Palmitoylation at Cys595 is essential for PECAM-1 localisation into membrane microdomains and for efficient PECAM-1-mediated cytoprotection

Caroline T. Sardjono; Stacey N. Harbour; Jana C. Yip; Cathy Paddock; Susheela Tridandapani; Peter J. Newman; Denise E. Jackson

doi:10.1160/TH06-08-0459

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 2006; 96(06): 756-766
DOI: 10.1160/TH06-08-0459

Platelets and Blood Cells

Schattauer GmbH

Palmitoylation at Cys⁵⁹⁵ is essential for PECAM-1 localisation into membrane microdomains and for efficient PECAM-1-mediated cytoprotection

Authors

Caroline T. Sardjono

¹Kronheimer Building, Burnet Institute incorporating the Austin Research Institute, Heidelberg, Victoria, Australia
Stacey N. Harbour

¹Kronheimer Building, Burnet Institute incorporating the Austin Research Institute, Heidelberg, Victoria, Australia
Jana C. Yip

¹Kronheimer Building, Burnet Institute incorporating the Austin Research Institute, Heidelberg, Victoria, Australia
Cathy Paddock

³Blood Research Institute, Blood Center of Wisconsin, Milwaukee, Wisconsin, USA
Susheela Tridandapani

²Department of Internal Medicine, The Ohio State University, Columbus, Ohio, USA
Peter J. Newman

³Blood Research Institute, Blood Center of Wisconsin, Milwaukee, Wisconsin, USA
Denise E. Jackson

¹Kronheimer Building, Burnet Institute incorporating the Austin Research Institute, Heidelberg, Victoria, Australia

Abstract

Summary

The Ig-ITIM superfamily member, PECAM-1 acts as a negative regulator of ITAM-signalling pathways in platelets involving GPVI/FcR gamma chain and FcγRIIa. This negative feedback loop involves regulation of collagen and GPVI-dependent aggregation events, platelet-thrombus-growth on immobilised collagen under flow and FcγRIIa-mediated platelet responses. In this study, we show that PECAM-1 is selectively palmitoylated involving a thioester linkage with an unpaired cysteine residue at amino acid position 595 in its cytoplasmic domain. As palmitoylation is known to target proteins to membrane microdomains, we investigated the microdomain localisation for PECAM-1 in platelets and nucleated cells. In unstimulated platelets, ∼20% of PECAM-1 is localised toTriton-insoluble microdomain fractions and it does not increase with platelet activation by collagen, collagen-related peptide, thrombin-or human-aggregated IgG. PECAM-1 is in close physical proximity with GPVI in platelet microdomains. Removal of platelet cytoskeleton prior to sucrose-density-gradient separation showed that PECAM-1 was associated with both theTriton-soluble and membrane skeleton in microdomain-associated fractions. Disruption of microdomains by membrane-cholesterol depletion resulted in loss of PECAM-1 localisation to membrane microdomains. Mutational analysis of juxtamembrane cysteine residue to alanine (C595A) of human PECAM-1 resulted in loss of palmitoylation and a sixfold decrease in association with membrane microdomains. Functionally, the palmitoylated cysteine 595 residue is required, in part, for efficient PECAM-1-mediated cytoprotection. These results show that cysteine 595 is required for constitutive association of PECAM-1 with membrane microdomains and PECAM-1-mediated cytoprotection, where it may act as a crucial regulator of signaling and apoptosis events.

Keywords

Adhesion receptors - signal transduction - platelet physiology - lipid rafts - GP VI

Full Text

References

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