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...
The small guanine-nucleotide-binding protein Rap1 plays a key role in platelet aggregation and hemostasis, and we recently identified Rap1GAP2 as the only GTPase-activating protein of Rap1 in platelets. In search of Rap1GAP2-associated proteins, we performed yeast-2-hybrid screening and found synaptotagmin-like protein 1 (Slp1) as a new binding partner. We confirmed the interaction of Rap1GAP2 and Slp1 in transfected COS-1 and HeLa cells and at endogenous level in human platelets. Mapping studies showed that Rap1GAP2 binds through amino acids T524-K525-X-T527 within its C-terminus to the C2A domain of Slp1. IntroductionBlood platelets are essential for hemostasis and play an important role in the development of thrombosis in the vasculature. 1 During primary hemostasis, platelets adhere to sites of endothelial damage and the initial platelet coat is soon reinforced by additional platelets forming a stable aggregate. At the same time, platelets secrete their intracellular granules containing substances that further activate platelets in an autocrine loop and affect local coagulation and endothelial and smooth muscle cell functions. 2 Two main types of secretory granules have been described in platelets, dense granules, and ␣-granules. Dense granules contain small molecules, including adenosine diphosphate, adenosine triphosphate, and 5-hydroxytryptamine (5-HT, serotonin), whereas ␣-granules store many proteins, such as fibrinogen, von Willebrand factor, various cytokines, and growth factors. 3,4 Dense granule secretion is regulated by the small GTPase Rab27 that is localized at the granule membrane. 5 Rab27 probably mediates its action by promoting granule motility or by enhancing tethering and fusion of granules with the plasma membrane. 6,7 For these functions, Rab27 needs to interact with effector proteins, and Munc13-4 was identified as the first Rab27-binding protein in platelets. 8 Other proteins involved in dense granule secretion are the small GTPase Ral 9 and membrane proteins of the soluble NSF attachment protein receptor family either bound to the granule, such as 10 or to the plasma membrane, such as SNAP-23 and syntaxin 2. 11 Granule secretion is accompanied by the development of tight cellular interactions between platelets. This aggregation of platelets is mediated by integrins and other adhesion receptors. 2 Integrin activation is tightly regulated by the small guanine-nucleotidebinding protein Rap1, and deletion of the platelet isoform Rap1b in mouse platelets results in a bleeding phenotype. 12 Rap1 activity is controlled by GTPase-activating proteins (GAPs) and guaninenucleotide exchange factors (GEFs). We recently discovered Rap1GAP2 as the only GTPase-activating protein of Rap1 in platelets. 13 Rap1GAP2 contains a conserved central catalytic GAP domain, an N-terminal 14-3-3-binding site, and a large C-terminal region of unknown function. Platelet activation results in phosphorylation of Rap1GAP2 on serine 9, binding of 14-3-3 protein, and probably inhibition of GAP function. 14 In parallel, ...
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