RESUMO
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.
Assuntos
Plaquetas/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Complexos Multiproteicos/metabolismo , Vesículas Secretórias/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Motivos de Aminoácidos/fisiologia , Animais , Células COS , Chlorocebus aethiops , Proteínas Ativadoras de GTPase/genética , Células HeLa , Humanos , Proteínas de Membrana , Complexos Multiproteicos/genética , Ligação Proteica/fisiologia , Estrutura Terciária de Proteína/fisiologia , Vesículas Secretórias/genética , Técnicas do Sistema de Duplo-Híbrido , Proteínas de Transporte Vesicular/genética , Proteínas rap1 de Ligação ao GTP/genética , Proteínas rap1 de Ligação ao GTP/metabolismoRESUMO
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.
Assuntos
Proteínas 14-3-3/metabolismo , Plaquetas/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Adesividade Plaquetária/fisiologia , Transdução de Sinais/fisiologia , Proteínas 14-3-3/genética , Difosfato de Adenosina/metabolismo , Difosfato de Adenosina/farmacologia , Animais , Plaquetas/citologia , Células COS , Chlorocebus aethiops , AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Endotélio Vascular/citologia , Endotélio Vascular/metabolismo , Epoprostenol/metabolismo , Proteínas Ativadoras de GTPase/genética , Células HeLa , Humanos , Óxido Nítrico/metabolismo , Fosforilação/efeitos dos fármacos , Adesividade Plaquetária/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Trombina/metabolismo , Trombina/farmacologia , Proteínas rap1 de Ligação ao GTP/genética , Proteínas rap1 de Ligação ao GTP/metabolismoRESUMO
The paper provides evidence that transforming growth factor-beta activated kinase 1 (TAK1, MEKK7), a downstream mediator of IL-1beta signal transduction, plays an important role in the regulation of catabolic events and inflammatory processes in the context of degenerative joint diseases. We investigated the expression of TAK1 in human articular chondrocytes and in the murine growth plate by cDNA array, quantitative RT-PCR and immunohistochemistry, respectively. The human chondrosarcoma cell line SW1353 was stimulated with the proinflammatory cytokine IL-1beta. The subsequent expression of proteolytic enzymes and proinflammatory cytokines was quantified. TAK1 specific siRNA was used to study the influence of TAK1 downregulation on the expression of MMP-13, MMP1 and TNF-alpha. As a result we demonstrated the expression of TAK1 in normal and osteoarthritic human articular cartilage. Expression of TAK1 in the hypertrophic zone of the growth plate gave us a first evidence for a catabolic function of TAK1 concerning cartilage metabolism. By gene suppression with RNAi technology we could show that TAK1 downregulation leads to a 60-70% reduced release of TNF-alpha, a 40-50% reduced release of MMP13, and a 20-30% reduction of MMP1 release. As TNF-alpha is a main player in inflammatory processes, and MMP13 is one of the major proteases involved in cartilage degradation, our results suggests that TAK1 has an important regulatory role in the context of degenerative joint diseases and thus is an attractive drug target in attempts to reduce inflammation and suppress structural changes in OA induced by IL-1beta.
