Signaling proteins in the response of macrophages to CSF-1: pp57, EF-1 alpha c-Cbl

Abstract

Regulation of mononuclear phagocyte survival, proliferation and differentiation by colony-stimulating factor-1 (CSF-1) is mediated by the CSF-1 receptor (CSF-1R) tyrosine kinase. CSF-1 binding initially causes non-covalent dimerization and tyrosine phosphorylation of the CSF-1R. This is followed by the tyrosine phosphorylation of other cellular proteins. The identities of many of the signaling proteins were unknown and their roles in the CSF-1 regulated pathways were not well characterized. To further study the mechanism of the CSF-1 action, this study has identified and characterized three proteins involved in CSF-1 signal transduction in macrophages.;A 57kDa tyrosine-phosphorylated protein (pp57) and a 55kDa non-tyrosine phosphorylated protein (p55) were purified from the cytosolic fraction of BAC1.2F5 cells. Sequence analysis of the purified proteins indicated that pp57 is a novel protein and p55 is elongation factor-1 alpha (EF-1alpha). Although EF-1alpha is not tyrosine-phosphorylated, its association with tyrosine-phosphorylated protein(s) suggests that it may be involved in CSF-1 signaling.;Addition of CSF-1 to macrophages stimulates the rapid, transient tyrosine phosphorylation, membrane association and multiubiquitination of Cbl, a 120kDa protooncoprotein. Kinetic analysis reveals that the tyrosine phosphorylation of Cbl is coincident with its plasma membrane translocation and association with signaling proteins and that these events precede the simultaneous multiubiquitination of Cbl and the CSF-1R. The multi-ubiquitinated Cbl is not degraded but recovered in deubiquitinated form in the cytosol.;The role of Cbl in the response of macrophages to CSF-1 was further studied in bone marrow-derived macrophages (BMM) from Cbl knockout mice (Cbl-/-). Compared with wild type BMM, Cbl-/- BMM had a more transformed colonial morphology, a lower degree of protein tyrosine phosphorylation and a reduced rate of the CSF-1R multiubiquitination and degradation. Treatment of Cbl-/- BMM with iodoacetic acid or pervanadate at 4°C restored the protein tyrosine phosphorylation but not the CSF-1R multiubiquitination. Our data suggest that the predicted negative regulatory role of Cbl is via its regulation of CSF-1R tyrosine phosphorylation and multiubiquitination that in turn regulate ligand-induced CSF-1R internalization and/or degradation.