Regulation of the erythropoietin receptor and involvement of JAK2 in differentiation of J2E erythroid cells.
In response to erythropoietin, J2E cells proliferate and differentiate into mature hemoglobin-producing erythroid cells. Here we show that following hormonal stimulation, between 10 and 17 proteins, including the erythropoietin receptor and JAK2, were tyrosine phosphorylated immediately after exposure to the hormone. Although the receptor was only phosphorylated to 15% of its maximum with 0.1 unit/ml erythropoietin, this was sufficient to induce peak hemoglobin synthesis. The importance of JAK2 to J2E cell maturation was demonstrated by inhibiting JAK2 protein synthesis with antisense oligonucleotides; not only was erythropoietin-stimulated mitogenesis inhibited by this procedure, but differentiation was also suppressed. In addition, the activation of STAT5 paralleled the kinetics of receptor phosphorylation. During differentiation, 94% decrease in surface erythropoietin receptors was detected 48 h after ligand binding, but transcription of the receptor gene, mRNA steady-state levels, protein content, and translation rates did not alter with hormonal stimulation. We concluded from these experiments that (a) sub-maximal receptor phosphorylation is sufficient for differentiation to proceed; (b) JAK2 is required for erythropoietin-induced cell division and maturation; and (c) post-translational processing, or translocation, play important roles in controlling surface erythropoietin receptor numbers.
Tilbrook
P A
PA
Department of Biochemistry, University of Western Australia, Nedlands.
Bittorf
T
T
Callus
B A
BA
Busfield
S J
SJ
Ingley
E
E
Klinken
S P
SP
D000818:Animals; D015152:Blotting, Northern; D015153:Blotting, Western; D002454:Cell Differentiation; D002461:Cell Line, Transformed; D004268:DNA-Binding Proteins / Q000378:metabolism; D015536:Down-Regulation; D015672:Erythroid Precursor Cells / Q000187:drug effects*; D004921:Erythropoietin / Q000494:pharmacology; D005786:Gene Expression Regulation; D006454:Hemoglobins / Q000096:biosynthesis; D053614:Janus Kinase 2; D008099:Liver / Q000166:cytology; D051379:Mice; D008894:Milk Proteins; D010766:Phosphorylation / Q000187:drug effects; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011518:Proto-Oncogene Proteins; D017467:Receptors, Erythropoietin / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D050799:STAT5 Transcription Factor; D015534:Trans-Activators / Q000378:metabolism
D004268:DNA-Binding Proteins; D006454:Hemoglobins; D008894:Milk Proteins; D011518:Proto-Oncogene Proteins; D017467:Receptors, Erythropoietin; D050799:STAT5 Transcription Factor; D015534:Trans-Activators; D004921:Erythropoietin; D011505:Protein-Tyrosine Kinases; C507926:Jak2 protein, mouse; D053614:Janus Kinase 2