Identification and subcellular localization of proteins that are rapidly phosphorylated in tyrosine in response to colony-stimulating factor 1.

Abstract

To investigate growth factor-mediated signal transduction, we have studied phosphorylation events that take place within seconds of the binding of colony-stimulating factor 1 (CSF-1) to its cell-surface receptor. CSF-1 stimulated rapid tyrosine phosphorylation of cellular proteins in murine BAC1.2F5 macrophages at 3TC and 4@C. The pattern of CSF-1-stimulated tyrosine phosphorylation of at least 15 different proteins at both temperatures was similar and unchanged by treatment of the lysate with reducing agent. With the exception ofthe 185-kDa CSF-1 receptor, a 260-kDa protein and a 133-kDa protein, the proteins were predominantly cytoplasmic. At 37°C, all the proteins were phosphorylated within 30 sec of addition of growth factor. At 4°C, CSF-1 receptor sites were saturated after 2 min of incubation in the presence of high concentrations of CSF-1 and differences in the order of appearance of phosphorylated proteins were observed: 185 kDa (CSF-1 receptor) (by 2 min); 99 kDa (by 4 min); 125 kDa (by 10 nmn); 61 kDa (by 30 min); and 260 kDa, 84 kDa, and 41 kDa (by 180 min). In addition to stimulating the phosphorylation of these proteins in tyrosine, CSF-1 caused dephosphorylation of phosphorylated serine residues on the receptor. As neither CSF-1 nor its receptor is internalized at 4°C, analysis of these early reactions and the phosphotyrosinecontaining proteins in intact cells under these conditions should lead to an understanding of the early events in growth factor receptor-mediated signal transduction.The purification and cloning, of the receptor for several growth factors, including epidermal growth factor, insulin, platelet-derived growth factor, colony-stimulating factor 1 (CSF-1), and insulin-like growth factor have indicated that they are tyrosine kinases, possessing an extracellular ligandbinding domain and an intracellular tyrosine kinase domain (reviewed in ref. 1). Furthermore, recent studies with the receptors for insulin and epidermal growth factor indicate that their tyrosine kinase activity is essential for signal transduction (2-4). Thus, a major question concerning the action ofthese growth factors is the nature and function ofthe intracellular physiological substrates of their receptor ki- (5, 6). The CSF-1 receptor is a tyrosine kinase (7), which has been shown to be the c-fms protooncogene product (8)(9)(10). The mechanism by which CSF-1 stimulates survival and proliferation can be conveniently studied in the CSF-1-dependent murine macrophage cell line BAC1.2F5 (11,12). In this paper, we describe conditions for CSF-1 stimulation at 4°C, under which the very early postreceptor tyrosine phosphorylation events in the CSF-1 response in BAC1.2F5 cells can be studied. This temperature not only slows down the rate of these phosphorylation reactions but also prevents ligand or receptor internalization (13). We also identify several proteins that are very rapidly phosphorylated in tyrosine in response to CSF-1 and that may play an important role in the regulation of cells by this growth factor....

Macrophage colony-stimulating factor is indispensable for both proliferation and differentiation of osteoclast progenitors.

“…Signal transduction pathways through M-CSF receptors have been investigated in bone marrow-derived macrophages and several established cell lines of the monocyte-macrophage lineage. In the M-CSF (CSF-1)-dependent mouse macrophage cell line BAC 1 .2F5, M-CSF stimulated the rapid tyrosine phosphorylation of several, predominantly cytoplasmic, proteins (18,28,29) that was followed by serine phosphorylation of the cytoplasmic protooncogene product, RAF-1, and activation of the RAF-1-associated seine kinase (30). Using a murine macrophage cell line, P388D 1, Varticovski et al reported that M-CSF receptors activated phosphatidylinositol-3 kinase, which may play an important role in the signal transduction pathway of M-CSF (31 ).…”

Macrophage colony-stimulating factor-induced tyrosine phosphorylation of c-fms proteins expressed in FDC-P1 and BALB/c 3T3 cells.

“…In intact cells, the tyrosine kinase activity of the c-fms protein is activated after M-CSF binding (5). Stimulation of the tyrosine kinase activity results in the phosphorylation of the c-fms protein plus several other cellular proteins which are thought to mediate the biological effects of M-CSF (5,35). These substrate proteins have not been identified other than on the basis of their molecular weight.…”

Colony-stimulating factor-1 induces cytoskeletal reorganization and c-src-dependent tyrosine phosphorylation of selected cellular proteins in rodent osteoclasts.

“…Ligand-dependent receptor dimerization and activation lead to rapid tyrosine phosphorylation of selected cellular proteins and the recruitment of signaling molecules to the receptor (20,21). Among these is c-src , a ubiquitous cellular tyrosine kinase (22).…”