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.
Highlights
The mechanisms mediating platelet inhibition downstream of the substrates are largely unknown
We studied the time course of Rap1GAP2 serine 7 phosphorylation in platelets induced by endothelium-derived cyclic nucleotide activators nitric oxide (NO) and prostacyclin (PGI2)
We identified 14-3-3 proteins as interaction partners of Rap1GAP2
Summary
Materials—Human thrombin, ADP, wortmannin, YC-1, DEA-NOate, DETA-NOate, sodium-nitroprusside (SNP), and forskolin were obtained from Sigma (Taufkirchen, Germany). To produce purified GST-tagged 14-3-3 and 14-3-3, full-size cDNAs, obtained from the yeast two-hybrid screen, were cloned into pGEX-4T3 vector (GE Healthcare, Freiburg, Germany). HeLa cells were transiently transfected using Metafectene (Biontex, Martinsried, Germany) according to the manufacturer’s instructions. In pulldown experiments 5 l of glutathione-Sepharose bead suspension (GE Healthcare) saturated with GST-143-3, GST-14-3-3 or GST-Rip were added to 500 l of platelet or cell lysate and incubated at 4 °C for 2 h or overnight. FLAG-tagged Rap1GAP2 proteins were purified from transfected HeLa cells using ANTIFLAG M2 Affinity Gel. Rap1GAP2 proteins bound to beads were incubated with purified [32P]GTP-loaded His-Rap1B in a buffer containing 30 mM Tris/HCl, pH 7.5, 5 mM MgCl2, and 1 mM DTE in 50-l aliquots at 25 °C. Numbers of bound cells were calculated and corrected for transfection efficiency by measuring luciferase activity of total input cells
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