXL-containing receptors.

GPVI and CLEC-2 in hemostasis and vascular integrity

The glycoprotein VI (GPVI)-FcR gamma-chain complex initiates powerful activation of platelets by the subendothelial matrix proteins collagen and laminin through an immunoreceptor tyrosine-based activation motif (ITAM)-regulated signaling pathway. ITAMs are characterized by two YxxL sequences separated by 6-12 amino acids and are found associated with several classes of immunoglobulin (Ig) and C-type lectin receptors in hematopoietic cells, including Fc receptors. Cross-linking of the Ig GPVI leads to phosphorylation of two conserved tyrosines in the FcR gamma-chain ITAM by Src family tyrosine kinases, followed by binding and activation of the tandem SH2 domain-containing Syk tyrosine kinase and stimulation of a downstream signaling cascade that culminates in activation of phospholipase Cgamma2 (PLCgamma2). In contrast, the C-type lectin receptor CLEC-2 mediates powerful platelet activation through Src and Syk kinases, but regulates Syk through a novel dimerization mechanism via a single YxxL motif known as a hemITAM. CLEC-2 is a receptor for podoplanin, which is expressed at high levels in several tissues, including type 1 lung alveolar cells, lymphatic endothelial cells, kidney podocytes and some tumors, but is absent from vascular endothelial cells and platelets. In this article, we compare the mechanism of platelet activation by GPVI and CLEC-2 and consider their functional roles in hemostasis and other vascular processes, including maintenance of vascular integrity, angiogenesis and lymphogenesis.

Endothelin-1 and fibroblast growth factors stimulate the mitogen-activated protein kinase signaling cascade in cardiac myocytes. The potential role of the cascade in the integration of two signaling pathways leading to myocyte hypertrophy.

Maximally effective concentrations of endothelin-1 (ET-1), acidic FGF (aFGF), or 12-O-tetradecanoylphorbol-13-acetate (TPA) activated mitogen-activated protein kinase (MAPK) by 3-4-fold in crude extracts of myocytes cultured from neonatal rat heart ventricles. Maximal activation was achieved after 5 min. Thereafter, MAPK activity stimulated by ET-1 or aFGF declined to control values within 1-2 h, whereas activation by TPA was more sustained. Two peaks of MAPK activity (a 42- and a 44-kDa MAPK) were resolved in cells exposed to ET-1 or aFGF by fast protein liquid chromatography on a Mono Q column. One major and one minor peak of MAPK kinase (MAPKK) was stimulated by ET-1 or aFGF. Cardiac myocytes expressed protein kinase C (PKC)-alpha, -delta, -epsilon and -zeta as shown immunoblotting. Exposure to 1 microM TPA for 24 h down-regulated PKC-alpha, -delta, and -epsilon, but not PKC-zeta. This maneuver wholly abolished the activation of MAPK on re-exposure to TPA but did not affect the response to aFGF. The effect of ET-1 was partially down-regulated. ET-1 stimulated phospho[3H]inositide hydrolysis 18-fold, whereas aFGF stimulated by only 30%. Agonists which initially utilize dissimilar signaling pathways may therefore converge at the level of MAPKK/MAPK and this may be relevant to the hypertrophic response of the heart.

Regulation of mitogen-activated protein kinase cascade in adult rat heart preparations in vitro.

The regulation of mitogen-activated protein kinase (MAPK) and MAPK kinase (MEK) was studied in freshly isolated adult rat heart preparations. In contrast to the situation in ventricular myocytes cultured from neonatal rat hearts, stimulation of MAPK activity by 1 mumol/L phorbol 12-myristate 13-acetate (PMA) was not consistently detectable in crude extracts. After fast protein liquid chromatography, MAPK isoforms p42MAPK and p44MAPK and two peaks of MEK were shown to be activated > 10-fold in perfused hearts or ventricular myocytes exposed to 1 mumol/L PMA for 5 minutes. The identities of MAPK or MEK were confirmed by immunoblotting and, for MAPK, by the "in-gel" myelin basic protein phosphorylation assay. In retrogradely perfused hearts, high coronary perfusion pressure (120 mm Hg for 5 minutes), norepinephrine (50 mumol/L for 5 minutes), or isoproterenol (50 mumol/L for 5 minutes) stimulated MAPK and MEK approximately 2- to 5-fold. In isolated myocytes, endothelin 1 (100 nmol/L for 5 minutes) also stimulated MAPK, but stimulation by norepinephrine or isoproterenol was difficult to detect. Immunoblotting showed that the relative abundances of MAPK and MEK protein in ventricles declined to < 20% of their postpartal abundances after 50 days. This may explain the difficulties encountered in assaying the activity of MAPK in crude extracts from adult hearts. We conclude that potentially hypertrophic agonists and interventions stimulate the MAPK cascade in adult rats and suggest that the MAPK cascade may be an important intracellular signaling pathway in this response.