Human c-fgr induces a monocyte-specific enzyme in NIH 3T3 cells.

Abstract

The mutant c-fgr protein (p58c-fgr/F523) containing Phe-523 instead of Tyr-523 exhibited transforming activity in NIH 3T3 cells like other protein-tyrosine kinases of the src family, but normal p58c-fgr (p5gc-fgIw't) did not.The mutant protein showed tyrosine kinase activity threefold higher than that of the normal protein in vitro. 58-, 62-, 75-, 120-, 200-, and 230-kDa proteins were commonly phosphorylated at tyrosine residues in NIH 3T3 cells transfected with normal and mutated c-fgr, while 95-kDa protein was significantly phosphorylated at tyrosine residues in cells transfected with the mutated c-fgr. These findings suggest that tyrosine phosphorylation of specific cellular substrate proteins is important in induction of NaF-sensitive a-NBE and cell transformation by p58-fg'.Nearly half the retroviral transforming genes described to date encode proteins with protein-tyrosine kinase activity. The protein-tyrosine kinases represented by the src protooncogene do not contain extracellular or transmembrane sequences, and so the physiological functions of their products have been extremely difficult to analyze (8,14). Clues to the functions of src kinases may be the properties of the cells in which they are expressed naturally; many src family kinases are found naturally in differentiated cell types with limited proliferative potentials (4,12,27). Therefore, it is tempting to postulate that these src family kinases play roles in controlling differentiated functions. Indeed, it was previously reported that the v-src gene causes PC12 pheochromocytoma cells to differentiate into neuronlike cells (2). Recently, the lck kinase was reported to be activated by association with CD4 or CD8 molecules in T lymphocytes (29,30 As the next step in elucidating the physiological functions of p58c-fgr, we examined the biological properties of cells that express high levels of p58c-fgr as a consequence of transfection with vectors containing the normal or mutated (i.e., the carboxy-terminal Tyr-523 substituted with Phe-523) version of the human c-fgr cDNA. We also analyzed tyrosine phosphorylation in these cells with antiphosphotyrosine antibodies. MATERUILS AND METHODSCells and cell culture. NIH 3T3 cells were cultured in Dulbecco's modified Eagle minimal essential medium with 5% calf serum. The murine myelomonocytic cell line WEHI-3B (31) was provided by the Japanese Cancer Research Resources Bank. Cells were cultured in Dulbecco's modified Eagle minimal essential medium with 5% fetal calf serum. la,25-Dihydroxyvitamin D3 was kindly provided by the Chugai Pharmaceutical Co., Ltd., Tokyo, Japan. In order to induce differentiation of WEHI-3B cells, la,25-dihydroxyvitamin D3 was solubilized in absolute ethanol (final concentration, 0.01%) and added to the growth medium (Dulbecco's modified Eagle minimal essential medium plus 1% fetal calf serum) to a final concentration of 10-8 M (1). Cells were collected 4 days later, when more than 50% of the cells were positive for a-naphthyl butyrate esterase (a-NBE).Induction of murine perit...

Association of immunoglobulin G Fc receptor II with Src-like protein-tyrosine kinase Fgr in neutrophils.

“…This fact suggests that the physiological role of Fgr is associated with functions in differentiated cells. Indeed, we previously found that Fgr induced a monocyte-specific enzyme, NaF-sensitive a-naphthyl butyrate esterase, in NIH 3T3 mouse cells (30). In the present study, we have found that Fgr is associated with and comodulated with Fc4Il and is activated after receptor engagement with specific mAbs or natural ligands such as heat-aggregated human IgG, suggesting that Fgr is physically and functionally associated with FcRII in neutrophils.…”