Human IL-3 induction of c-jun in normal monocytes is independent of tyrosine kinase and involves protein kinase C.
We have used normal human monocytes as a model system to begin elucidating the signal transduction mechanism associated with the IL-3R. Normal human monocytes deprived of human serum and CSF become quiescent in vitro. Stimulation of these cells with rIL-3 induces expression of the c-jun protooncogene, as detected by Northern blotting of total monocyte RNA. This protooncogene is also induced in these cells by phorbol ester through direct stimulation of protein kinase C. Concentrations of the protein kinase C inhibitor I-(5-isoquindinyl-sulfonyl)-2 methylpiperazine (H-7) between 30 and 100 microM (5-20 x Ki) inhibit this induction by phorbol ester. The same concentration-range of H-7 completely inhibited the induction of c-jun by human IL-3. A structural analog of H-7 designated HA-1004 preferentially inhibits cyclic nucleotide-dependent protein kinase rather that protein kinase C. HA-1004 at 5 to 20 x Ki did not inhibit IL-3-induced c-jun mRNA accumulation. Further 30 microM genistein that is an effective inhibitor of cellular tyrosine kinases did not inhibit IL-3-induced c-jun expression. Immunoprecipitation of lysates from [32P]orthophosphate labeled cells with antiphosphotyrosine polyclonal antibody showed that IL-3-stimulated phosphorylation of a 70-kDa protein and a 110-kDa protein on tyrosine, and that these protein phosphorylations were completely inhibited by 30 microM genistein. As further confirmation that IL-3 is stimulating protein kinase C in human monocytes we have found that IL-3 stimulates phosphorylation of the unique protein kinase C substrate myristoylated alanine-rich C kinase substrate in these cells. It is therefore likely that the interaction of IL-3 with its receptor generates diacylglycerol and stimulates the Ca2+/phospholipid-dependent protein kinase C.
Mufson
R A
RA
American Red Cross, Holland Laboratory for Biomedical Sciences, Rockville, MD 20892.
Szabo
J
J
Eckert
D
D
D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D004789:Enzyme Activation; D005786:Gene Expression Regulation / Q000187:drug effects*; D016758:Genes, jun; D019833:Genistein; D006801:Humans; D007377:Interleukin-3 / Q000494:pharmacology*; D007529:Isoflavones / Q000494:pharmacology; D007546:Isoquinolines / Q000494:pharmacology; D009000:Monocytes / Q000187:drug effects / Q000378:metabolism*; D010766:Phosphorylation; D010879:Piperazines / Q000494:pharmacology; D011493:Protein Kinase C / Q000502:physiology*; D011505:Protein-Tyrosine Kinases / Q000502:physiology*; D015398:Signal Transduction; D014443:Tyrosine / Q000378:metabolism
D007377:Interleukin-3; D007529:Isoflavones; D007546:Isoquinolines; D010879:Piperazines; D014443:Tyrosine; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D019833:Genistein; D011505:Protein-Tyrosine Kinases; D011493:Protein Kinase C