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

Der NO/cGMP-Signalweg reguliert das kleine G-Protein Rap1B in humanen Thrombocyten

Die Aggregation von Thrombocyten ist ein wichtiger physiologischer Schutzmechanismus zur primären Blutstillung nach Gefäßverletzungen. Dieser Vorgang kann jedoch unter pathologischen Bedingungen zu Herzinfarkten und Schlaganfällen führen. Der Aggregationsprozeß ist durch Ausbildung sogenannter "Fibrinogenbrücken" zwischen verschiedenen Thrombocyten gekennzeichnet. Dies wird durch Bindung von Fibrinogen an das aktivierte Integrin alphaIIbbeta3 auf der Thrombocytenoberfläche ausgelöst. Das kleine G-Protein Rap1B aus der Ras-Superfamilie reguliert den Aktivitätszustand von Integrinen und besitzt damit eine zentrale Rolle bei der Aggregation von Thrombocyten. Die Aktivierung von Rap1B wird durch eine Vielzahl von Plättchenagonisten innerhalb von wenigen Sekunden ausgelöst. Der von Thrombocyten und Gefäßendothelzellen gebildete Botenstoff Stickstoffmonoxid (NO) kann die Thrombocytenaggregation über den NO/cGMP-Signalweg hemmen. Das Signalmolekül NO aktiviert in Thrombocyten die NO-sensitive Guanylyl-Cyclase (sGC), hierdurch wird die Synthese des sekundären Botenstoffes cGMP angeregt. Das cGMP-Molekül aktiviert nachfolgend die cGMP-abhängige Proteinkinase-Ibeta (cGK-Ibeta), welche die aggregationshemmende NO-Wirkung vermittelt. Die verantwortlichen Zielproteine der cGK-Ibeta wurden bis heute jedoch nicht hinreichend aufgeklärt. In der vorliegenden Arbeit sollten verschiedene Aspekte der NO-induzierten Hemmung der Thrombocytenaggregation untersucht werden. Dabei wurden neue Mechanismen dieser Inhibition identifiziert. Zum einen konnte eine kinetisch schnelle Hemmung der Rap1B-Aktivierung in Thrombocyten nachgewiesen werden. Zum anderen konnten einer cGK-Ibeta-vermittelten, kinetisch langsamen Rap1B-Phosphorylierung hemmende Effekte auf die Membranlokalisation von Rap1B in MDCK-Zellen und auf die Zellausbreitung von Hela-Zellen zugeordnet werden. Weiterhin wurde im Rahmen dieser Arbeit eine neue Proteininteraktion zwischen dem mitochondrialen CGI-51-Protein und Rap1B identifiziert und verifiziert. Zur Aufklärung eines Einflusses des NO/cGMP-Signalweges auf die Aktivierung von Rap1B in Thrombocyten wurde die NO/sGC/cGMP/cGK-Ibeta-Signalkaskade auf verschiedenen Stufen aktiviert oder gehemmt, bevor anschließend die Rap1GTPBildung mit verschiedenen Plättchenagonisten induziert wurde. Das aktive Rap1B wurde unter Verwendung eines Rap1GTP-bindenden Fusionsproteins präzipitiert und nachgewiesen. Durch NO-freisetzende Substanzen konnte eine Hemmung der Rap1BAktivierung erreicht werden. Auch die Aktivierung der sGC mit einem spezifischen Aktivator führte zur Inhibition von Rap1B. Die direkte Aktivierung der cGK-Ibeta konnte Rap1B ebenfalls hemmen, während eine Blockade der cGK-Ibeta die NO-induzierte Hemmung der Rap1-Aktivierung verhinderte. Die genannten Effekte des NO/cGMP-Signalwegs waren unabhängig vom Stimulus, der zur Rap1B-Aktivierung genutzt wurde, sowohl die Aktivierung über verschiedene G-Protein-gekoppelte Rezeptoren (GPCR) als auch die Aktivierung über Tyrosin-Kinasen wurden gehemmt. Eine detailliertere Untersuchung ergab, daß cGK-Ibeta die Ca2+-unabhängige Aktivierung von Rap1B hemmen konnte. Die Rolle der cGK-Ibeta wurde abschließend im unabhängigen Zellsystem der Megakaryocyten abgesichert. Die Hemmung der Rap1B-Aktivierung durch den NO/cGMP-Signalweg stellt einen schnellen Regulationsmechanismus zur Inhibition der Thrombocytenaggregation dar. Aus der Literatur ist eine kinetisch langsame Phosphorylierung von Rap1B an Serin-179 durch cGK-Ibeta bekannt. Zur Ermittlung ihrer Funktion wurden mikroskopische Untersuchungen der subzellulären Rap1B-Lokalisation in lebenden MDCK-Zellen durchgeführt. Hierbei konnte gezeigt werden, daß eine nicht-phosphorylierbare Rap1BMutante eine ausgeprägte Membranlokalisation aufweist, während eine phosphomimetische Rap1B-Mutante bevorzugt cytoplasmatisch lokalisiert ist. In einer weiterführenden Studie wurde der Effekt dieser Rap1B-Mutanten auf das Zellausbreitungsverhalten von Hela-Zellen analysiert. Die Expression der nichtphosphorylierbaren Rap1B-Mutante führte dabei zu einer signifikant gesteigerten Zellausbreitung, welche hingegen durch eine phosphomimetische Rap1B-Mutante deutlich abgeschwächt war. Dies impliziert einen zusätzlichen Mechanismus, über den der NO/cGMP-Signalweg die Adhäsion bzw. die Aggregation von Thrombocyten regulieren kann. Zur Identifizierung von neuen Interaktionspartnern, die spezifisch an phosphoryliertes Rap1B binden und dessen subzelluläre Verteilung oder Aktivität regulieren, wurde das Yeast-Two-Hybrid-System eingesetzt. Hierbei konnte das mitochondriale CGI-51-Protein als neuer Bindepartner von Rap1B identifiziert und in Säugerzellen verifiziert werden. Eine phosphospezifische Interaktion konnte allerdings nicht nachgewiesen werden. Das CGI-51-Protein spielt eine wichtige Rolle bei der Proteinsortierung in der äußeren Mitochondrienmembran. Die Funktion der Interaktion von CGI-51-Protein mit Rap1B wurde im Rahmen dieser Arbeit nicht untersucht. Zusammenfassend kann gesagt werden, daß in der vorliegenden Arbeit erstmalig neue Erkenntnisse zur Regulation des kleinen G-Proteins Rap1B durch den NO/cGMP-Signalweg dargestellt sind. Dieser Regelmechanismus besitzt eine physioplogische Bedeutung bei der Inhibition der Thrombocytenaggregation.The aggregation of platelets is a crucial event in normal hemostasis following vascular injuries. Under pathological conditions, clotting can cause thrombotic or embolial events leading to myocardial infarction or stroke. The process of platelet aggregation requires binding of fibrinogen to activated integrin alphaIIbbeta3 receptors on the surface of platelets. Since fibrinogen can bind two alphaIIbbeta3 receptors simultaneously, it functions as a solid link between two platelets. These platelet-fibrinogen connections initiate platelet aggregation. Rap1B, a small GTPase of the Ras-superfamily, controls integrin alphaIIbbeta3 activity and is therefore critical for platelet aggregation. Many platelet agonists can rapidly trigger a massive Rap1B activation. Platelet aggregation can be blocked by nitric oxide (NO) which is derived either from platelets or endothelial cells. NO binds to and activates NO-sensitive guanylyl cyclase (sGC) resulting in the production of cGMP. The main target for cGMP in platelets is cGMP-dependent protein kinase Ibeta (cGK-Ibeta). Studies of both cGK-Ibeta-deficient human and murine platelets suggest that cGK-Ibeta is an important mediator of NO-induced inhibition of platelet aggregation. However, the responsible targets of cGK-Ibeta in platelets have not been sufficiently resolved yet. This thesis presents novel and different aspects of NO-induced inhibition of platelet aggregation: First, a kinetically fast inhibition of Rap1B activation was discovered in platelets. Second, a kinetically slow inhibition of Rap1B attachment to cellular membranes as well as an inhibition of cell spreading mediated by cGK-Ibeta-dependent Rap1B phosphorylation were revealed in cell models (MDCK and Hela cells respectively). Third, a new protein interaction between the mitochondrial CGI-51 protein and Rap1B was identified and confirmed. In order to study the influence of the NO/cGMP signaling pathway on the activation of Rap1B in platelets, different steps along the NO/sGC/cGMP/cGK-Ibeta pathway were activated or blocked. Subsequently, the activation of Rap1B was induced by various platelet agonists. The active GTP-bound form of Rap1B was pulled down using an activation-specific probe and assessed by Western blot. Treatment of platelets with NO-donors strongly inhibited activation of Rap1B. An NO-independent activator of sGC reduced Rap1GTP-levels as well, whereas blocking of sGC-dependent synthesis of cGMP abolished the NO effect on Rap1GTP-formation. Direct activation of cGK-Ibeta also inhibited Rap1B activation, while an inhibitor of cGK-Ibeta kinase function abrogated NO-induced inhibition of Rap1B. The effects of the NO/cGMP signaling cascade were independent of the stimuli used for Rap1B activation: RapGTP-formation induced via both G protein coupled receptors (GPCR) and tyrosine kinases was inhibited. Furthermore Rap1GTP-levels were reduced by cGK-Ibeta independently of its effects on Ca2+-signaling. Ultimately, the role of cGK-Ibeta was confirmed in megakaryocytes, i.e. platelet progenitor cells. The regulation of Rap1B activation by the NO/sGC/cGMP/cGK-Ibeta cascade is a kinetically fast mechanism, which inhibits platelet aggregation. A kinetically slow phosphorylation of Rap1B by cGK-Ibeta is known from the literature. To clarify cellular functions of this phosphorylation, a microscopic study of the subcellular Rap1B localisation was performed with living MDCK cells. A non-phosphorylated Rap1B mutant was located predominantly in cellular membranes, whereas a phosphomimetic Rap1B mutant was found in the cytosol. Further, the effects on cell spreading of these two mutants were examined in Hela cells. The expression of the non-phosphorylated Rap1B mutant increased cell spreading significantly, whereas a phosphomimetic Rap1B mutant abolished this effect. These results lead to the hypothesis that the NO-induced phosphorylation of Rap1B by cGK-Ibeta is an additional kinetically slow regulatory mechanism inhibiting platelet adhesion and aggregation. Another major focus of this thesis was the identification of novel binding proteins of phosphorylated Rap1B which might regulate its spatial distribution and activity. Using a yeast-two-hybrid approach, the mitochondrial CGI-51-protein was found to interact with Rap1B. This binding was verified in mammalian cells. CGI-51-protein has an important role in the sorting and assembly machinery of the outer mitochondrial membrane. However, the CGI-51 protein and Rap1B interaction is not phosphospecific. In summary, this thesis presents novel regulatory mechanisms of the small GTPase Rap1B by the NO/cGMP signaling pathway, leading to the inhibition of platelet aggregation

The NO/cGMP pathway inhibits Rap1 activation in human platelets via cGMP-dependent protein kinase I

The 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 a 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 a IIbß 3 , we propose that Rap1 inhibition might play a central role in the anti-aggregatory actions of NO/cGMP

NO/cGMP/cGKI inhibit Rap1

The 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 Rap 1, an abundant guanine-nucleotidebinding protein in platelets. Rap 1-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) Rap 1 inhibition correlated with cGKI activity as measured by the phosphorylation state of VASP, 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-,ADP- and collagen-induced formation of Rap 1-GTP in platelets as well as turbulence-induced Rap 1 activation in megakaryocytes were inhibited. Furthermore, cGKI inhibited ADP-induced Rap 1 activation induced by the Galpha(i)-coupled P2Y12 receptor alone, i.e. independently of effects on Ca2+-signalling. From these studies we conclude that NO/cGMP inhibit Rap 1 activation in human platelets and that this effect is mediated by cGKI. Since Rap1 controls the function of integrin alpha(IIb)beta3, we propose that Rap 1 inhibition might play a central role in the anti-aggregatory actions of NO/cGMP.Deutsche Forschungsgemeinschaf

Identification of Rap1GAP2

The Ras-like guanine-nucleotide-binding protein Rap1 controls integrin alpha(IIb)beta3 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 GTPase-activating 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 discs-large 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. Rap1GAP2 is the first GTPase-activating protein of Rap1 found in platelets and is likely to have an important regulatory role in platelet aggregation.Deutsche Forschungsgemeinschaf

Quantitative analysis of the cardiac fibroblast transcriptome-implications for NO/cGMP signaling

Cardiac fibroblasts regulate tissue repair and remodeling in the heart. To quantify transcript levels in these cells we performed a comprehensive gene expression study using serial analysis of gene expression (SAGE). Among 110,169 sequenced tags we could identify 30,507 unique transcripts. A comparison of SAGE data from cardiac fibroblasts with data derived from total mouse heart revealed a number of fibroblast-specific genes. Cardiac fibroblasts expressed a specific collection of collagens, matrix proteins and metalloproteinases, growth factors, and components of signaling pathways. The NO/cGMP signaling pathway was represented by the mRNAs for α1 and β1 subunits of guanylyl cyclase, cGMP-dependent protein kinase type I (cGK I), and, interestingly, the G-kinase-anchoring protein GKAP42. The expression of cGK I was verified by RT-PCR and Western blot. To establish a functional role for cGK I in cardiac fibroblasts we studied its effect on cell proliferation. Selective activation of cGK I with a cGMP analog inhibited the proliferation of serum-stimulated cardiac fibroblasts, which express cGK I, but not higher passage fibroblasts, which contain no detectable cGK I. Currently, our data suggest that cGK I mediates the inhibitory effects of the NO/cGMP pathway on cardiac fibroblast growth. Furthermore the SAGE library of transcripts expressed in cardiac fibroblasts provides a basis for future investigations into the pathological regulatory mechanisms underlying cardiac fibrosis.Deutsche Forschungsgemeinschaf

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