CSF-1 stimulated association ofcbl with signaling proteins in macrophages

We have reported that CSF-1 stimulation of macrophages leads to the transient tyrosine phosphorylalion, membrane localization and ubiquitination of the proto-oncogene product Cbl. To further study the kinetic relationships among these events, we stimulated cells for various times with CSF-1 at 4°C and analyzed Cbl immunoprecipiiations from cytosolic and membrane fractions. As early as 10 min of stimulation, Cbl was tyrosine phosphorylated and translocated from cytosolic to membrane fraction where it associated with CSF-1 R, Sos, p85 subunit of PI3 Kinase, She and an increased amount of Grb2. At about l h of stimulation Cbl ubiquitination was detected in the membrane fraction, and some of the phosphorylated and ubquitinated Cbl was also found in the cytosolic fraction. The association of Cbl with She and Grb2 persisted over 5 h of stimulation while the association with CSF-1R, Sos and some of p85 was transient and decreased over 20-40 min. Our results suggest that tyrosine phosphorylation, preceding ubquitination, is associated with Cbl translocation and association with signaling proteins in the membrane fraction. The function of Cbl ubquitination is not clear. However this kinetic analysis indicates that ubquitination of Cbl could be important for its dissociation from those signaling proteins and/or its movement back from the membrane to the cytoplasm.