Role of protein phosphorylation in EPO-mediated early signal transduction: analysis in the EPO-reactive cell line ELM-I-1 transfected with a c-fos-enhancer/promoter-luciferase reporter gene.
To investigate the role of protein phosphorylation in the early phase of EPO-mediated signal transduction, we EPO-stimulated a murine erythroid cell line ELM-I-1 transformed by plasmids comprised of the c-fos enhancer/promoter linked to the luciferase gene. Using this reporter gene system, we previously showed that EPO-induced activation of the c-fos promoter can be detected rapidly and sensitively as an elevation of cellular luciferase activity. In this study, we first examined the role of protein tyrosine phosphorylation. The tyrosine phosphatase inhibitor orthovanadate not only induced luciferase activity by itself but enhanced the action of EPO. On the other hand, the tyrosine kinase inhibitors erbstatin and herbimycin suppressed the effect of EPO. Next, the role of protein kinase C (PKC) in the EPO response was assessed. The PKC activator phorbol myristate acetate (PMA) not only induced luciferase activity by itself but enhanced the action of Epo. On the other hand, the PKC inhibitor 1-(5-isoquinolynyl-sulfonyl)-2-methylpiperazine (H7) suppressed the effect of Epo and PMA, whereas a nonspecific protein kinase inhibitor, N-(2-Guanidinoethyl)-5-Isoquinolinesulfornamine (HA1004) inhibited the action of neither Epo nor PMA. Another known PKC inhibitor staurosporine (STSP) did not inhibit but rather enhanced the effect of Epo. This action of STSP was blocked by H7 but not by HA1004. These results suggest that the EPO-mediated early signal transduction pathway leading to c-fos expression involves protein-tyrosine phosphorylation, is modulated by tyrosine phosphatase activity and is positively regulated by PKC.
Tsuda
H
H
Division of Clinical Hematology/Immunology, Kumamoto City Hospital, Japan.
Huang
R W
RW
Takatsuki
K
K
D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D000470:Alkaloids / Q000494:pharmacology; D000818:Animals; D016227:Benzoquinones; D002460:Cell Line; D004921:Erythropoietin / Q000494:pharmacology*; D016762:Genes, fos; D006873:Hydroquinones / Q000494:pharmacology; D066298:In Vitro Techniques; D007546:Isoquinolines / Q000494:pharmacology; D047029:Lactams, Macrocyclic; D008156:Luciferases; D051379:Mice; D010750:Phosphoproteins / Q000378:metabolism; D010879:Piperazines / Q000494:pharmacology; D011401:Promoter Regions, Genetic; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000502:physiology*; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000502:physiology*; D011809:Quinones / Q000494:pharmacology; D017828:Rifabutin / Q000031:analogs & derivatives; D015398:Signal Transduction; D019311:Staurosporine; D014638:Vanadates / Q000494:pharmacology
D000470:Alkaloids; D016227:Benzoquinones; D006873:Hydroquinones; D007546:Isoquinolines; D047029:Lactams, Macrocyclic; D010750:Phosphoproteins; D010879:Piperazines; D011809:Quinones; D004921:Erythropoietin; D017828:Rifabutin; D014638:Vanadates; C020754:herbimycin; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D008156:Luciferases; D011505:Protein-Tyrosine Kinases; D011493:Protein Kinase C; D019311:Staurosporine; C048212:erbstatin