Reactive oxygen species induce reversible PECAM-1 tyrosine phosphorylation and SHP-2 binding

Abstract

Platelet endothelial cell adhesion molecule-1 (PECAM-1, CD31) functions to control the activation and survival of the cells on which it is expressed. Many of the regulatory functions of PECAM-1 are dependent on its tyrosine phosphorylation and subsequent recruitment of the Src homology (SH2) domain containing protein tyrosine phosphatase SHP-2. The recent demonstration that PECAM-1 tyrosine phosphorylation occurs in cells exposed to the reactive oxygen species hydrogen peroxide (H2O2) suggested that this form of oxidative stress may also support PECAM-1/SHP-2 complex formation. In the present study, we show that PECAM-1 tyrosine phosphorylation in response to exposure of cells to H2O2 is reversible, involves a shift in the balance between kinase and phosphatase activities, and supports binding of SHP-2 and recruitment of this phosphatase to cell-cell borders. We speculate, however, that the unique ability of H2O2 to reversibly oxidize the reactive site cysteine residues of protein tyrosine phosphatases may result in transient inactivation of the SHP-2 that is bound to PECAM-1 under these conditions. Finally, we provide evidence that PECAM-1 tyrosine phosphorylation and SHP-2 binding in endothelial cells requires exposure to an "oxidative burst" of H2O2, but that exposure of these cells to sufficiently high concentrations of H2O2 for a sufficiently long period of time abrogates binding of SHP-2 to tyrosine-phosphorylated PECAM-1. These findings support a role for PECAM-1 as a sensor of oxidative stress, perhaps most importantly during the process of inflammation.

Evidence for the involvement of PECAM-1 in a receptor mediated signal-transduction pathway regulating capacitation-associated tyrosine phosphorylation in human spermatozoa

“…This, in turn, stimulates the recruitment and activation of various downstream signalling molecules including Src family kinases (Ohmori et al, 2001) in keeping with the suggested role of Src in mediating the WGA-activated, SU6656-inhibited increase in tyrosine phosphorylation observed in human spermatozoa. The fact that PECAM-1 can be activated by hydrogen peroxide is also perfectly in keeping with the ability of this oxidant to activate tyrosine phosphorylation in capacitating human spermatozoa (Aitken et al, 1998;Maas et al, 2003).…”

MAPKs (ERK½, p38) and AKT Can Be Phosphorylated by Shear Stress Independently of Platelet Endothelial Cell Adhesion Molecule-1 (CD31) in Vascular Endothelial Cells

“…PECAM-1 has important intracellular signaling/scaffolding capacities that function to control the activation and cell survival (22,38). The best described signaling property of PECAM-1 is the phosphorylation of the two ITAM tyrosine residues at positions 663 and 686 of the PECAM-1 cytoplasmic domain, which occur in response to exposure of cells to a number of different chemical and mechanical stimuli (13,19,38,39). Functionally, PECAM-1 tyrosine phosphorylation has been shown to play a role in the migration of EC, neutrophil transmigration, and angiogenesis (23,24,38) In cultured endothelial cells, PECAM-1 is distributed diffusely in the plasma mem- FIG.…”

An alternatively spliced isoform of PECAM-1 is expressed at high levels in human and murine tissues, and suggests a novel role for the C-terminus of PECAM-1 in cytoprotective signaling

“…Cells were cultured in DMEM (HEK293) or RPMI media containing 10% heat-inactivated fetal bovine serum and 40 mg/ml gentamicin. Human umbilical vein endothelial cells (HUVECs) were cultured as described previously (Maas et al, 2003).…”

“…Lysates were prepared from HEK293 cells stably transfected with either human PECAM-1 or Δ15 PECAM-1 (Maas et al, 2003). PECAM-1 was immunoprecipitated with PECAM-1.3 (Jackson et al, 1997a) incubated overnight at 4°C.…”

“…Cells were prepared as described previously (Maas et al, 2003). Slides were examined with a Leica TCS SP2 confocal microscope with a 100ϫ oil-immersion lens and analyzed with MetaMorph software (Molecular Devices, Sunnyvale, CA).…”