Vasodilator-stimulated phosphoprotein (VASP) is phosphorylated on Ser 157 by protein kinase C-dependent and -independent mechanisms in thrombin-stimulated human platelets

Department of Pharmacology, School of Medical Sciences, University Walk, Bristol BS8 1TD, UK.
Biochemical Journal (Impact Factor: 4.4). 02/2006; 393(Pt 2):555-64. DOI: 10.1042/BJ20050796
Source: PubMed

ABSTRACT VASP (vasodilator-stimulated phosphoprotein) is an actin- and profilin-binding protein that is expressed in platelets at high levels and plays a major role in negatively regulating secretory and adhesive events in these cells. VASP is a major substrate for cAMP- and cGMP-regulated protein kinases and it has been shown to be directly phosphorylated on Ser157 by PKC (protein kinase C). In the present paper, we show that, in human platelets, VASP is phosphorylated by PKC on Ser157, but not Ser239, in response to phorbol ester stimulation, in a manner blocked by the PKC inhibitor BIM I (bisindolylmaleimide I). In response to thrombin, VASP was also phosphorylated on Ser157, but this response was only partially inhibited by BIM I, indicating PKC-dependent and -independent pathways to VASP phosphorylation by thrombin. Using inhibitors, we have ruled out the possibility that the PKC-independent pathway acts through guanylate cyclase generation of cGMP, or through a phosphoinositide 3-kinase-dependent kinase. Inhibition of Rho kinase, however, substantially reduced Ser157 VASP phosphorylation, and its effects were additive with BIM I. This implicates Rho kinase and PKC as the major kinases that phosphorylate VASP Ser157 in response to thrombin in platelets.

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Available from: Giordano Pula, Sep 25, 2015
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    • "Mitogen activated protein kinases [MAPKs (ERK, P38, and JNK)] and phosphatidylinositide 3-kinase (PI-3K)/Akt signaling pathways have been identified in agonist induced platelet activation and aggregation [17]. In contrast, cyclic nucleotide monoamine phosphates such as cyclic adenosine monophosphate (cAMP) have shown broad spectrum antiplatelet activity through vasodilator-stimulated phosphoprotein (VASP) phosphorylation [33] and subsequent integrin αIIbβ3 inactivation [24]. Therefore, agents that enhance cAMP production with subsequent VASP phosphorylation, in one hand, and inhibit MAPK and Akt phosphorylation on the other are important to prevent intravascular platelet aggregation and thrombus formation. "
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    ABSTRACT: Mushrooms possess untapped source of enormous natural compounds showing anti-inflammatory, antioxidant and anti-platelet activities. Paxillus curtisii, wild mushroom, is a rich source of curtisian E (CE) reported for neuroprotective effects; however, its anti-platelet effect was unknown. Here, therefore, we investigated the anti-platelet activity of CE in rat platelets. Curtisian E (12.5 - 200 μM) attenuated collagen (2.5 μg/ml), thrombin (0.1 U/ml) and ADP (10 μM) induced platelet aggregation in vitro. Likewise, CE diminished intracellular calcium and adenosine triphosphate (ATP) release in collagen activated platelets. Fibrinogen binding and fibronectin adhesion to platelets were also inhibited. While CE downregulated c-jun N-terminal kinase (JNK), extracellular related kinase (ERK), p38, and Akt dose dependently in collagen stimulated platelets, it upregulated intraplatelet cyclic adenosine monophosphate (cAMP) and vasodilator-stimulated-phosphoprotein (VASP) phosphorylation. Protein kinase A inhibitor (H-89) markedly inhibited p-VASP(157) protein expression, suggesting cAMP-PKA-VASP(157) pathway may mediate its anti-platelet effect and thus CE could be considered as a potential anti-thrombotic agent.
    Vascular Pharmacology 07/2013; DOI:10.1016/j.vph.2013.07.002 · 3.64 Impact Factor
    • "Additionally, Ena/VASP proteins are targeted downstream of numerous guidance receptors (Bashaw et al., 2000; Evans et al., 2007; Lebrand et al., 2004), with the misregulation of many of these implicated in cancer (Brantley-Sieders et al., 2008; Kaufmann et al., 2009; Legg et al., 2008). Members of the Ena/VASP family are also regulated by the kinases Abl and PKC (Gertler et al., 1990; Wentworth et al., 2006), which are themselves heavily implicated in cancer progression (Mauro et al., 2010; Srinivasan et al., 2008). "
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    ABSTRACT: It is seldom the primary tumour that proves fatal in cancer, with metastasis the fundamental pathological process for disease progression. Upregulation of Mena, a member of the evolutionarily conserved Ena/VASP family of actin cytoskeletal regulators, promotes metastasis and invasive motility of breast cancer cells in vivo. To complement in vitro studies of Ena/VASP function in fibroblasts, we manipulated levels of Ena, the Drosophila homologue of Mena, in migrating embryonic macrophages (haemocytes). Consistent with data from fibroblasts in vitro, Ena localises to regions of actin dynamics within migrating haemocytes, stimulates lamellipodial dynamics and positively regulates the number and length of filopodia. However, whereas Ena overexpression in fibroblasts reduces migration speeds, overexpressing Ena in haemocytes leads to a dramatic increase in migration speeds, more closely resembling the increased motility of breast cancer cells that overexpress Mena. We provide evidence that this key difference is due to spatial constraints imposed on cells within the three-dimensional environment of the embryo; this might explain how Mena can be used to promote aggressive migratory behaviour during cancer progression.
    Disease Models and Mechanisms 11/2010; 4(1):126-34. DOI:10.1242/dmm.005694 · 4.97 Impact Factor
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    • "PKCs play a critical role in platelet activation as pharmacological inhibitors of PKC inhibit aggregation and secretion by the majority of agonists [3], [4]. Many PKC substrates have been identified in platelets including components of the secretory machinery and signalling molecules [5], [6], [7], [8]. "
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    ABSTRACT: Background Increasing evidence suggests that individual isoforms of protein kinase C (PKC) play distinct roles in regulating platelet activation.Methodology/Principal FindingsIn this study, we focus on the role of two novel PKC isoforms, PKCδ and PKCε, in both mouse and human platelets. PKCδ is robustly expressed in human platelets and undergoes transient tyrosine phosphorylation upon stimulation by thrombin or the collagen receptor, GPVI, which becomes sustained in the presence of the pan-PKC inhibitor, Ro 31-8220. In mouse platelets, however, PKCδ undergoes sustained tyrosine phosphorylation upon activation. In contrast the related isoform, PKCε, is expressed at high levels in mouse but not human platelets. There is a marked inhibition in aggregation and dense granule secretion to low concentrations of GPVI agonists in mouse platelets lacking PKCε in contrast to a minor inhibition in response to G protein-coupled receptor agonists. This reduction is mediated by inhibition of tyrosine phosphorylation of the FcRγ-chain and downstream proteins, an effect also observed in wild-type mouse platelets in the presence of a PKC inhibitor.Conclusions These results demonstrate a reciprocal relationship in levels of the novel PKC isoforms δ and ε in human and mouse platelets and a selective role for PKCε in signalling through GPVI.
    PLoS ONE 11/2008; 3(11). DOI:10.1371/journal.pone.0003793 · 3.23 Impact Factor
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