Crosslinking of membrane-bound IMmuno- (5-7). Associations of the interleukin 2 receptor f3 chain and T-cell antigen receptor with the Src-like kinases Lck and Fyn, respectively, were also reported (8-10). However, the physiological substrates or targets of these Src-like kinases in receptor-mediated signaling have yet to be identified. In recent studies on the targets of the tyrosine kinases, much information has been obtained about the plateletderived growth factor receptor (PDGF-R). The binding of PDGF to PDGF-R (J3 type) activates the receptor's kinase activity and induces its association with tyrosine-phosphorylated proteins such as phosphatidylinositol 3-kinase (PI 3-kinase), Ras GTPase-activating protein, and phospholipase C-y upon ligand-mediated signaling (11). One possible target of the tyrosine kinase, PI 3-kinase, is also associated with ligand-stimulated receptors for colony-stimulating factor 1 and insulin (11)(12)(13) and with an activated form of Src, p60-src (14). The levels ofproducts of PI 3-kinase are elevated in cells carrying these activated tyrosine kinases. Therefore, PI 3-kinase is likely to be an important target oftyrosine kinases.The lyn gene is a member of the src family and encodes two forms of protein-tyrosine kinase (Lyn), p531Yfl and p56IYn. 1118The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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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...

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