Kinetic Regulation of β3 Integrin Tyrosine Phosphorylation

Scott D Blystone

doi:10.1074/jbc.m209506200

Abstract

Tyrosine phosphorylation of beta(3) integrins is a permissive stage in the activation of alpha(IIb)beta(3) and alpha(v)beta(3) in platelets and leukocytes, respectively. In this study we demonstrated direct phosphorylation of beta(3) integrins as a result of interaction with soluble monomeric ligand, and we characterized the differential kinetics of beta(3) phosphorylation as a consequence of alpha subunit pairing. We found that beta(3) phosphorylation is initiated by RGD peptide binding in a dose-dependent and saturable fashion with alpha(IIb)beta(3) becoming phosphorylated and dephosphorylated more rapidly than alpha(v)beta(3). Site mapping of phosphate incorporation reveals significant phosphorylation at Tyr-747 in both beta(3) integrin species with incorporation at Tyr-759 found at significant levels only in alpha(IIb)beta(3). Mutation of cytoplasmic beta(3) tyrosine residues in a transfection model prevents cell adhesion via these integrins. These data demonstrate that recognition of ligand is sufficient to induce beta(3) tyrosine phosphorylation and suggests that this event is regulated by the alpha subunit pairing of beta(3).

Highlights

The ␤3 integrin subunit has been shown to become tyrosinephosphorylated either as ␣v␤3 or as ␣IIb␤3 in monocytes, endothelial cells, platelets, and some cell lines (1–3)
Incorporation of phosphate into ␤3 in transfected cell models was similar to that seen in both murine and human primary macrophages and occurred in approximately half of the recovered ␤3 in RGD-stimulated suspended cells. The effects of these ␤3 mutations on adhesions both in vitro and in vivo combined with significant phosphorylation stoichiometry suggests that tyrosine phosphorylation of ␤3 is a prominent event in hematopoietic cell adhesion via ␤3 integrins
K␣v␤3 was incubated with increasing concentrations of GRGDSP peptide, and samples were evaluated for ␤3 tyrosine phosphorylation as described under “Experimental Procedures” (Fig. 1). ␤3 phosphorylation was increased in a dose

Summary

EXPERIMENTAL PROCEDURES

Transfections, Cells, and Flow Cytometry—K562 cells were stably transfected with cDNA that encoded human integrin subunits ␣v or ␣IIb with either ␤3 wild-type or ␤3 in which tyrosine residues at position [747, 759], or both, were mutated to phenylalanine as described previously (2). ␤3 Phosphorylation—For visual analysis of ␤3 tyrosine phosphorylation, 1 ϫ 107 K562 cells that expressed ␤3 integrins were suspended in Iscove’s modifed Dulbecco’s medium that contained 75 ␮M NaVO4 and incubated with or without the indicated concentrations of Gly-Arg-GlyAsp-Ser-Pro (GRGDSP) peptide at room temperature for the indicated. To determine the site and stoichiometry of ␤3 tyrosine phosphorylation, ␤3 was immunoprecipitated from human monocyte-derived macrophages, murine bone marrow-derived macrophages, and K562 cells that expressed either ␣v␤3 or ␣IIb␤3. Shown is the average Ϯ S.D. of no fewer than five determinations performed in triplicate for each group

RESULTS

Integrin Phosphorylation

DISCUSSION