GTPase activating protein Rap1GAP2 and synaptotagmin-like protein 1 interact and are involved in platelet dense granule secretion

Platelets are anucleate cells that play a major role in hemostasis and thrombosis in the vasculature. During primary hemostasis platelets adhere to sites of vascular damage and the initial platelet coat is 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 and paracrine fashion and affect local coagulation and endothelial smooth muscle cell function. The small guanine nucleotide binding protein Rap1 regulates the activity of the platelet integrin alphaIIbbeta3 and thus platelet aggregation. Rap1 activity is controlled by guanine nucleotide exchange factors and GTPase activating proteins. In platelets, Rap1GAP2 is the only GTPase activating protein of Rap1. In order to identify Rap1GAP2-associated proteins, a genetic two-hybrid screening in yeast was performed and synaptotagmin-like protein 1 (Slp1, also called JFC1) was found as a new putative binding partner of Rap1GAP2. Slp1 is a tandem C2 domain containing protein and is known to bind to Rab27, a small GTPase involved in platelet dense granule secretion. The direct interaction between Rap1GAP2 and Slp1 was confirmed in yeast and in transfected cells. More importantly, Slp1 is expressed in platelets and binding of endogenous Rap1GAP2 and Slp1 was verified in these cells. The Rap1GAP2 and Slp1 interaction sites were mapped by mutational analysis. Rap1GAP2 binds through the -TKXT- motif within its C-terminus to the C2A domain of Slp1. Moreover, the Slp1 binding -TKXT- motif of Rap1GAP2 was confirmed by complementary approaches using short synthetic Rap1GAP2 peptides. The C2A domain of Slp1 is a phospholipid binding domain and thus mediates binding of Slp1 to the plasma membrane. Phospholipid overlay assays revealed that simultaneous binding of Slp1 via its C2A domain to Rap1GAP2 and to phospholipids can occur. In addition, the interaction between Rap1GAP2 and Slp1 is regulated by cAMP-dependent protein kinase (cAK or PKA), and kinase activation in platelets enhanced binding of endogenous Rap1GAP2 to Slp1. In-vitro phosphorylation assays revealed that Slp1 is a substrate of PKA, and serine 111 was identified as phosphorylation site. Since Slp1 is a Rab27 binding protein, a trimeric complex of Slp1, Rab27 and Rap1GAP2 is conceivable. The association of Slp1, Rab27 and Rap1GAP2 was investigated by immunofluorescence and co-immuno-precipitation experiments in both, transfected cells and platelets. By Slp1 affinity chromatography and subsequent mass spectrometric analysis additional Slp1 binding proteins were identified in platelets, and binding of Slp1 to Rab8 was confirmed in pull-down assays. To investigate the functional significance of the interaction between Rap1GAP2 and Slp1, an assay system was established to determine serotonin secretion of streptolysin-O permeabilized platelets. Addition of recombinant Slp1 protein to permeabilized platelets strongly inhibited platelet dense granule secretion, whereas addition of recombinant Rap1GAP2 protein or synthetic Rap1GAP2 peptide enhanced secretion. Deleting the Slp1 binding -TKXT- motif abolished the stimulatory effect of Rap1GAP2 on secretion. Addition of Rap1 to permeabilized platelets had no effect on secretion. These findings indicate that the Rap1GAP2 effect on platelet secretion does not depend on the GTPase activating function of Rap1GAP2, but is rather dependent on the -TKXT- mediated interaction of Rap1GAP2 with Slp1. In addition, in-vitro GAP assays revealed that Slp1 binding to Rap1GAP2 does not affect the Rap1GAP activity of Rap1GAP2, and adhesion assays excluded a role for the Rap1GAP2/Slp1 interaction in cell adhesion. Altogether, the results of the present study demonstrate that besides its function in platelet aggregation by controlling the activity of the small guanine nucleotide binding protein Rap1, Rap1GAP2 is involved in platelet dense granule secretion by the new -TKXT- mediated interaction with the Rab27 and membrane binding protein Slp1. In addition, the interaction between Rap1GAP2 and Slp1 is embedded into an elaborate network of protein-protein interactions in platelets which appear to be regulated by phosphorylation. Future studies will in particular aim to dissect the molecular details of Rap1GAP2 and Slp1 action in platelet secretion and investigate the potential biochemical and pharmacological value of the unique protein binding -TKXT- motif of Rap1GAP2.Blutplättchen, auch Thrombozyten genannt, sind kernlose Zellen, die unter physiologischen Umständen eine wichtige Rolle in der primären Hämostase spielen. Nach einer Gefäßverletzung adhärieren Thrombozyten an Strukturen der freigelegten subendothelialen Matrix. Bei dieser primären Adhäsion werden Thrombozyten aktiviert und bilden durch Wechselwirkung untereinander ein zunächst fragiles Thrombozytenaggregat. Dieser initial gebildete Thrombus wird in der sekundären Hämostase durch Fibrin verfestigt und ermöglicht dann die stabile Abdichtung der Gefäßwandläsion gegenüber dem Blutstrom (Kehrel 2003). Die Aktivierung von Thrombozyten durch Adhäsion oder durch lösliche Plättchenaktivatoren bewirkt eine Formveränderung der Thrombozyten. Gleichzeitig werden intrazelluläre Speichergranula von Thrombozyten ausgeschüttet. Thrombozyten enthalten drei verschiedene Arten von Granula: alpha-Granula, dichte Granula und Lysosomen. Die Granula in Thrombozyten dienen als Speicherorte für Proteine und Substanzen, welche sowohl autokrin die Thrombozytenaktivierung verstärken als auch parakrin noch ruhende Thrombozyten aus der Blutzirkulation rekrutieren. Die dichten Granula in Thrombozyten enthalten niedermolekulare Substanzen wie zum Beispiel ADP, ATP, Ca2+ und Serotonin. In den alpha-Granula dagegen befinden sich vor allem Proteine, die für die Adhäsion, Aggregation und die Blutgerinnung wichtig sind. Zusätzlich enthalten alpha-Granula Zytokine und Wachtumsfaktoren. Die lysosomalen Granula in Thrombozyten enthalten hydrolytische Enzyme wie beispielsweise beta-Hexosaminidase und Heparitinase (Gawaz 1999). ... Zielsetzung dieser Arbeit: Um weitere Einblicke in die Funktion und Regulation von Rap1GAP2 in Thrombozyten zu gewinnen, wurde ein Hefe-zwei-Hybrid-Screening durchgeführt (O. Danielewski und A. Smolenski, IBCII, Frankfurt). Neben 14-3-3-Proteinen (Hoffmeister et al. 2008), wurde das Rab27-bindende Protein synaptotagmin-like protein 1 (Slp1, auch JFC1 genannt) als neuer putativer Interaktionspartner von Rap1GAP2 gefunden. Ausgehend von den Ergebnissen dieses Hefe-zwei-Hybrid-Screenings waren die konkreten Ziele der vorliegenden Arbeit (1) die Bindung von Slp1 an Rap1GAP2 zu verifizieren, (2) die für die Bindung verantwortlichen Bindebereiche zu identifizieren, (3) die mögliche Komplexbildung von Rap1GAP2, Slp1, Rab27 und anderen Proteinen zu untersuchen sowie (4) die funktionale Bedeutung der Interaktion zwischen Rap1GAP2 und Slp1 zu bestimmen. ... Fazit Die Ergebnisse der vorliegenden Arbeit zeigen, dass das Rab27-bindende Protein Slp1 ein neuer direkter Interaktionspartner des GTPase-aktivierenden Proteins Rap1GAP2 in Thrombozyten ist. Die Expression von Slp1 wurde in Thrombozyten nachgewiesen. Damit ist Slp1 neben Munc13-4 das zweite bisher bekannte Rab27-bindende Protein in diesen Zellen. Der Nachweis der Interaktion von Slp1 mit GTP-gebundenen Rab27 legte eine Funktion von Slp1 als Rab27-Effektorprotein nahe und es konnte in dieser Arbeit gezeigt werden, dass Slp1 die Sekretion der dichten Granula in Thrombozyten beeinflusst. Darüber hinaus zeigen die Ergebnisse der vorliegenden Arbeit, dass Rap1GAP2, zusätzlich zu seiner bereits bekannten Funktion bei der Thrombozytenaggregation durch die Regulation des kleinen G-Proteins Rap1, auch an der Sekretion der dichten Granula in Thrombozyten beteiligt ist. Der Rap1GAP2 Effekt auf die Sekretion in Thrombozyten beruht auf der -TKXT- 111 vermittelten Wechselwirkung mit Slp1, welche Teil eines komplexen Netzwerks von Protein-Protein-Interaktionen in Thrombozyten ist. Rap1GAP2 könnte somit eine zentrale Rolle bei der Koordination der wichtigen Funktionen Aggregation und Sekretion in Thrombozyten spielen

cGMP-dependent protein kinase regulates Rap1 signaling in platelets : poster presentation

cGMP- and cAMP-dependent protein kinases (cGK and cAK) mediate the inhibitory effects of endothelium-derived messenger molecules nitric oxide and prostacyclin on platelets. To understand the mechanisms involved in platelet inhibition we searched for new substrates of cGK and cAK. We identified Rap1GAP2, the only GTPase-activating protein of Rap1 in platelets. Rap1 is a guanine-nucleotide binding protein that controls integrin activity, platelet adhesion and aggregation. Rap1GAP2 is required to turn over Rap1-GTP to Rap1-GDP resulting in the inactivation of integrins and reduced cellular adhesion. Using phospho-specific antibodies we demonstrate phosphorylation of endogenous Rap1GAP2 on serine 7 by cGK and cAK in intact platelets. Yeast-two-hybrid screening revealed an interaction of the phosphoserine/-threonine binding adapter protein 14-3-3 with Rap1GAP2, and we mapped the 14-3-3 binding site to the N-terminus of Rap1GAP2 close to the cGK/cAK phosphorylation site. We could show that 14-3-3 binding to Rap1GAP2 requires phosphorylation of serine 9. Platelet activation by ADP and thrombin treatment induces Rap1GAP2 serine 9 phosphorylation and enhances the attachment of 14-3-3 to Rap1GAP2. In contrast, phosphorylation of serine 7 by cGK/cAK leads to the detachment of 14-3-3. Furthermore, Rap1GAP2 serine 7 phosphorylation correlates with the inhibition of Rap1-GTP formation by cGMP and cAMP in platelets. Cell adhesion experiments provide additional evidence that Rap1GAP2 is activated by the detachment of 14-3-3. Point mutants of Rap1GAP2 deficient in 14-3-3 binding inhibit Rap1-mediated cell adhesion significantly stronger than a Rap1GAP2 mutant that binds 14-3-3 constitutively. Our findings define a novel regulatory mechanism that might contribute to both platelet activation and endothelial inhibition of platelet adhesion and aggregation

Cyclic nucleotide-dependent Protein Kinases Inhibit Binding of 14-3-3 to the GTPase-activating Protein Rap1GAP2 in Platelets

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.Deutsche Forschungsgemeinshaf

Slp1 regulates platelet secretion

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. Slp1 contains a Rab27-binding domain, and we demonstrate that Rap1GAP2, Slp1, and Rab27 form a trimeric complex in transfected cells and in platelets. Purified Slp1 dose-dependently decreased dense granule secretion in streptolysin-O–permeabilized platelets stimulated with calcium or guanosine 5′-O-[gamma-thio] triphosphate. The isolated C2A domain of Slp1 had a stimulatory effect on granule secretion and reversed the inhibitory effect of full-length Slp1. Purified Rap1GAP2 augmented dense granule secretion of permeabilized platelets, whereas deletion of the Slp1-binding TKXT motif abolished the effect of Rap1GAP2. We conclude that Slp1 inhibits dense granule secretion in platelets and that Rap1GAP2 modulates secretion by binding to Slp1.Science Foundation IrelandDeutsche ForschungsgemeinschaftExcellence Cluster Cardio-Pulmonary Syste