Flow Activates ERK1/2 and Endothelial Nitric Oxide Synthase via a Pathway Involving PECAM1, SHP2, and Tie2

Lung-Kuo Tai,Qinlei Zheng,Shi Pan,Zheng-Gen Jin,Bradford C Berk

doi:10.1074/jbc.m501243200

Lung-Kuo Tai, Qinlei Zheng + Show 3 more

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https://doi.org/10.1074/jbc.m501243200

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Abstract

Blood flow modulates endothelial cell (EC) functions through specific signaling events. Previous data show that flow stimulates SHP2 translocation to cell membranes and binding to phosphotyrosine proteins. Flow-induced ERK1/2 phosphorylation depends on SHP2 phosphatase activity and SHP2 binding to phospho-PECAM1 (platelet endothelial adhesion molecule 1), suggesting that SHP2 forms a signaling module with PECAM1. We hypothesized that flow induces assembly of the multi-protein complexes with SHP2 that are required for downstream signaling. ECs were exposed to flow for 10 min, and endogenous SHP2 was immunoprecipitated. SHP2-associated proteins were analyzed by SDS-PAGE and identified by mass spectrometry. Tie2 and several known SHP2-binding proteins were identified in flow-induced SHP2 complexes. Flow significantly increased tyrosine phosphorylation of both Tie2 and PECAM1 and their association with SHP2. To evaluate their functional roles, ECs were treated with Tie2 or PECAM1 small interfering RNA (siRNA). Tie2 and PECAM1 expression decreased >80% after siRNA treatment, and flow-stimulated phosphorylation of ERK1/2, Akt, and endothelial nitric oxide synthase was significantly inhibited by Tie2 and PECAM1 siRNA. Tie2 phosphorylation by flow was significantly inhibited by PECAM1 siRNA treatment. These results establish Tie2 transactivation via PECAM1 as an early event in flow-mediated mechanotransduction and suggest an important role for a PECAM1-SHP2-Tie2 pathway in flow-mediated signal transduction.

Highlights

Fluid shear stress, the frictional force from blood flow acting on the vessel wall, regulates vascular remodeling, arterial tone, and atherogenesis [1,2,3]
Flowinduced ERK1/2 phosphorylation depends on SHP2 phosphatase activity and SHP2 binding to phospho-PECAM1, suggesting that SHP2 forms a signaling module with PECAM1
Human umbilical vein endothelial cells (HUVECs) were treated with flow for 10 min, and endogenous SHP2 was immunoprecipitated with three different antibodies (B1, C18, and N16; Fig. 1)

Summary

Introduction

The frictional force from blood flow acting on the vessel wall, regulates vascular remodeling, arterial tone, and atherogenesis [1,2,3]. Recent data show that tyrosine-phosphorylated PECAM1 binds to the phosphatase SHP2 and that the PECAM1/SHP2 complex is required for activation of ERK1/2 by flow [13]. In addition to its phosphatase activity, SHP2 serves as a scaffold and binds to several membrane proteins, including the epidermal growth factor receptor [21], the platelet-derived growth factor receptor [22], and PECAM1 (in response to flow) [13]. We found several known SHP2-associated membrane proteins and other adaptor proteins Among these proteins we focused on Tie (tyrosine kinase with immunoglobulin and epidermal growth factor homology domain 2), which is a receptor tyrosine kinase expressed predominantly on ECs and hematopoietic cells. We show that flow-induced Tie phosphorylation requires PECAM1 and that interactions among PECAM1, Tie, and SHP2 mediate activation of ERK1/2, Akt, and eNOS by flow

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