Interleukin-3, GM-CSF, and TPA Induce Distinct Phosphorylation Events in an Interleukin 3-Dependent Multipotential Cell Line

• Published in: Blood 1989-02, Vol.73 (2), p.406-418
• Main Authors: Sorensen, Poul H.B., Mui, Alice L-F., Murthy, Sudish C., Krystal, Gerald
• Format: Article
• Language: English
• Subjects: Amino Acids - analysis; Animals; Biological and medical sciences; Bone Marrow - metabolism; Cell Division - drug effects; Cell Line; Colony-Stimulating Factors - pharmacology; Fundamental and applied biological sciences. Psychology; Granulocyte-Macrophage Colony-Stimulating Factor; Growth Substances - pharmacology; Hematopoietic Stem Cells - metabolism; Humans; Interleukin-3 - pharmacology; Kinetics; Mice; Mice, Inbred C57BL; Phosphoproteins - metabolism; Phosphorus Radioisotopes; Phosphorylation; Recombinant Proteins - pharmacology; Tetradecanoylphorbol Acetate - pharmacology; Tyrosine - metabolism; Vertebrates: blood, hematopoietic organs, reticuloendothelial system
• ISSN: 0006-4971; 1528-0020
• DOI: 10.1182/blood.V73.2.406.406

The mechanism of action of the hemopoietic growth factor, murine interleukin-3 (mIL-3), was investigated using an mIL-3-dependent multipotential hematopoietic cell line, B6SUtA1. Murine granulocyte-macrophage colony-stimu-lating factor (mGM-CSF) was as potent as mIL-3 in stimulating these cells. In addition, sodium orthovanadate, an inhibitor of phosphotyrosine phosphatase, and 12-O-tetradecanoyl-phorbol-13-acetate (TPA), a known activator of protein kinase C, also 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 B6SUtA1 cells exposed for brief periods to mIL-3, mGM-CSF, and TPA were analyzed for changes in phosphorylation patterns using metabolic 32P-labeling and antibodies to phosphoty- rosine. Both mIL-3 and mGM-CSF induced the serine-specific phosphorylation of a 68-Kd cytosolic protein, whereas all three agents stimulated the serine-specific phosphorylation of a 68-Kd membrane protein. Furthermore, mIL-3 stimulated tyrosine phosphorylation of the 68-Kd membrane protein, as well as of 140-, 90-, 55, and 40-Kd proteins. The 90-Kd protein was also tyrosine phosphorylated in response to mGM-CSF. These phosphotyro-sine containing proteins were not detected in TPA-treated cells. These results indicate that protein phosphorylations on tyrosine and serine residues occur in B6SUtA1 cells following short-term incubation with mIL-3 or mGM-CSF and that most of these phosphorylation events are mediated by kinases other than protein kinase C (PkC).