Granulocyte‐Macrophage colony‐stimulating factor and steel factor induce phosphorylation of both unique and overlapping signal transduction intermediates in a human factor‐dependent hematopoietic cell line

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Abstract
Steel factor (SF), the ligand for the proto‐oncogene c‐kit, acts synergistically with GM‐CSF or IL‐3 to support the growth of normal human hematopoietic progenitor cell. We examined the effect of SF on GM‐CSF or IL‐3 induced proliferation of a human factor‐dependent cell line, MO7. SF supported MO7 cell proliferation as well as IL‐3 or GM‐CSF alone, and its addition dramatically enhanced (three‐to sixfold) maximal GM‐CSF or IL‐3 stimulated proliferation. SF did not increase the number of affinity of cell surface GM‐CSF receptors. We examined several early events of signal tranduction in an effort to elucidate the biochemical mechanisms of synergy of these factors. Since each of these three cytokines is believed to function in part through activation of a tyrosine kinse, we examined their effects on cellular phosphotyrosine containing proteins. Each cytokine induced rapid, transient, and concentration dependent tyrosine phosphorylation of a number of substrates. For GM‐CSF and IL‐3, these phosphoproteins were indistinguishable (150, 125, 106, 93, 80, 79, 73, 44, 42, and 36 kDa), while SF induced major or minor tyrosine phosphorylation of 205, 140–150, 116, 106, 90, 90, 80, 79, 73, 44, 42, 39, 36, 32 kDa phosphoproteins. Two other signal tranduction intermediates known to be phosphorylated and activated by GM‐CSF and IL‐3, the 70–75 kDa Raf‐1 kinase, and p42 mitogen‐activated protein kinase‐2 (MAPK) were also phosphorylated by SF. Combinations of GM‐CSF or IL‐3 with SF did not further increase the phosphorylation of Raf‐1 or p42 MAPK when compared to any of the factors alone. In contrast SF, but not GM‐CSF or IL‐3, induced tyrosine phosphorylation of phospholipase C‐γ (PLC‐γ). These results indicate that SF and GM‐CSF/IL‐3 have partially overlapping effects on early signal tranducing events, as well as striking differences, such as tyrosine phophorylation of PLC‐γ. This cell line should provide a useful model system to investigate the complicated process of hematopoietic growth factor synergy.