Biochemical and Biophysical Research Communications
Nitration of PECAM-1 ITIM tyrosines abrogates phosphorylation and SHP-2 binding
Section snippets
Cell lines and cell culture
The acute T cell leukemia line, Jurkat E6-1, was obtained from the American Type Culture Collection (ATCC, Manassas, VA) and cultured, as previously described [15]. The human embryonic kidney (HEK-293) cell transfectants stably expressing wild-type PECAM-1 have been described previously [17].
Generation of mAb 235.1
A peptide corresponding to the C-terminal 15 amino acids of human PECAM-1 was synthesized in the Peptide Synthesis Core of the Blood Research Institute with an additional cysteine residue at its amino
Results
Previous studies have shown that tyrosine nitration interferes with tyrosine phosphorylation and therefore might be expected to interfere with cell signaling. PECAM-1 is an inhibitory receptor that is expressed at high density on the surfaces of endothelial cells and its inhibitory function depends upon phosphorylation of two key tyrosine residues within a cytoplasmic ITIM. Therefore, we sought to determine whether (1) PECAM-1 becomes nitrated on ITIM tyrosine residues, (2) nitration of
Discussion
When doubly phosphorylated on two specific tyrosine residues, the PECAM-1 dual ITIM supports binding and activation of SHP-2, resulting in the inhibition of signal transduction by several ITAM-containing activating receptors expressed on vascular cells. Since previous studies have shown that nitrated tyrosine residues are poor substrates for phosphorylation, we sought to directly assess the extent to which nitration occurs on PECAM-1 ITIM tyrosine residues and the impact of nitration on the
Acknowledgements
The authors thank Trudy Holyst (Peptide Core Facility, BRI) for peptide synthesis, Michael Pereckas (Protein and Nucleic Acid Shared Facility, MCW) for mass spectrometric analysis, and Rolf Jakobi (Department of Pharmacology, MCW) for helpful discussions in establishing an in vitro kinase assay. This work was supported in part by the Blood Center Research Foundation (DKN and PJN) and by National Institutes of Health Grants HL44612 (DKN and PJN), HL63119 (BK), and HL68769 (PJN, DKN, and BK).
References (32)
- et al.
Peroxynitrite-mediated tyrosine nitration catalyzed by superoxide dismutase
Arch. Biochem. Biophys.
(1992) - et al.
Kinetics of superoxide dismutase- and iron-catalyzed nitration of phenolics by peroxynitrite
Arch. Biochem. Biophys.
(1992) - et al.
Formation of reactive nitrogen species during peroxidase-catalyzed oxidation of nitrite. A potential additional mechanism of nitric oxide-dependent toxicity
J. Biol. Chem.
(1997) - et al.
Mass spectrometric quantification of 3-nitrotyrosine, ortho-tyrosine and O,O′-dityrosine in brain tissue of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated mice, a model of oxidative stress in Parkinson's disease
J. Biol. Chem.
(1999) - et al.
Peroxynitrite formation from macrophage-derived nitric oxide
Arch. Biochem. Biophys.
(1992) - et al.
Effects of peroxynitrite-induced protein modifications on tyrosine phosphorylation and degradation
FEBS Lett.
(1996) - et al.
Regulation of mouse PECAM-1 tyrosine phosphorylation by the Src and Csk families of protein-tyrosine kinases
J. Biol. Chem.
(1998) - et al.
Inhibition of antigen-receptor signaling by Platelet Endothelial Cell Adhesion Molecule-1 (CD31) requires functional ITIMs, SHP-2, and p56lck
Blood
(2001) - et al.
The protein-tyrosine phosphatase SHP-2 binds PECAM-1 and forms a distinct signaling complex during platelet aggregation. Evidence for a mechanistic link between PECAM-1 and integrin-mediated cellular signaling
J. Biol. Chem.
(1997) - et al.
Characterization of phosphotyrosine binding motifs in the cytoplasmic domain of platelet/endothelial cell adhesion molecule-1 (PECAM-1) that are required for the cellular association and activation of the protein-tyrosine phosphatase, SHP-2
J. Biol. Chem.
(1997)