Mac-1-dependent tyrosine phosphorylation during neutrophil adhesion

Abstract

Activated neutrophils display an array of physiological responses, including initiation of the oxidative burst, phagocytosis, and cell migration, that are associated with cellular adhesion. Under conditions that lead to cellular adhesion, we observed rapid tyrosine phosphorylation of an intracellular protein with an approximate relative molecular mass of 92 kDa (p92). Phosphorylation of p92 was inducible when Mac-1 was activated by phorbol 12-myristate 13-acetate, the β2-specific activating antibody CBR LFA-1/2, or interleukin-8 (77 amino acids). In addition, tyrosine phosphorylation of p92 was dependent on engagement of Mac-1 with ligand. Several observations suggest that this event may be an important step in the signaling pathway initiated by Mac-1 binding. p92 phosphorylation was specifically blocked with antibodies to CD11b, the α-subunit of Mac-1, and was rapidly reversible on disengagement of the integrin ligand interaction. Integrin-stimulated phosphorylation of p92 created binding sites that were recognized in vitro by the SH2 domains of c-CrkII and Src. Our observations suggest that neutrophil adhesion mediated through the binding of the β2-integrin Mac-1 initiates a signaling cascade that involves the activation of protein tyrosine kinases and leads to the regulation of protein-protein interactions via SH2 domains, a key process shared with growth factor signaling pathways.

Structural Basis of Integrin Regulation and Signaling

“…Extensive, physiologically relevant studies of β2 integrins on intact cells have shown that CBR LFA-1/2 mAb induce the high-affinity state and that, depending on the experimental system, KIM127 mAb can either stabilize or report the high-affinity state (35,45,54,57,(70)(71)(72)(73)(74)(75)(76). The binding sites for KIM127 and CBR LFA-1/2 antibodies have been mapped to the I-EGF2 and I-EGF3 domains, respectively (45,77).…”

“…Furthermore, KIM127 Fab bound only when extension was induced by another agent such as CBR LFA-1/2 Fab or an α/β allosteric antagonist. In combination with the following cited functional studies, the EM study (43) established that (a) extension is sufficient to activate ligand-binding competence by β2 integrins (35,45,54,(72)(73)(74)(75)(76)78), (b) ligand-bound β2 integrins on cell surfaces are extended (70,71), (c) binding to soluble ligand induces extension (57), and (d ) extracellular activation of integrins by Mn 2+ and inside-out activation of integrins stimulated by protein kinase C or cytoplasmic domain mutations induce the extended conformation in the absence of ligand binding (35,45,75).…”

Signaling Pathways Involved in IL-8-Dependent Activation of Adhesion Through Mac-1

“…1, A and B) or EDTA (data not shown). In addition, both of these agents have been shown to reverse protein tyrosine phosphorylation resulting from integrin-ligand binding (31). Neither MAPK (Fig.…”

“…There is also evidence that IL-8 stimulates phospholipase D activity (27), phosphatidylinositol-3 kinase (PI3K) activity (28), and GTP loading of RhoA (29) in human neutrophils. IL-8 has been shown to activate mitogen-activated protein kinase (MAPK) in human neutrophils as well as in enucleate neutrophil cytoplasts (28,30,31). In addition, IL-8 has been shown to stimulate GTP loading of Ras and Raf activation in human neutrophils with a time course that indicates that these events are upstream of MAPK activation.…”

Haemodialysis through a cellulose membrane induces dephosphorylation of CD11b and promotes leukocyte adhesion to endothelial cells

“…Integrins also activate intracellular signalling pathways in a process termed 'outside-in' signalling and enhanced cell adhesion. [9][10][11][12] The balance between phosphorylation and dephosphorylation of integrins might be a mechanism by which both integrin activation and signal transduction could be regulated. 13,14 Phosphorylation events are believed to mediate 'outside-in' signal transduction by integrins and are generally induced by cell adhesion.…”