title: Signaling functions of L-selectin. Enhancement of tyrosine phosphorylation and activation of MAP kinase.
abstract: L-selectin is a leukocyte cell surface glycoprotein involved in carbohydrate-specific ligand binding which mediates rolling of leukocytes along endothelial surfaces. In addition to its role in adhesion, an intracellular signaling role for L-selectin has recently been recognized. In particular, cross-linking L-selectin leads to increased cytosolic Ca2+ levels and potentiation of the oxidative burst. As several cell surface glycoproteins have been shown to be linked to tyrosine kinases, we examined the hypothesis that L-selectin may be linked to pathways involving tyrosine phosphorylation in human neutrophils. Ligation of L-selectin by three different antibodies recognizing separate epitopes led to increased tyrosine phosphorylation of several cellular proteins as judged by anti-phosphotyrosine immunoblots of whole cell lysates with prominent bands at 40-42, 55-60, 70-72, and 105-120 kDa. The 42-kDa band comigrated with mitogen-activated protein (MAP) kinase as determined by immunoblotting with anti-MAP kinase antibody. This effect was specific for L-selectin, because antibodies against CD18, CD45, and CD10 did not increase tyrosine phosphorylation. Phosphorylation was not due to Fc binding, since F(ab')2 fragments of the anti-L-selectin antibodies were similarly effective, and the response was unaffected by Fc receptor blockade. Cross-linking of L-selectin was not required for enhanced tyrosine phosphorylation, because monovalent Fab fragments also increased tyrosine phosphorylation. The response to L-selectin antibodies was not inhibited by cytochalasin, suggesting that reorganization of the actin cytoskeleton was not required for this response. Sulfatides, sulfated glycolipids which may be natural ligands for L-selectin, also induced a rapid, dose-dependent increase in tyrosine phosphorylation. In addition, sulfatides, but not control glycolipids, resulted in enhanced tyrosine phosphorylation of MAP kinase. Both sulfatides and anti-L-selectin antibodies increased kinase activity of MAP kinase as determined by gel renaturation assay. The tyrosine kinase inhibitor, genistein, blocked the transient increase in intracellular Ca2+ and the oxidative burst induced by sulfatides, suggesting that this tyrosine phosphorylation is functionally important. We conclude that L-selectin is able to transmit intracellular signals, including increased tyrosine phosphorylation and activation of MAP kinase in neutrophils. We speculate that these events may contribute to the activation of neutrophils during adhesion.
authors.affiliation: Department of Medicine, University of Toronto, Ontario, Canada.
authors.forename: T K
authors.initials: TK
authors.lastname: Waddell
authors.forename: L
authors.initials: L
authors.lastname: Fialkow
authors.forename: C K
authors.initials: CK
authors.lastname: Chan
authors.forename: T K
authors.initials: TK
authors.lastname: Kishimoto
authors.forename: G P
authors.initials: GP
authors.lastname: Downey
mesh_terms: D000818:Animals; D000906:Antibodies / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D015815:Cell Adhesion Molecules / Q000276:immunology / Q000502:physiology*; D003600:Cytosol / Q000378:metabolism; D004789:Enzyme Activation; D005434:Flow Cytometry; D006801:Humans; D015151:Immunoblotting; D066298:In Vitro Techniques; D019041:L-Selectin; D008562:Membrane Glycoproteins / Q000097:blood / Q000502:physiology; D051379:Mice / Q000276:immunology; D009504:Neutrophils / Q000502:physiology*; D010750:Phosphoproteins / Q000097:blood / Q000302:isolation & purification; D010766:Phosphorylation; D019000:Phosphotyrosine; D011961:Receptors, Fc / Q000502:physiology; D015398:Signal Transduction; D013481:Superoxides / Q000097:blood; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000378:metabolism
chemical_list: D000906:Antibodies; D015815:Cell Adhesion Molecules; D008562:Membrane Glycoproteins; D010750:Phosphoproteins; D011961:Receptors, Fc; D013481:Superoxides; D019041:L-Selectin; D019000:Phosphotyrosine; D014443:Tyrosine; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002118:Calcium