Abstract
A recent report (Wu, H., Klingmuller, U., Besmer, P., and Lodish, H. F. (1995) Nature 377, 242-246) documents the interaction of the erythropoietin (EPO) receptor (EPOR) with the stem cell factor (SCF) receptor (c-KIT) and suggests that SCF acts through the EPOR. To elucidate the ability of SCF to affect the erythropoietin signaling pathway, we studied the effect of SCF on EPOR phosphorylation, SHC/ERK-1 activity, and cell proliferation and apoptosis in EPO-dependent HCD57 cells. Treatment of these cells with SCF resulted in phosphorylation of the EPOR. However, SCF-dependent phosphorylation of the EPOR did not initiate an EPO-like intracellular signal. SCF induced proliferation, SHC phosphorylation, and activation of ERK-1 but did not activate the JAK/STAT pathway. SCF stimulated SHC phosphorylation and ERK-1 activation independent of the EPOR in cells where the EPOR was down-regulated; the presence of the EPOR appeared to facilitate SCF activation of SHC and ERK-1. Furthermore, treatment of HCD57 cells with SCF increased cell number over a 3-day treatment, but apoptosis was observed in these cells. These data may illustrate two distinct pathways for erythroid cell proliferation and prevention of apoptosis in response to EPO, thereby providing a system to discriminate these intracellular signals.
Highlights
A tyrosine-phosphorylated band was detected which was similar to the molecular mass of JAK2 (130 kDa) [16]; re-probing this blot of Tyr(P)-containing proteins with anti-JAK2 antibody revealed that JAK2 was only phosphorylated in response to EPO (Fig. 1A, lane E); no phosphorylation of JAK2 was observed in cells treated with stem cell factor (SCF) (Fig. 1A, lane F)
Immunoprecipitation of HCD57 cellular proteins with antiEPOR antibody and Western blotting with anti-phosphotyrosine antibody demonstrated that the EPO receptor (EPOR) was phosphorylated in response to SCF (Fig. 1B, lanes 2, 5, and 6); this phosphorylation was greatest at 5 min but maintained for 10 min or longer
We report here that the EPOR was phosphorylated in response to SCF to 10% or less than that observed in response to EPO
Summary
To elucidate the ability of SCF to affect the erythropoietin signaling pathway, we studied the effect of SCF on EPOR phosphorylation, SHC/ERK-1 activity, and cell proliferation and apoptosis in EPO-dependent HCD57 cells. Treatment of these cells with SCF resulted in phosphorylation of the EPOR. Wu and co-workers [8] in Dr Harvey Lodish’s laboratory showed that SCF induced proliferation and phosphorylation of the EPOR in these EPO-dependent cells This erythroleukemia cell line is a model for erythropoietin-dependent proliferation and erythroid cell survival [9, 10]. The proliferative and cell survival signals transduced from either receptor appeared to be distinct
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