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Abstract
BackgroundFcγ receptors mediate important biological signals in myeloid cells including the ingestion of microorganisms through a process of phagocytosis. It is well-known that Fcγ receptor (FcγR) crosslinking induces the tyrosine phosphorylation of CBL which is associated with FcγR mediated phagocytosis, however how signaling molecules coordinate to desensitize these receptors is unclear. An investigation of the mechanisms involved in receptor desensitization will provide new insight into potential mechanisms by which signaling molecules may downregulate tyrosine phosphorylation dependent signaling events to terminate important signaling processes.ResultsUsing the U937IF cell line, we observed that FcγR1 crosslinking induces the tyrosine phosphorylation of CBL, which is maximal at 5 min. followed by a kinetic pattern of dephosphorylation. An investigation of the mechanisms involved in receptor desensitization revealed that pretreatment of U937IF or J774 cells with PMA followed by Fcγ receptor crosslinking results in the reduced tyrosine phosphorylation of CBL and the abrogation of downstream signals, such as CBL-CRKL binding, Rac-GTP activation and the phagocytic response. Pretreatment of J774 cells with GF109203X, a PKC inhibitor was observed to block dephosphorylation of CBL and rescued the phagocytic response. We demonstrate that the PKC induced desensitization of FcγR/ phagocytosis is associated with the inactivation of Rac-GTP, which is deactivated in a hematopoietic specific phosphatase SHP1 dependent manner following ITAM stimulation. The effect of PKC on FcγR signaling is augmented by the transfection of catalytically active SHP1 and not by the transfection of catalytic dead SHP1 (C124S).ConclusionsOur results suggest a functional model by which PKC interacts with SHP1 to affect the phosphorylation state of CBL, the activation state of Rac and the negative regulation of ITAM signaling i.e. Fcγ receptor mediated phagocytosis. These findings suggest a mechanism for Fcγ receptor desensitization by which a serine-threonine kinase e.g. PKC downregulates tyrosine phosphorylation dependent signaling events via the reduced tyrosine phosphorylation of the complex adapter protein, CBL.
Highlights
Fcγ receptors mediate important biological signals in myeloid cells including the ingestion of microorganisms through a process of phagocytosis
We have investigated the interplay between Protein kinase C (PKC) and SHP1 to regulate the deactivation of the Fcγ receptor, which leads to inhibition of phagocytic signal transduction cascade
Our results show the kinetics of phosphorylation and dephosphorylation of CBL immunoprecipitated from resting and FcγR1 stimulated U937IF cells differentiated with human interferon gamma (250 U/ml) for 5 days
Summary
Fcγ receptors mediate important biological signals in myeloid cells including the ingestion of microorganisms through a process of phagocytosis It is well-known that Fcγ receptor (FcγR) crosslinking induces the tyrosine phosphorylation of CBL which is associated with FcγR mediated phagocytosis, how signaling molecules coordinate to desensitize these receptors is unclear. FcγRI, the high-affinity Fc receptor for monomeric IgG (CD64) and FcγRIIIA (CD16), the low affinity Fc receptor for IgG, are members of the immunoglobulin gene super family, which includes the T cell receptor, the B cell receptor, and Fc receptors such as the multi-subunit immune receptors (IRs) for IgE and IgG [1] Both FcγRI and FcγRIIIA receptors signal through the 7 kd FcγRIγ subunit ITAM (termed the gamma subunit), whereas the FcγRIIA receptor contains a receptor intrinsic ITAM motif and the FcγRIIb receptor contains an immunoreceptor tyrosine inhibitory motif, ITIM [2]. It has been suggested that CBL can serve as a linker for phosphatidyl-inositol-3 kinase (PI3Kinase) via Y731 site [16,17,18]
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