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

Abstract

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...

Interaction with 14-3-3 Adaptors Regulates the Sorting of hMex-3B RNA-binding Protein to Distinct Classes of RNA Granules

“…Similarly, 14-3-3 proteins did not coimmunoprecipitate with a mutant form of hMex-3B where Ser-462 has been replaced by a phosphomimetic residue such as glutamic acid (hMex-3B mS462E, Fig. 2E), as has been described for other proteins (27,28). We next employed a mouse monoclonal antibody that recognizes phosphoserine residues within a 14-3-3 binding consensus sequence (anti-phosphoserine 14-3-3 binding).…”

Thrombin and Collagen Induce a Feedback Inhibitory Signaling Pathway in Platelets Involving Dissociation of the Catalytic Subunit of Protein Kinase A from an NFκB-IκB Complex

“…S1). We determined that VASP is not the only PKA substrate phosphorylated in response to thrombin and collagen stimulation of platelets by examining the phosphorylation of Rap1GAP2, which has been recently identified as a substrate of PKA and PKG in platelets (21,27). In our experiments, Rap1GAP2 was indeed phosphorylated in thrombin-and collagen-stimulated platelets (Fig.…”

“…Ser7 antibodies were described previously (21 Blood was collected into ACD solution (12 mM citric acid, 15 mM sodium citrate, 25 mM D-glucose, final concentrations). Platelet-rich plasma (PRP) was obtained by 5 min of centrifugation at 330 ϫ g, and then apyrase (0.01 unit/ml, final concentration) was added.…”

Arachidonic acid depletion extends survival of cold-stored platelets by interfering with the [glycoprotein Ib - 14-3-3 ] association

“…Factors that control COX-1 Ser-phosphorylation might, therefore, contribute to 14-3-3ζ translocation. Apart from COX-1, 14-3-3 proteins are known to bind pro-apoptotic Bax, 43 members of the GPIb-V-IX complex, 2,44 GTPase-activating protein Rap1GAP2, 45 phosphoinositide 3-kinase, 46 c-Raf-1 and insulin receptor substrate-1. 47 Trapping of 14-3-3ζ by accumulated AA might, therefore, affect many steps that control platelet functions.…”