Phosphorylation of leukocyte PECAM and its association with detergent-resistant membranes regulate transendothelial migration.
Leukocyte migration across the endothelial lining is a critical step in the body's response to infection and inflammation. The homophilic interaction between endothelial PECAM and leukocyte PECAM is essential for this process. The molecular events that are triggered in the endothelial cell by PECAM engagement have been well characterized; however, the function of leukocyte PECAM remains to be elucidated. To study this, we first blocked leukocyte transmigration using anti-PECAM Ab and then specifically activated leukocyte PECAM. This was sufficient to overcome the block and promote transmigration, suggesting an active signaling role for leukocyte PECAM. Consistent with this, we found that ligation of leukocyte PECAM induces phosphorylation of two tyrosine residues on its cytoplasmic tail. By performing RNA interference-rescue experiments, we demonstrate that these phosphorylation events are indispensable for transendothelial migration. Finally, we show that leukocyte PECAM translocates to a detergent-resistant membrane (DRM) during transmigration. PECAM localized in DRMs displays reduced phosphorylation and does not support transmigration. Together, these data support a model whereby engagement of leukocyte PECAM induces its transient tyrosine phosphorylation and induction of downstream signals that drive transmigration. These signals are then downregulated following PECAM translocation to DRMs.
Florey
Oliver
O
Department of Pathology, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611, USA. floreyo@mskcc.org
Durgan
Joanne
J
Muller
William
W
D019138:Antibodies, Blocking / Q000378:metabolism; D057134:Antibodies, Neutralizing / Q000378:metabolism; D002460:Cell Line; D002465:Cell Movement / Q000276:immunology; D003432:Cross-Linking Reagents / Q000378:metabolism; D003902:Detergents / Q000378:metabolism; D015536:Down-Regulation / Q000276:immunology; D004730:Endothelium, Vascular / Q000166:cytology* / Q000276:immunology / Q000378:metabolism*; D006801:Humans; D008563:Membrane Lipids / Q000378:metabolism* / Q000502:physiology; D009000:Monocytes / Q000166:cytology / Q000276:immunology / Q000378:metabolism*; D017830:Octoxynol / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation; D019408:Platelet Endothelial Cell Adhesion Molecule-1 / Q000276:immunology / Q000378:metabolism*; D021381:Protein Transport / Q000276:immunology; D015398:Signal Transduction / Q000276:immunology; D014443:Tyrosine / Q000378:metabolism; D020298:U937 Cells; D047392:beta-Cyclodextrins / Q000378:metabolism*
D019138:Antibodies, Blocking; D057134:Antibodies, Neutralizing; D003432:Cross-Linking Reagents; D003902:Detergents; D008563:Membrane Lipids; D019408:Platelet Endothelial Cell Adhesion Molecule-1; D047392:beta-Cyclodextrins; C108732:methyl-beta-cyclodextrin; D014443:Tyrosine; D017830:Octoxynol