Regulated Proteolytic Processing of Tie1 Modulates Ligand Responsiveness of the Receptor-tyrosine Kinase Tie2

Marie B Marron,Harprit Singh,Tariq A Tahir,Jais Kavumkal,Hak-Zoo Kim,Gou Young Koh,Nicholas P.J Brindle

doi:10.1074/jbc.m702535200

Abstract

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.

Highlights

Degradation of transmembrane proteins [1, 2]. Such processing has been described for a number of transmembrane proteins, including Notch, amyloid precursor protein and the receptortyrosine kinase ErbB-4 and involves an initial metalloproteasemediated ectodomain shedding followed by secondary cleavage of the remaining membrane-associated fragment [3,4,5,6,7,8]
The initiating and key regulatory step in RIP is ectodomain cleavage, and in most cases this is catalyzed by members of a disintegrin and metalloprotease (ADAM) family, matrix metalloproteases and the aspartyl proteases ␤-site amyloid precursor protein-cleaving enzymes 1 and 2 have been implicated to a lesser degree [9, 10]
Similar observations were made with Human umbilical vein endothelial cells (HUVEC) after activation with vascular endothelial growth factor (VEGF) (Fig. 1B)

Summary

Introduction

Degradation of transmembrane proteins [1, 2]. Such processing has been described for a number of transmembrane proteins, including Notch, amyloid precursor protein and the receptortyrosine kinase ErbB-4 and involves an initial metalloproteasemediated ectodomain shedding followed by secondary cleavage of the remaining membrane-associated fragment [3,4,5,6,7,8]. The ␥-secretase inhibitor further increased 45-kDa endodomain levels and caused loss of the 42-kDa form of Tie1 in endothelial cells stimulated with VEGF (Fig. 2C). Cells were stimulated with COMP-Ang1, and ectodomain cleavage was induced by the addition of PMA before cell lysis and immunoprecipitation of Tie1.

Results

Conclusion