Differential interaction of Crkl with Cbl or C3G, Hef-1, and γ subunit immunoreceptor tyrosine-based activation motif in signaling of myeloid high affinity Fc receptor for IgG (FcγRI)

Cbl-Crkl and Crkl-C3G interactions have been implicated in T cell and B cell receptor signaling and in the regulation of the small GTPase, Rap1. Recent evidence suggests that Rap1 plays a prominent role in the regulation of immunoreceptor tyrosine-based activation motif (ITAM) signaling. To gain insight into the role of Crkl in myeloid ITAM signaling, we investigated Cbl- Crkl and Crkl-C3G interactions following FcγRI aggregation in U937IF cells. FcγRI cross-linking of U937IF cells results in the tyrosine phosphorylation of Cbl, Crkl, and Hef-1, an increase in the association of Crkl with Cbl via direct SH2 domain interaction and increased Crkl-Hef-1 binding. Crkl constitutively binds to the guanine nucleotide-releasing protein, C3G, via direct SH3 domain binding. Our data show that distinct Cbl-Crkl and Crkl-C3G complexes exist in myeloid cells, suggesting that these complexes may modulate distinct signaling events. Anti-Crkl immunoprecipitations demonstrate that the ITAM-containing γ, subunit of FcγRI is induced to form a complex with the Crkl protein, and Crkl binds to the cytoskeletal protein, Hef-1. The induced association of Crkl with Cbl, Hef-1, and FcγRIγ after FcγRI activation and the constitutive association between C3G and Crkl provide the first evidence that a FcγRIγ-Crkl-C3G complex may link ITAM receptors to the activation of Rap1 in myeloid cells.