Signaling through the erythropoietin receptor (EPO-R) is crucial for proliferation, differentiation, and survival of erythroid progenitor cells. EPO induces homodimerization of the EPO-R, triggering activation of the receptor-associated kinase JAK2 and activation of STAT5. By mutating the eight tyrosine residues in the cytosolic domain of the EPO-R, we show that either Y343 or Y41 is sufficient to mediate maximal activation of STAT5; tyrosine residues Y429 and Y431 can partially activate STAT5. Comparison of the sequences surrounding these tyrosines reveals YXXL as the probable motif specifying recruitment of STAT5 to the EPO-R. Expression of a mutant EPO-R lacking all eight tyrosine residues in the cytosolic domain supported a low but detectable level of EPO-induced STAT5 activation, indicating the existence of an alternative pathway for STAT5 activation independent of any tyrosine in the EPO-R. The kinetics of STAT5 activation and inactivation were the same, regardless ofwhich tyrosine residue in the EPO-R mediated its activation or whether the alternative pathway was used. The ability of mutant EPO-Rs to activate STAT5 did not directly correlate with their mitogenic potential.Erythropoietin (EPO), a hormone synthesized by the kidney, is the primary regulator of mammalian erythropoiesis. Hormone binding to its cognate receptor is crucial for proliferation, differentiation, and survival of erythroid progenitor cells. The erythropoietin receptor (EPO-R) (1) belongs to the large cytokine receptor superfamily, which is characterized by a WSXWS motif in the extracellular domain and by the absence of enzymatic activity in the cytoplasmic domain. To transmit a signal these receptors couple to protein tyrosine kinases and other signal transduction proteins. Ligand induced homodimerization of the EPO-R (2) triggers the activation of the receptor-bound protein tyrosine kinase JAK2 (3), resulting in a transient increase in tyrosine phosphorylation of several cellular proteins including the EPO-R. Since phosphotyrosine residues are specifically recognized by Src homology 2 (SH2) domains, this event enables the EPO-R to recruit a number of signal transduction proteins to the receptor, including SH-PTP1 (4), SH-PTP-2 (5), and P13 kinase (6).Little is known about how signals from the activated receptor complex assembled at the plasma membrane are transmitted to the nucleus and converted into gene activation. One system involves activation of the JAK-STAT pathway (for reviews see refs. 7 and 8). At least six STAT proteins, STAT1-STAT6, have been cloned (for review see ref. 9); all have an SH2 domain that allows it to bind to a phosphorylated tyrosine on a receptor or other protein. Following tyrosine phosphorylation by a Janus kinase or other kinase(s), STAT proteins dissociate from the receptor and form homo-or heterodimers via their SH2 domains. They then move to the nucleus and bind to and presumably activate specific genes. STAT proteins enable a signal to be transduced within minutes from the cell surface to the ...

Homodimerization of the erythropoietin (EPO) receptor (EPO-R) in response to EPO binding transiently activates the receptor-associated protein tyrosine kinase JAK2. Tyrosine phosphorylation of the EPO-R creates ''docking sites'' for SH2 domain(s) in signaling molecules such as the protein tyrosine phosphatases SH-PTP1 and SH-PTP2, phosphoinositide 3-kinase (PI3 kinase), and STAT5. However, little is known about the specific intracellular signals essential for proliferation and differentiation of erythroid progenitors. Here we show that an EPO-R containing only one cytosolic (phospho)tyrosine residue, Y479, induces a signal transduction pathway sufficient for proliferation and differentiation of fetal liver progenitors of erythroid colony-forming units from EPO-R ؊͞؊ mice as well as for proliferation of cultured hematopoietic cells. This cascade involves sequential EPOinduced recruitment of PI3 kinase to the EPO-R and activation of mitogen-activated protein kinase activity, independent of the Shc͞Grb2-adapter pathway and of STAT5. Protein kinase C may be one of the mediators connecting PI3 kinase with the mitogen-activated protein kinase signaling cascade. Our results identify a signaling cascade important in vivo for erythroid cell proliferation and differentiation.Erythropoietin (EPO), a hormone synthesized by the kidney, is the primary regulator of mammalian erythropoiesis. Signaling through the EPO receptor (EPO-R) (1), which is a member of the cytokine receptor family, regulates proliferation, differentiation and survival of erythroid progenitor cells. Homodimerization of the receptor in response to EPO binding (2, 3) transiently activates the receptor-associated protein tyrosine kinase JAK2 (4). Tyrosine phosphorylation of the EPO-R creates ''docking sites'' for SH2 domain(s) in signaling molecules such as the protein tyrosine phosphatases SH-PTP1 (5) and SH-PTP-2 (6), phosphoinositide 3-kinase (PI3 kinase) (7), and STAT5 (8-10). However, little is known about the specific intracellular signals essential for proliferation and differentiation of erythroid progenitors.In a number of growth factor receptor systems recruitment of PI3 kinase correlates with mitogenic signaling (11). This lipid kinase is a heterodimeric enzyme composed of the regulatory p85 subunit and the catalytic p110 subunit that phosphorylates phosphoinositides at the D3 position of the inositol ring. Stimulation by many cytokines including EPO (7, 12, 13) results in a transient increase in PI3 kinase activity. Mediated by the SH2-domains of p85, recruitment of PI3 kinase to specific phosphotyrosine residues within the cytoplasmic domain of receptors places the enzyme in close proximity to substrates. Recent evidence suggests that the lipid products act as second messengers and directly activate enzymes such as Akt (protein kinase B) (14) and certain isoforms of protein kinase C (PKC) (15, 16). Proposed downstream targets of PI3 kinase include p70 S6 kinase and MEK [mitogen-activated protein (MAP) kinase (MAPK) kinase]. The latter wo...

Production of mature erythrocytes requires multiple growth factors, but we do not know how their actions are coordinated. Here we show that erythroid progenitors from erythropoietin receptor (Epo-R) ؊͞؊ fetal livers, infected in vitro with a retrovirus expressing the wild-type Epo-R, require addition of both Epo and stem cell factor (SCF) to form colony-forming unit erythroid (CFU-E) colonies. Thus, a functional interaction between KIT and the Epo-R, similar to what we reported in cultured cells, is essential for the function of CFU-E progenitors. In contrast, CFU-E colony formation in vitro by normal fetal liver progenitors requires only Epo; the essential interaction between activated KIT and the Epo-R must have occurred in vivo before or at the CFU-E progenitor stage. Using truncated dominant-negative mutant Epo-Rs, we show that KIT does not activate the Epo-R by inducing its dimerization, but presumably does so by phosphorylating tyrosine residue(s) in its cytosolic domain. By expressing mutant Epo-Rs containing only one of eight cytosolic tyrosines, we show that either tyrosine residue Y464 or Y479 suffices for Epo-dependent cell proliferation. However, only Epo-R F7Y479 is capable of supporting erythroid colony formation when expressed in Epo-R ؊͞؊ fetal liver cells, indicating that Y464 either cannot send a differentiation signal or fails to respond to SCF͞KIT activation. This work employs a novel experimental system to study the function of growth factors and their receptors in normal hematopoiesis.Hematopoietic progenitors require multiple growth factors for their optimal development and terminal differentiation. Previous studies suggested that stem cell factor (SCF), interleukin 3 (IL-3), and granulocyte-macrophage colony-stimulating factor (GM-CSF) are important for the generation and proliferation of erythroid progenitors. However, analysis of mice with null mutations in either GM-CSF (1, 2) or IL-3 receptor (3) genes indicated that GM-CSF and IL-3 are not crucial for erythropoiesis, or that other factors can compensate for their function. Mice deficient in either SCF or its receptor (KIT), however, suffer from severe anemia (4). Even though normal numbers of early erythroid progenitors [burst-forming-unit erythroids (BFU-Es)] are present in the fetal liver, the numbers of late progenitors [colony-forming-unit erythroids (CFU-Es)] are significantly reduced (5). Mice deficient in erythropoietin (Epo; ref. 6) or its receptor (Epo-R) (6-8) accumulate fetal liver CFU-Es that cannot differentiate and eventually undergo apoptosis. Thus SCF͞KIT and Epo͞Epo-R function as key switches for erythropoiesis: SCF and KIT are essential for the proliferation and differentiation of BFU-Es to CFU-Es, and Epo and the Epo-R are crucial for survival of CFU-Es and for their proliferation and irreversible terminal differentiation.Using cultured erythroleukemia cells, we showed previously that activation of the KIT protein tyrosine kinase induces tyrosine phosphorylation of the Epo-R, and that KIT interacts with the...