ICAM-1-mediated, Src- and Pyk2-dependent vascular endothelial cadherin tyrosine phosphorylation is required for leukocyte transendothelial migration.
Leukocyte transendothelial migration (TEM) has been modeled as a multistep process beginning with rolling adhesion, followed by firm adhesion, and ending with either transcellular or paracellular passage of the leukocyte across the endothelial monolayer. In the case of paracellular TEM, endothelial cell (EC) junctions are transiently disassembled to allow passage of leukocytes. Numerous lines of evidence demonstrate that tyrosine phosphorylation of adherens junction proteins, such as vascular endothelial cadherin (VE-cadherin) and beta-catenin, correlates with the disassembly of junctions. However, the role of tyrosine phosphorylation in the regulation of junctions during leukocyte TEM is not completely understood. Using human leukocytes and EC, we show that ICAM-1 engagement leads to activation of two tyrosine kinases, Src and Pyk2. Using phospho-specific Abs, we show that engagement of ICAM-1 induces phosphorylation of VE-cadherin on tyrosines 658 and 731, which correspond to the p120-catenin and beta-catenin binding sites, respectively. These phosphorylation events require the activity of both Src and Pyk2. We find that inhibition of endothelial Src with PP2 or SU6656 blocks neutrophil transmigration (71.1 +/- 3.8% and 48.6 +/- 3.8% reduction, respectively), whereas inhibition of endothelial Pyk2 also results in decreased neutrophil transmigration (25.5 +/- 6.0% reduction). Moreover, overexpression of the nonphosphorylatable Y658F or Y731F mutants of VE-cadherin impairs transmigration of neutrophils compared with overexpression of wild-type VE-cadherin (32.7 +/- 7.1% and 38.8 +/- 6.5% reduction, respectively). Our results demonstrate that engagement of ICAM-1 by leukocytes results in tyrosine phosphorylation of VE-cadherin, which is required for efficient neutrophil TEM.
Allingham
Michael J
MJ
Department of Cell and Developmental Biology, University of North Carolina, Chapel Hill, NC 27599, USA. michael_allingham@med.unc.edu
van Buul
Jaap D
JD
Burridge
Keith
K
D022005:Adherens Junctions / Q000201:enzymology / Q000378:metabolism; D015820:Cadherins / Q000378:metabolism* / Q000502:physiology; D051177:Catenins; D002448:Cell Adhesion / Q000276:immunology; D015815:Cell Adhesion Molecules / Q000378:metabolism; D002465:Cell Movement / Q000276:immunology*; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000201:enzymology / Q000378:metabolism; D004730:Endothelium, Vascular / Q000166:cytology* / Q000201:enzymology / Q000378:metabolism; D004789:Enzyme Activation / Q000276:immunology; D051418:Focal Adhesion Kinase 2 / Q000502:physiology*; D006801:Humans; D018799:Intercellular Adhesion Molecule-1 / Q000378:metabolism / Q000502:physiology*; D007962:Leukocytes / Q000166:cytology / Q000201:enzymology / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding / Q000276:immunology; D015398:Signal Transduction / Q000276:immunology; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D014443:Tyrosine / Q000378:metabolism*; D015854:Up-Regulation / Q000276:immunology; D051176:beta Catenin / Q000378:metabolism; D019061:src-Family Kinases / Q000502:physiology*; D000095002:Delta Catenin