Influence of tyrosine phosphorylation on protein interaction with FcγRIIa

Abstract

The cytoplasmic tail of Fc(gamma)RIIa present on human neutrophils shares with other antigen receptors a common amino acid sequence called ITAM (Immunoreceptor Tyrosine-based Activation Motif). After receptor ligation, the tyrosine residues within this motif become phosphorylated. We prepared a recombinant fusion protein of the cytoplasmic tail of Fc(gamma)RIIa (containing the ITAM) with glutathione-S-Transferase (GST-CT) to characterize the phosphorylation of Fc(gamma)RIIa and its ability to interact with other proteins involved in signal transduction. The GST-CT became phosphorylated in the presence of Lyn, Hck and Syk (immunoprecipitated from human neutrophils), but not in the presence of Fgr. Of the active kinases, only Lyn (mainly present in the membrane fraction) was found to associate with the GST-CT in the absence of ATP. This association was also observed in immunoprecipitates of Fc(gamma)RIIa from resting neutrophils, suggesting that Lyn might be the kinase responsible for the initial Fc(gamma)RIIa phosphorylation. Moreover, we observed specific association of Syk and the p85 subunit of PI 3-kinase after incubation of the GST-CT with neutrophil cytosol. This interaction was dependent on tyrosine phosphorylation of the GST-CT. Substitution of 269Tyr by Phe almost completely abolished tyrosine phosphorylation of the fusion protein. Substitution of either 253Tyr or 269Tyr eliminated Syk binding, but only 253Tyr appeared to be essential for p85 binding. We hypothesize that, upon activation, the membrane-associated Lyn is responsible for the initial tyrosine phosphorylation of Fc(gamma)RIIa, thus creating a docking site for Syk and PI 3-kinase.

The Ubiquitin Ligase c-Cbl Down-Regulates FcγRIIa Activation in Human Neutrophils

“…1C only). 3) CT10 is an IgG fraction of a polyclonal rabbit serum against the cytoplasmic domain of Fc␥RIIa raised against the synthetic peptide whose sequence was published by Ibarrola et al (43). It was used for immunoblotting.…”

Activation of Syk in Neutrophils by Antineutrophil Cytoplasm Antibodies Occurs via Fcγ Receptors and CD18

“…Syk is recruited to Fc␥RIIa after phosphorylation of critical immunoreceptor tyrosine activation motif (ITAM) tyrosine residues by a Src kinase constitutively associated with the receptor at the plasma membrane (26). Syk itself is then tyrosine phosphorylated, leading to its activation.…”

Modulation of Human Neutrophil Responses to CD32 Cross-Linking by Serine/Threonine Phosphatase Inhibitors: Cross-Talk Between Serine/Threonine and Tyrosine Phosphorylation

“…The observation that Cbl and Syk are rapidly tyrosine phosphorylated in response to calyculin is of potential physiological relevance because these two proteins are thought to be intimately involved in neutrophil activation (39,46,47). In neutrophils, Cbl is tyrosine phosphorylated in response to a variety of agonists including CD32 cross-linking, opsonized bacteria and zymosan, GM-CSF, monosodium urate, and calcium pyrophosphate microcrystals (46).…”

“…It has a membrane-spanning domain and an immunoreceptor tyrosine-based activation motif-containing cytoplasmic tail which has been shown to play an important signaling role (39). Although the signal transduction pathway in response to the ligation of this receptor is poorly understood in neutrophils, the data accumulated in other systems favor a model in which Cbl is a direct downstream target of Syk/Zap70, whereas members of the Src family of tyrosine kinases contribute indirectly to this process by the activation of Syk/ Zap70.…”

“…The polyclonal anti-phosphoserine (AB1603) and the monoclonal anti-Syk (MAB88906) Abs were obtained from Chemicon International (Mississauga, Ontario, Canada). The anti-CD32 polyclonal Ab against the cytoplasmic tail of CD32 was generated in our laboratory as described previously (39).…”