Protein kinases and phosphatases are involved in erythropoietin-mediated signal transduction.
To study the role of protein phosphorylation in erythropoietin (EPO)-mediated signal transduction, we examined the effects of tyrosine phosphatase and tyrosine and serine-threonine kinase inhibitors as well as activators of serine kinases on DNA synthesis and cell proliferation in the murine EPO-dependent cell line HCD-57. HCD-57 cells were obtained synchronized in G0 by centrifugal elutriation, and DNA synthesis was measured by incorporation of labeled thymidine into DNA. Half-maximal DNA synthesis was stimulated by 0.001 U/ml of EPO. Sodium orthovanadate (Na3VO4), a tyrosine phosphatase inhibitor, at 5 microM potentiated a subsaturating concentration of EPO. Na3VO4 alone stimulated HCD-57 DNA synthesis at concentrations of 0.1-20 microM. Zinc chloride, another tyrosine phosphatase inhibitor, also stimulated HCD-57 DNA synthesis at concentrations of 50-100 microM. Genistein, a tyrosine kinase inhibitor, blocked the effect of EPO at a concentration of 5 micrograms/ml. Bryostatin, a protein kinase C (PKC) activator, stimulated DNA synthesis in HCD-57 cells at concentrations of 10(-9)-10(-10) M, whereas the phorbol ester, phorbol 12,13-dibutyrate (PDBu), was stimulatory only at a concentration of 10(-11) M. Staurosporine, a PKC inhibitor, blocked the effect of EPO at a concentration of 10(-7) M, and H-7, a nonspecific protein kinase inhibitor, was not inhibitory. These agents also had similar effects on the in vitro proliferation of HCD-57 cells. Taken together, the data indicate that the EPO-mediated transition from G0 to S phase in HCD-57 cells involves the activation of both tyrosine and serine-threonine kinases and is modulated by tyrosine phosphatase activity.
Spivak
J L
JL
Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland.
Fisher
J
J
Isaacs
M A
MA
Hankins
W D
WD
D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D000470:Alkaloids / Q000494:pharmacology; D000818:Animals; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D004920:Erythropoiesis; D004921:Erythropoietin / Q000494:pharmacology*; D019833:Genistein; D066298:In Vitro Techniques; D007529:Isoflavones / Q000494:pharmacology; D007546:Isoquinolines / Q000494:pharmacology; D051379:Mice; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology; D010749:Phosphoprotein Phosphatases / Q000502:physiology*; D010879:Piperazines / Q000494:pharmacology; D011494:Protein Kinases / Q000502:physiology*; D017027:Protein Tyrosine Phosphatases / Q000037:antagonists & inhibitors; D015398:Signal Transduction; D019311:Staurosporine; D014638:Vanadates / Q000494:pharmacology; D015032:Zinc / Q000494:pharmacology
D000470:Alkaloids; D007529:Isoflavones; D007546:Isoquinolines; D010879:Piperazines; D004921:Erythropoietin; D015240:Phorbol 12,13-Dibutyrate; D014638:Vanadates; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D019833:Genistein; D011494:Protein Kinases; D010749:Phosphoprotein Phosphatases; D017027:Protein Tyrosine Phosphatases; D019311:Staurosporine; D015032:Zinc