The mechanism of action of the hemopoietic growth factor, murine interleukin-3 (mIL-3), was investigated using an mIL-3-dependent multipotential hemopoietic cell line, B6SUtA. These cells were first used to identify the mIL-3 surface receptor as a monomeric 67 kDa protein with a pI of approximately 6.2. Further studies suggested the presence of an additional mIL-3 binding protein with an apparent molecular mass of 140 kDa. Then, in an attempt to gain some insights into the mechanism of action of mIL-3, molecules other than mIL-3 were tested to determine their effects on cell proliferation. Murine granulocyte -macrophage colony-stimulating factor (mGM-CSF) was found to be as potent as mIL-3 in stimulating B6SUtA cells. In addition, sodium orthovanadate, an inhibitor of phosphotyrosine phosphatase, and 12-0-tetradecanoyl-phorbol-13-acetate (TPA), a known activator of protein kinase C, both stimulated DNA synthesis in these cells, suggesting that protein phosphorylation might be involved in the mechanism of action of mIL-3 and mGM-CSF. To assess this possibility, intact B6SUtA cells exposed for brief periods to mIL-3, mGM-CSF or TPA were analyzed for changes in phosphorylation patterns using metabolic [superscript]32p-labeling. Both mIL-3 and mGM-CSF induced the serine-specific phosphorylation of a 68 kDa cytosolic protein, while all three agents stimulated the serine-specific phosphorylation of a 67 kDa membrane protein. Furthermore, using antibodies to phosphotyrosine, it appeared that mIL-3 stimulated tyrosine phosphorylation of 67 kDa and 140 kDa membrane proteins, as well as of 40, 55 and 90 kDa cytosolic proteins. The 90 kDa protein was also tyrosine phosphorylated in response to mGM-CSF, suggesting that this phosphorylation results from a common step in mIL-3 and mGM-CSF-stimulated signaling pathways. These phosphotyrosine containing proteins were not detected in TPA-treated cells. Moreover, evidence from a variety of studies is presented that the 140 kDa but not the 67 kDa mIL-3 receptor becomes phosphorylated on tyrosine residues when B6SUtA cells bind mIL-3. / Medicine, Faculty of / Pathology and Laboratory Medicine, Department of / Graduate