PKC epsilon is involved in granulocyte-macrophage colony-stimulating factor signal transduction: evidence from microphysiometry and antisense oligonucleotide experiments.
We have used microphysiometry and antisense methodology to show that the epsilon isoenzyme of protein kinase C (PKC) is involved in the signal transduction pathway of granulocyte-macrophage colony-stimulating factor (GM-CSF) in a human bone marrow cell line, TF-1. These cells require GM-CSF or a related cytokine for proliferation. When the cells are appropriately exposed to GM-CSF, they exhibit a burst of metabolic activity that can be detected on the time scale of minutes in the microphysiometer, a biosensor-based instrument that measures the rate at which cells excrete protons. These cells express PKC alpha and -epsilon, as determined by Western blot analysis. Treatment with isoenzyme-specific antisense oligonucleotides inhibits expression appropriately, but only inhibition of PKC epsilon appreciably diminishes the burst of metabolic activity induced by GM-CSF. Consistent with the involvement of PKC epsilon, GM-CSF appears to activate phospholipase D and does not cause a detectable increase in cytosolic [Ca2+].
Baxter
G T
GT
Molecular Devices Corporation, Menlo Park, California 94025.
Miller
D L
DL
Kuo
R C
RC
Wada
H G
HG
Owicki
J C
JC
D000470:Alkaloids / Q000494:pharmacology; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D002413:Cations, Divalent; D002794:Choline / Q000378:metabolism; D004789:Enzyme Activation; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000378:metabolism*; D006801:Humans; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000378:metabolism*; D008969:Molecular Sequence Data; D016376:Oligonucleotides, Antisense / Q000737:chemistry*; D010739:Phospholipase D / Q000378:metabolism; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051381:Rats; D015398:Signal Transduction; D019311:Staurosporine
D000470:Alkaloids; D002413:Cations, Divalent; D007527:Isoenzymes; D016376:Oligonucleotides, Antisense; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D011493:Protein Kinase C; D010739:Phospholipase D; D019311:Staurosporine; D002794:Choline; D002118:Calcium