Interleukin-3, GM-CSF, and TPA induce distinct phosphorylation events in an interleukin 3-dependent multipotential cell line.
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-stimulating 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 phosphotyrosine. 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 phosphotyrosine 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).
Sorensen
P H
PH
Terry Fox Laboratory, B.C. Cancer Research Centre, Vancouver, Canada.
Mui
A L
AL
Murthy
S C
SC
Krystal
G
G
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001853:Bone Marrow / Q000378:metabolism; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D003115:Colony-Stimulating Factors / Q000494:pharmacology*; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D006133:Growth Substances / Q000494:pharmacology*; D006412:Hematopoietic Stem Cells / Q000378:metabolism*; D006801:Humans; D007377:Interleukin-3 / Q000494:pharmacology*; D007700:Kinetics; D051379:Mice; D008810:Mice, Inbred C57BL; D010750:Phosphoproteins / Q000378:metabolism; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D011994:Recombinant Proteins / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D014443:Tyrosine / Q000378:metabolism
D000596:Amino Acids; D003115:Colony-Stimulating Factors; D006133:Growth Substances; D007377:Interleukin-3; D010750:Phosphoproteins; D010761:Phosphorus Radioisotopes; D011994:Recombinant Proteins; D014443:Tyrosine; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D013755:Tetradecanoylphorbol Acetate