Erythropoietin modulation of intracellular calcium: a role for tyrosine phosphorylation.
We have reported that erythropoietin induces a dose-dependent increase in cytosolic calcium ([Cai]) in single human peripheral blood BFU-E derived erythroblasts which is specific for stage of differentiation and that this increase is modulated by erythropoietin through an ion channel permeable to Ca2+. Here, the role of protein phosphorylation in the increase in intracellular free calcium [Cai] stimulated by erythropoietin was studied with digital video imaging. Preincubation of day 10 erythroblasts with a broad inhibitor of serine/threonine and tyrosine kinases, staurosporine (100 nM), blocked the increase in [Cai] over 20 min following erythropoietin stimulation. However, erythropoietin-induced calcium influx was unaffected by preincubation of cells with specific inhibitions of protein kinase C (calphostin C) or the cAMP- or cGMP-dependent kinases (KT 5720, HA 1004), and [Cai] did not increase following stimulation with phorbol 12-myristate 13-acetate (PMA) or dibutyryl cAMP. These results suggest that neither protein kinase C nor protein kinase A mediate the erythropoietin-induced [Cai] increase. In contrast, preincubation with genistein, a tyrosine kinase inhibitor, blocked the erythropoietin induced increase in [Cai]. To further study calcium entry in erythroblasts, we determined mastoparan, a peptide from wasp venom, induced a dose-dependent rise in [Cai] in erythroblasts which required external calcium. Stimulation of erythroid precursors with 10 microM mastoparan resulted in an increase in [Cai] from 52 +/- 3 nM to 214 +/- 36 nM which peaked at 20 min. The mastoparan-induced [Cai] increase was also dependent on tyrosine phosphorylation since it was blocked by preincubation with genistein. These results demonstrate that both erythropoietin and mastoparan stimulate calcium entry by a mechanism which has a genistein sensitive step and suggest that tyrosine kinase activation is required for the rise in [Cai] to occur.
Miller
B A
BA
Department of Pediatrics, Milton S. Hershey Medical Center, Pennsylvania State University, Hershey.
Bell
L L
LL
Lynch
C J
CJ
Cheung
J Y
JY
D000328:Adult; D000470:Alkaloids / Q000494:pharmacology; D000703:Analog-Digital Conversion; D001692:Biological Transport / Q000187:drug effects; D003994:Bucladesine / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D002227:Carbazoles; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors; D015672:Erythroid Precursor Cells / Q000187:drug effects* / Q000378:metabolism; D004921:Erythropoietin / Q000494:pharmacology*; D019833:Genistein; D006801:Humans; D007091:Image Processing, Computer-Assisted; D007211:Indoles / Q000494:pharmacology; D036341:Intercellular Signaling Peptides and Proteins; D007424:Intracellular Fluid / Q000187:drug effects / Q000378:metabolism; D007529:Isoflavones / Q000494:pharmacology; D007546:Isoquinolines / Q000494:pharmacology; D009281:Naphthalenes; D010455:Peptides; D010766:Phosphorylation; D011083:Polycyclic Compounds / Q000494:pharmacology; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D011499:Protein Processing, Post-Translational; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000502:physiology*; D011758:Pyrroles / Q000494:pharmacology; D015398:Signal Transduction; D019311:Staurosporine; D013449:Sulfonamides; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014743:Videotape Recording; D014862:Wasp Venoms / Q000494:pharmacology
D000470:Alkaloids; D002227:Carbazoles; D007211:Indoles; D036341:Intercellular Signaling Peptides and Proteins; D007529:Isoflavones; D007546:Isoquinolines; D009281:Naphthalenes; D010455:Peptides; D011083:Polycyclic Compounds; D011758:Pyrroles; D013449:Sulfonamides; D014862:Wasp Venoms; D004921:Erythropoietin; C057416:KT 5720; D003994:Bucladesine; C037500:mastoparan; C043204:N-(2-guanidinoethyl)-5-isoquinolinesulfonamide; D019833:Genistein; D011505:Protein-Tyrosine Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D019311:Staurosporine; C058819:calphostin C; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium