Intracluster Restriction of Fc Receptor γ-Chain Tyrosine Phosphorylation Subverted by a Protein-tyrosine Phosphatase Inhibitor

Lorraine C Pfefferkorn,Sharon L Swink

doi:10.1074/jbc.271.19.11099

Abstract

This study shows that aggregation of U937 cell high affinity IgG Fc receptor (Fc gamma RI) results in the transient tyrosine phosphorylation of Fc gamma RI gamma-chain but not the phosphorylation of gamma-chains associated with nonaggregated IgA Fc receptors (Fc alpha R) on the same cells. Thus, normally, tyrosine phosphorylation of gamma-chains is limited to FcR in aggregates. In contrast, aggregation of Fc gamma RI in the presence of vanadate induced the sustained tyrosine phosphorylation of Fc gamma RI gamma-chains and the rapid and extensive phosphorylation of nonaggregated Fc alpha R gamma-chains and low affinity IgG Fc receptors (Fc gamma RII). This global phosphorylation of motifs on nonaggregated FcR was also detected upon aggregation of Fc alpha R or Fc gamma RII, which induced the phosphorylation of nonaggregated Fc gamma RI gamma-chains. Vanadate prevented dephosphorylation of proteins and increased kinase activity in stimulated cells. Evidence failed to support alternative explanations such as acquisition of phospho-gamma through subunit exchange or a coalescence of nonaggregated with aggregated FcR. It is likely, therefore, that activated kinases interacted with nonaggregated FcR in stimulated cells. Pervanadate induced the tyrosine phosphorylation of gamma-chains in the absence of FcR cross-linking, indicating that the kinases could be activated by phosphatase inhibition and could react with nonaggregated substrates. We conclude that under normal conditions there is a vanadate-sensitive mechanism that prevents tyrosine phosphorylation of nonaggregated FcR gamma-chain motifs in activated cells, restricting their phosphorylation to aggregates.

Highlights

Sequences [11] are substrates for Src family kinases [12] that are capable of reacting with the nonphosphorylated motif and of binding through SH2 domains to the tyrosine-phosphorylated product Y*XXL
Antibodies and Fc receptors (FcR) ligands used for experiments included anti-Fc␥RI mAb 197 (Medarex, Annendale, NJ), mAb HB63, anti-Fc␥RI mAb 32.2 (Medarex), anti-Fc receptors (Fc␣R) mAbs A62 and A77
Transient Tyrosine Phosphorylations and the Effect of Vanadate—An early response by monocytic cells triggered through Fc␥RI is the tyrosine phosphorylation of several proteins including Fc␥RI ␥-chains [6, 7]

Summary

Introduction

Sequences [11] are substrates for Src family kinases [12] that are capable of reacting with the nonphosphorylated motif and of binding through SH2 domains to the tyrosine-phosphorylated product Y*XXL. Limitation to intracluster units is evidenced by sustained binding of Lyn to Fc⑀RI in isolated aggregates and phosphorylation of aggregate subunits in preference to an exogenously supplied substrate [14, 18]. Both kinds of evidence suggest a spatial restriction of kinase activity to aggregates. It has been suggested that restriction is due to a requirement for aggregated receptors as sites for kinase activation [14, 19] and to a requirement that substrate be in the aggregated state [18] Another possibility is that kinase activity is under a positive control preventing activity in nonaggregated receptors. The data in this report implicate phosphatases as necessary to prevent global FcR involvement and suggest that normal intracluster restriction of ␥-chain phosphorylation may be due to this vanadate-sensitive mechanism

Methods

Results

Conclusion