Cbl functions downstream of Src kinases in FcγRI signaling in primary human macrophages

Cbl is a cytosolic protein that is rapidly tyrosine phosphorylated hi response to Fc receptor activation and binds to the adaptor proteins Grb2, CrkL, and Nck, A few reports describe CLI interactions in primary human hematopoietic cells, We show evidence that Cbl participates in signaling initiated by Fc gamma RI receptor cross-linking in human primary macrophages, and functions downstream of Src family kinases in this pathway, Fc gamma RI stimulation in human macrophages was associated with rapid and transient tyrosine phosphorylation of the Cbl adaptor protein, Immunoprecipitated Cbl was complexed with several tyrosine phosphorylated proteins, the most prominent of which was a 38-kDa band identified as the CrkL adaptor protein, CrkL associated with tyrosine-phosphorylated Cbl and itself became tyrosine phosphorylated after Fc gamma RI cross-linking. SLP-76, a recently cloned Grb2-associated protein, was strongly tyrosine phosphorylated after Fc gamma RI stimulation and was associated with both Cbl and Grb2, Grb2 and Cbl binding to SLP-76 were inducible after Fc gamma RI stimulation of the macrophages. Nck was inducibly bound to Cbl after Fc gamma RI stimulation, whereas Grb2 was constitutively associated with it. She was also inducibly tyrosine phosphorylated and bound to Grb2 after Fc gamma RI stimulation of the macrophages, PP1, a specific inhibitor of Src kinases, inhibited the Fc gamma RI-induced respiratory hurst, as well as the tyrosine phosphorylation of Cbl and its inducible association with CrkL, These results suggest a fundamental role for the tyrosine phosphorylation of Cbl, CrkL, SLP-76, and She and the association of Cbl with CrkL, SLP-76, and Nck in Fc gamma RI signaling in human macrophages. Experiments performed with PP1, the specific Src kinase inhibitor, demonstrate the first evidence that Cbl and the Cbl-Crkl interaction are downstream targets for myeloid Src kinases required for the activation of myeloid NADPH oxidase activity.