RESUMO
Regulated ectodomain shedding followed by intramembrane proteolysis has recently been recognized as important in cell signaling and for degradation of several type I transmembrane proteins. The receptor-tyrosine kinase Tie1 is known to undergo ectodomain cleavage generating a membrane-tethered endodomain. Here we show Tie1 is a substrate for regulated intramembrane proteolysis. After Tie1 ectodomain cleavage the newly formed 45-kDa endodomain undergoes additional proteolytic processing mediated by gamma-secretase to generate an amino-terminal-truncated 42-kDa fragment that is subsequently degraded by proteasomal activity. This sequential processing occurs constitutively and is stimulated by phorbol ester and vascular endothelial growth factor. To assess the biological significance of regulated Tie1 processing, we analyzed its effects on angiopoietin signaling. Activation of ectodomain cleavage causes loss of phosphorylated Tie1 holoreceptor and generation of phosphorylated receptor fragments in the presence of cartilage oligomeric protein angiopoietin 1. A key function of gamma-secretase is in preventing accumulation of these phosphorylated fragments. We also find that regulated Tie1 processing modulates ligand responsiveness of the Tie-1-associated receptor Tie2. Activation of Tie1 ectodomain cleavage increases cartilage oligomeric protein angiopoietin 1 activation of Tie2. This correlates with increased ability of Tie2 to bind ligand after shedding of the Tie1 extracellular domain. A similar enhancement of ligand activation of Tie2 is seen when Tie1 expression is suppressed by RNA interference. Together these data indicate that Tie1, via its extracellular domain, limits the ability of ligand to bind and activate Tie2. Furthermore the data suggest that regulated processing of Tie1 may be an important mechanism for controlling signaling by Tie2.
Assuntos
Angiopoietina-1/metabolismo , Células Endoteliais/enzimologia , Processamento de Proteína Pós-Traducional/fisiologia , Receptor de TIE-1/metabolismo , Receptor TIE-2/metabolismo , Transdução de Sinais/fisiologia , Secretases da Proteína Precursora do Amiloide/metabolismo , Carcinógenos/farmacologia , Células Cultivadas , Células Endoteliais/citologia , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , Humanos , Ligantes , Ésteres de Forbol/farmacologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Interferência de RNA , Transdução de Sinais/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/farmacologiaRESUMO
Tie2 is a receptor tyrosine kinase expressed predominantly in endothelial cells and is essential for blood vessel formation and maintenance. The receptor has potent antiinflammatory effects on endothelial cells, suppressing vascular endothelial growth factor- and tumor necrosis factor-induced expression of leukocyte adhesion molecules and procoagulant tissue factor and inhibiting vascular leakage. To delineate the signaling pathways utilized by Tie2, we performed yeast two-hybrid screening of a human endothelial cell cDNA library and identified a novel protein interacting with the intracellular domain of the receptor. This protein was found to be human A20 binding inhibitor of NF-kappaB activation-2, ABIN-2, an inhibitor of NF-kappaB-mediated inflammatory gene expression. Coexpression of Tie2 and ABIN-2 in CHO cells confirmed the interaction occurs in mammalian cells. In contrast, Tie1 did not interact with ABIN-2 in the yeast two-hybrid system or mammalian cells. Deletion analysis identified the Tie2 binding motif to be encompassed between residues 171 and 272 in ABIN-2. Interaction was dependent on Tie2 autophosphorylation but ABIN-2 was not tyrosine phosphorylated by Tie2. Furthermore, in endothelial cells the interaction was stimulated by the Tie2 ligand angiopoietin-1. Expression of ABIN-2 deletion mutants in endothelial cells suppressed the ability of angiopoietin-1 to inhibit phorbol ester-stimulated NF-kappaB-dependent reporter gene activity. These findings provide the first direct link between Tie2 and a key regulator of inflammatory responses in endothelial cells. Interaction between Tie2 and ABIN-2 may be important in the vascular protective antiinflammatory actions of Tie2.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas de Transporte/metabolismo , Endotélio Vascular/enzimologia , NF-kappa B/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Sequência de Aminoácidos , Indutores da Angiogênese/farmacologia , Angiopoietina-1 , Animais , Anti-Inflamatórios/metabolismo , Sítios de Ligação , Células CHO , Proteínas de Transporte/química , Proteínas de Transporte/genética , Células Cultivadas , Cricetinae , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Humanos , Glicoproteínas de Membrana/farmacologia , Camundongos , Dados de Sequência Molecular , NF-kappa B/antagonistas & inibidores , Fosfotirosina/metabolismo , Estrutura Terciária de Proteína , Receptor TIE-2 , Deleção de Sequência , Homologia de Sequência , Técnicas do Sistema de Duplo-HíbridoRESUMO
The receptor tyrosine kinase Tie-1 is expressed predominantly in endothelial cells where it physically associates with the related receptor Tie-2. Positive signalling through Tie-2 is associated with microvessel stability and suppression of this signal is thought to be required for vascular endothelial growth factor (VEGF)-induced microvessel remodelling or growth. Here we examine the effects of VEGF on Tie-1 and the Tie-2:Tie-1 complex. We show that VEGF induces generation of the Tie-1 endodomain and loss of the full-length receptor. The effects of VEGF on endodomain formation are not suppressed by inhibitors of protein kinase C and do not involve the nitric oxide signalling pathway. Tyrosine kinase inhibitors, in contrast, do abolish endodomain generation in response to the endothelial growth factor. VEGF stimulation of cells does not cause dissociation of the Tie-2:Tie-1 complex; rather the complex is converted to a form comprising the full-length-Tie-2 and Tie-1 endodomain. VEGF can therefore switch the Tie-2:Tie-1 complex between two different forms in endothelial cells. The ability of VEGF to modulate Tie-1 and the Tie-2:Tie-1 complex provides a mechanism whereby this initiator of vessel growth and remodelling can directly modulate receptors involved in vessel stabilization. Such cross-talk is likely to be important in the coordinate control of blood vessel formation during development and in postnatal angiogenesis.