Proteomic analysis of macrophage differentiation - p46/52(Shc) tyrosine phosphorylation is required for CSF-1-mediated macrophage differentiation

Macrophage colony stimulating factor (M-CSF or CSF-1) acts to regulate the development and function of cells of the macrophage lineage. Murine myeloid FDC-P1 cells transfected with the CSF-1 receptor (FD/WT) adopt a macrophag e-like morphology when cultured in CSF-1, This process is abrogated in FDC- P1 cells transfected with the CSF-1 receptor with a tyrosine to phenyalanin e substitution at position 807 (PD/807), suggesting that a molecular intera ction critical to differentiation signaling is lost (Bourette, R. P., Myles , G. M., Carlberg, K., Chen, A. R., and Rohrschneider, L. R. (1995) Cell Gr owth Differ, 6, (631-645). A detailed examination of lysates of CSF-1-treat ed FD/807 cells by two-dimensional SI)S-polyacrylamide gel electrophoresis (PAGE) revealed a number of proteins whose degree of tyrosine phosphorylati on was modulated by the Y807F mutation. Included in this category were thre e phosphorylated proteins that co-migrated with p46/52(Sch), Immunoprecipit ation, Western blotting, and in vitro binding studies suggest that they are indeed p46/52(Shc). A key regulator of differentiation in a number of cell systems, ERK was observed to exhibit an activity that correlated with the relative degree of differentiation induced by CSF-1 in the two cell types. Transfection of cells with a non-tyrosine-phosphorylatable form of p46/52(S hc) prevented the normally observed CSF-1-mediated macrophage differentiati on as determined by adoption of macrophage-like morphology and expression o f the monocyte/macrophage lineage cell surface marker, Mac-1. These results are the first to suggest that p46/52(Shc) may play a role in CSF-1-induced macrophage differentiation. Additionally, a number of proteins were identi fied by two-dimensional SDS-PAGE whose degree of tyrosine phosphorylation i s also modulated by the Y807F substitution. This group of molecules may con tain novel signaling molecules important in macrophage differentiation.