Human IL-3 receptor signaling: rapid induction of phosphatidylcholine hydrolysis is independent of protein kinase C but dependent on tyrosine phosphorylation in transfected NIH 3T3 cells.
Although tyrosine kinases are clearly activated after ligand engagement of the human IL-3R in both hematopoietic and nonhematopoietic cytoplasmic environments, a role for phospholipid hydrolysis and protein kinase C in IL-3R signal transduction is emerging. We have used NIH 3T3 cells transiently transfected with human IL-3R alpha- and beta-subunits to study phosphatidylcholine hydrolysis in response to human IL-3. We have found that NIH 3T3 cells expressing the complete human IL-3R respond to human IL-3 with a rapid and sustained increase in sn-1'2'-diacylglycerol. Accompanying this was a rapid increase in intracellular levels of phosphorylcholine. The protein kinase C inhibitor H-7, however, was not effective in inhibiting phosphatidylcholine hydrolysis in response to human IL-3 in NIH 3T3 cells expressing the human receptor. Thus the human IL-3R induces a rapid protein-kinase-C-independent hydrolysis induced by the murine receptor. Simultaneous with the increase in phosphatidylcholine hydrolysis induced by the murine receptor. Simultaneous with the increase in diacylglycerol levels was an increase in membrane-bound protein kinase C enzyme activity. Immunoblotting with isoform-specific Abs against protein kinase C showed that, whereas the zeta-isoform is constitutively membrane bound, the alpha-isoform of protein kinase C is translocated to the membrane in response to IL-3. Activation of phosphatidylcholine hydrolysis and protein kinase C activation required both alpha- and beta-receptor subunits. To determine the relationship of tyrosine phosphorylation to the activation of phosphatidylcholine hydrolysis and protein kinase C translocation, we used the specific and structurally unrelated tyrosine kinase inhibitors genistein and herbimycin. Both inhibitors effectively blocked human IL-3-induced tyrosine phosphorylation. In addition, both inhibitors blocked phosphatidylcholine hydrolysis and protein kinase C translocation. These data, combined with our previous report showing that c-jun induction by IL-3 is dependent on protein kinase C, suggest that, in hematopoietic and nonhematopoietic cells expressing the human IL-3R, phosphatidylcholine hydrolysis and protein kinase C are downstream effectors of tyrosine phosphorylation in the IL-3 signal transduction cascade resulting in immediate early response gene induction.
Holland Laboratory for BioMedical Science, American Red Cross, Rockville, MD 20855.
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P
Rao
T
T
Kitamura
A
A
Miyajima
R A
RA
Mufson
D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D016475:3T3 Cells; D000818:Animals; D016227:Benzoquinones; D018076:DNA, Complementary / Q000235:genetics; D004075:Diglycerides / Q000096:biosynthesis; D005786:Gene Expression Regulation; D017781:Genes, Immediate-Early; D016758:Genes, jun; D019833:Genistein; D006868:Hydrolysis; D007377:Interleukin-3 / Q000494:pharmacology; D007529:Isoflavones / Q000494:pharmacology; D007546:Isoquinolines / Q000494:pharmacology; D047029:Lactams, Macrocyclic; D051379:Mice; D010713:Phosphatidylcholines / Q000378:metabolism*; D010766:Phosphorylation; D010879:Piperazines / Q000494:pharmacology; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000502:physiology; D011499:Protein Processing, Post-Translational; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000502:physiology*; D011809:Quinones / Q000494:pharmacology; D016185:Receptors, Interleukin-3 / Q000502:physiology*; D011994:Recombinant Proteins / Q000494:pharmacology; D017828:Rifabutin / Q000031:analogs & derivatives; D015398:Signal Transduction / Q000502:physiology*; D015533:Transcriptional Activation; D014162:Transfection
C011439:1,2-diacylglycerol; D016227:Benzoquinones; D018076:DNA, Complementary; D004075:Diglycerides; D007377:Interleukin-3; D007529:Isoflavones; D007546:Isoquinolines; D047029:Lactams, Macrocyclic; D010713:Phosphatidylcholines; D010879:Piperazines; D011809:Quinones; D016185:Receptors, Interleukin-3; D011994:Recombinant Proteins; D017828:Rifabutin; C020754:herbimycin; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D019833:Genistein; D011505:Protein-Tyrosine Kinases; D011493:Protein Kinase C