Nitration of PECAM-1 ITIM tyrosines abrogates phosphorylation and SHP-2 binding

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Abstract

Platelet–endothelial cell adhesion molecule-1 (PECAM-1) is a cell adhesion molecule with a cytoplasmic immunoreceptor tyrosine-based inhibitory motif (ITIM) that, when phosphorylated, binds Src homology 2 domain-containing protein-tyrosine phosphatase (SHP-2). PECAM-1 is expressed at endothelial cell junctions where exposure to inflammatory intermediates may result in post-translational amino acid modifications that affect protein structure and function. Reactive nitrogen species (RNS), which are produced at sites of inflammation, nitrate tyrosine residues, and several proteins modified by tyrosine nitration have been found in diseased tissue. We show here that the RNS, peroxynitrite, induced nitration of both full-length cellular PECAM-1 and a purified recombinant PECAM-1 cytoplasmic domain. Mass spectrometric analysis of tryptic fragments revealed quantitative nitration of ITIM tyrosine 686. A synthetic peptide containing 3-nitrotyrosine at position 686 could not be phosphorylated nor bind SHP-2. These data suggest that ITIM tyrosine nitration may represent a mechanism for modulating phosphotyrosine-dependent signal transduction pathways.

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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).

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