CalDAG-GEFI and platelet activation

ArticleinPlatelets 21(4):239-43 · March 2010with16 Reads
DOI: 10.3109/09537101003639931 · Source: PubMed
The regulated activation of platelets is an essential component of thrombosis and hemostasis. Platelet stimulation through G protein-coupled and immunoglobulin-like receptors leads to the activation of phospholipase C (PLC) beta 2/3 and PLC gamma 2, respectively, which in turn mediate the generation of the second messengers calcium (Ca(2+)) and diacylgy-cerol (DAG). DAG is critical for protein kinase C (PKC) activation, a key event in platelet granule release and integrin activation [1]. Ca(2+) triggers many intracellular signaling processes important for various platelet responses such as integrin activation and the generation of autocrine agonists, such as ADP and TxA(2). In our recent work, we identified the guanine nucleotide exchange factor, CalDAG-GEFI, as a critical molecule in Ca(2+) signaling in platelets [2-5]. CalDAG-GEFI activates the small GTPase Rap1, a central molecular switch that drives platelet activation by directly regulating integrin-mediated aggregation [2-4, 6] and the release of autocrine agonists [5]. Here, we review key findings that highlight how one molecule, CalDAG-GEFI, coordinates various Ca(2+)-dependent platelet responses ensuring the rapid activation of platelets at the site of vascular injury.
    • "(For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) two EF hands and iv) an atypical C1 domain (Fig. 1A) [15] . Posttranslational modifications to RasGRP2 have previously been identified and are available from several databases that provide mass spectral data [21,22]. "
    [Show abstract] [Hide abstract] ABSTRACT: The dynamic regulation of ERK1 and -2 (ERK1/2) is required for precise signal transduction controlling cell proliferation, differentiation, and survival. However, the underlying mechanisms regulating the activation of ERK1/2 are not completely understood. In this study, we show that phosphorylation of RasGRP2, a guanine nucleotide exchange factor (GEF), inhibits its ability to activate the small GTPase Rap1 that ultimately leads to decreased activation of ERK1/2 in cells. ERK2 phosphorylates RasGRP2 at Ser394 located in the linker region implicated in its autoinhibition. These studies identify RasGRP2 as a novel substrate of ERK1/2 and define a negative-feedback loop that regulates the BRaf-MEK-ERK signaling cascade. This negative-feedback loop determines the amplitude and duration of active ERK1/2.
    Full-text · Article · Apr 2016
    • "The mechanism of integrin a IIb b 3 inside-out regulation is extremely complex and involves, as a key step, the activation of the small GTPase Rapb1 regulated by GEFs (mainly CalDAGGEFI) and GAP factors (including Rap1GAP2 and Rasa3). Rap1b activation modulates the interaction of several structural proteins, such as talin and kindlin, with integrin cytosolic domains, resulting in a conformational modification of the receptor that increases its affinity for fibrinogen (Guidetti and Torti, 2012; Shattil, 2009; Stefanini and Bergmeier, 2010; Stefanini et al., 2015). The adaptor protein RIAM was thought to represent an additional link between Rap1b and integrin a IIb b 3 activation, however, the recent analysis of RIAM-null mice suggested that this molecule is dispensable for an effective integrin inside-out stimulation (Stritt et al., 2015). "
    [Show abstract] [Hide abstract] ABSTRACT: Blood platelets are anucleated circulating cells that play a critical role in hemostasis and are also implicated in arterial thrombosis, a major cause of death worldwide. The biological function of platelets strongly relies in their reactiveness to a variety of extracellular agonists that regulate their adhesion to extracellular matrix at the site of vascular injury and their ability to form rapidly growing cell aggregates. Among the membrane receptors expressed on the cell surface, integrins are crucial for both platelet activation, adhesion and aggregation. Integrin affinity for specific ligands is regulated by intracellular signaling pathways activated in stimulated platelets, and, once engaged, integrins themselves generate and propagate signals inside the cells to reinforce and consolidate platelet response and thrombus formation. Phosphatidylinositol 3-Kinases (PI3Ks) have emerged as crucial players in platelet activation, and they are directly implicated in the regulation of integrin function. This review will discuss the contribution of PI3Ks in platelet integrin signaling, focusing on the role of specific members of class I PI3Ks and their downstream effector Akt on both integrin inside-out and outside-in signaling. The contribution of the PI3K/Akt pathways stimulated by integrin engagement and platelet activation in thrombus formation and stabilization will also be discussed in order to highlight the possibility to target these enzymes in effective anti-thrombotic therapeutic strategies. Copyright © 2015 Elsevier Ltd. All rights reserved.
    Full-text · Article · Jun 2015
    • "The same observation was also confirmed by analysing the immunoprecipitated proteins by immunoblotting with the anti-PKA (phospho)substrates antibody (Figure 2B). We next analysed whether the phosphorylation of CalDAG- GEFI also occurred in platelets, where this protein represents the major regulator of Rap1b activation [19,20]. Washed human platelets were treated with forskolin for increasing periods of time and endogenous CalDAG-GEFI was immunoprecipitated with a specific antibody and subsequently analysed by immunoblotting with the anti-PKA (phospho)substrates antibody. "
    [Show abstract] [Hide abstract] ABSTRACT: In blood platelets, the small GTPase Rap1b is activated by cytosolic Ca2+, and promotes integrin aIIbb3 inside-out activation and platelet aggregation. cAMP is the major inhibitor of platelet function and antagonizes Rap1b stimulation through a mechanism that remains unclear. In this work we demonstrate that the Ca2+-dependent exchange factor for Rap1b, CalDAG-GEFI, is a novel substrate for the cAMP-activated protein kinase A (PKA). CalDAG-GEFI phosphorylation occurred in intact platelets treated with the cAMP-increasing agent forskolin, and was inhibited by the PKA inhibitor H89. Purified recombinant CalDAG-GEFI was also phosphorylated in vitro by the PKA catalytic subunit. By screening a panel of specific serine to alanine mutants, we identified serine 116 and serine 586 as PKA phosphorylation sites in CalDAG-GEFI. In transfected HEK293 cells, as well as in platelets, forskolin-induced phosphorylation of CalDAG-GEFI prevented activation of Rap1b induced by the Ca2+ ionophore A23187. In platelets, this effect was associated to the inhibition of aggregation. Moreover, cAMP-mediated inhibition of Rap1b was lost in HEK293 cell transfected with a double mutant of CalDAG-GEFI unable to be phosphorylated by PKA. These results demonstrate that phosphorylation of CalDAG-GEFI by PKA affects its activity, and represents a novel mechanism for cAMP-mediated inhibition of Rap1b in platelets.
    Article · Apr 2013
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