The Ras-like guanine-nucleotide-binding protein Rap1 controls integrin ␣ IIb  3 activity and platelet aggregation. Recently, we have found that Rap1 activation can be blocked by the nitric oxide/cyclic guanosine monophosphate (NO/cGMP) signaling pathway by type 1 cGMP-dependent protein kinase (cGKI). In search of possible targets of NO/cGMP/cGKI, we studied the expression of Rap1-specific GTPaseactivating proteins (GAPs) and guanine nucleotide exchange factors (GEFs) in platelets. We could detect mRNAs for a new protein most closely related to Rap1GAP and for postsynaptic density-95 discslarge and zona occludens protein 1 (PDZ)-GEF1 and CalDAG-GEFs I and III. Using 5-rapid amplification of cDNA ends (RACE), we isolated the complete cDNA of the new GAP encoding a 715-amino acid protein, which we have termed Rap1GAP2. Rap1GAP2 is expressed in at least 3 splice variants, 2 of which are detectable in platelets. Endogenous Rap1GAP2 protein partially colocalizes with Rap1 in human platelets. In transfected cells, we show that Rap1GAP2 exhibits strong GTPase-stimulating activity toward Rap1. Rap1GAP2 is highly phosphorylated, and we have identified cGKI as a Rap1GAP2 kinase. cGKI phosphorylates Rap1GAP2 exclusively on serine 7, a residue present only in the platelet splice variants of Rap1GAP2. Phosphorylation of Rap1GAP2 by cGKI might mediate inhibitory effects of NO/cGMP on Rap1. IntroductionPlatelets are of great physiologic importance as regulators of clot formation and inflammation in the vasculature, and they have been established as major therapeutic targets in cardiovascular disease. 1 Platelets contain high levels of Rap1, a Ras-like guanine-nucleotidebinding protein. Recently, Rap1 was identified as a potent regulator of integrin function. [2][3][4][5] For example, the regulation of lymphocyte and macrophage adhesion by integrins ␣L 2 and ␣B M2 , respectively, is controlled by Rap1. 6,7 Rap1 also facilitates the activation of platelet integrin ␣ IIb  3 , which is required for fibrinogen binding and aggregation. 8,9 Recently, Rap1 was shown to control the aggregation of mouse platelets. 10 Many different platelet agonists, including thrombin, adenosine diphosphate (ADP), collagen, and thromboxane, induce Rap1 activation. 11,12 However, the exact pathways involved in Rap1 activation in platelets are unknown. Data from other cell types suggest that receptor-mediated formation of second messengers can lead to the activation of Rap1-specific guanine nucleotide exchange factors (GEFs). Calcium and diacylglycerol activate CalDAG-GEFI (also termed RasGRP2), a protein detected in mouse megakaryocytes and platelets, 10,13 and CalDAG-GEFIII (also termed RasGRP3), involved in neuronal differentiation and B-cell development. 14,15 Cyclic adenosine monophosphate (cAMP) activates cAMP-dependent Epacs, 16,17 and C3G is regulated by tyrosine phosphorylation 18 and by binding to adaptor proteins. 19,20 Postsynaptic density-95 discs-large and zona occludens protein 1 (PDZ)-GEF1 is a ubiquitously expressed GEF of Rap1 and co...
GTPase-activating proteins are required to terminate signaling by Rap1, a small guanine nucleotide-binding protein that controls integrin activity and cell adhesion. Recently, we identified Rap1GAP2, a GTPase-activating protein of Rap1 in platelets. Here we show that 14-3-3 proteins interact with phosphorylated serine 9 at the N terminus of Rap1GAP2. Platelet activation by ADP and thrombin enhances serine 9 phosphorylation and increases 14-3-3 binding to endogenous Rap1GAP2. Conversely, inhibition of platelets by endothelium-derived factors nitric oxide and prostacyclin disrupts 14-3-3 binding. These effects are mediated by cGMP-and cAMP-dependent protein kinases that phosphorylate Rap1GAP2 at serine 7, adjacent to the 14-3-3 binding site. 14-3-3 binding does not change the GTPase-activating function of Rap1GAP2 in vitro. However, 14-3-3 binding attenuates Rap1GAP2 mediated inhibition of cell adhesion. Our findings define a novel crossover point of activatory and inhibitory signaling pathways in platelets.Platelets are involved in many physiological and pathological events in the vascular system including hemostasis and thrombosis as well as inflammation, angiogenesis, and metastasis. Endothelium-derived messenger molecules nitric oxide (NO) and prostacyclin (PGI 2 ) 3 initiate two major inhibitory signaling pathways in platelets. NO and PGI 2 activate platelet guanylyl and adenylyl cyclases to produce cGMP and cAMP. In consequence cGMP and cAMP activate cGMP-and cAMP-dependent protein kinases (cGK/PKG and cAK/PKA) that phosphorylate substrate proteins leading to inhibition of platelet activation, adhesion and aggregation (1-3). To date only few substrates of cGK/cAKs in platelets have been characterized and the mechanisms mediating platelet inhibition downstream of the substrates are largely unknown. We and others (4, 5) have recently detected that cGK and cAK efficiently block agonistinduced formation of Rap1-GTP in platelets. Rap1 is a guanine nucleotide-binding protein that regulates integrin functions and plays a pivotal role in adhesion of many cell types (6 -9). In platelets Rap1 is required for adhesion, aggregation and thrombus formation (10). Because Rap1 controls integrin function and platelet aggregation, Rap1 inhibition might play a central role in platelet inhibition by NO and PGI 2 . Rap1 cycles between an active GTP-bound and an inactive GDP-bound conformation. The transition between these two states is controlled by unique guanine nucleotide exchange factors (GEF) and GTPase-activating proteins (GAP).In a previous study we identified Rap1GAP2, the only known GAP of Rap1 in platelets (11). Furthermore, in vitro experiments provided preliminary evidence for cGK-and cAK-mediated phosphorylation of Rap1GAP2 (11). Rap1GAP2 contains a central conserved GAP domain as well as large N-and C-terminal regions of unknown function. We hypothesized that these regions might be involved in protein/protein interactions and performed a genetic screening in yeast to identify Rap1GAP2-interacting proteins. In the p...
SummaryThe NO/cGMP signalling pathway strongly inhibits agonist-induced platelet aggregation. However, the molecular mechanisms involved are not completely defined. We have studied NO/cGMP effects on the activity of Rap1, an abundant guanine-nucleotidebinding protein in platelets. Rap1-GTP levels were reduced by NO-donors and activators of NO-sensitive soluble guanylyl cyclase. Four lines of evidence suggest that NO/cGMP effects are mediated by cGMP-dependent protein kinase (cGKI): (i) Rap1 inhibition correlated with cGKI activity as measured by the phosphorylation state ofVASP, an established substrate of cGKI, (ii) 8-pCPT-cGMP, a membrane permeable cGMP-analog and activator of cGKI, completely blocked Rap1 activation, (iii) Rp- 8pCPT-cGMPS, a cGKI inhibitor, reversed NO effects and (iv) expression of cGKI in cGKI-deficient megakaryocytes inhibited Rap1 activation. NO/cGMP/cGKI effects were independent of the type of stimulus used for Rap1 activation. Thrombin-,ADPand collagen-induced formation of Rap1-GTP in platelets as well as turbulence-induced Rap1 activation in megakaryocytes were inhibited. Furthermore, cGKI inhibited ADP-induced Rap1 activation induced by the G α i -coupled P2Y12 receptor alone, i.e. independently of effects on Ca2+-signalling. From these studies we conclude that NO/cGMP inhibit Rap1 activation in human platelets and that this effect is mediated by cGKI. Since Rap1 controls the function of integrin α IIbβ 3 , we propose that Rap1 inhibition might play a central role in the anti-aggregatory actions of NO/cGMP.
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