Identification of a novel pathway important for proliferation and differentiation of primary erythroid progenitors

Abstract

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...

The role of STATs in myeloid differentiation and leukemia

“…These discrepancies may be due to di erences in cell lines and highlight the problems of an in vitro, cell line-based analysis of STAT function, where cells may be lacking other critical components of the di erentiation process. Indeed, the expression of mutant EpoRs into fetal liver cells derived from EpoR (7/7) mice has revealed that the receptor that best reconstitutes erythropoiesis does not correlate with the ability to activate STAT5 but rather the phosphatidylinositol lipid kinase, PI3K (Klingmuller et al, 1997).…”

Growth factor independence and BCR/ABL transformation: promise and pitfalls of murine model systems and assays

“…48 Mutant EpoRs capable of activating only a subset of these signal transduction proteins, for instance only STAT5 or only PI-3 kinase, are capable of supporting normal in vitro differentiation of primary erythroid progenitors from EpoR −/− mice. [49][50][51][52][53] Thus, many of the signaling pathways activated by the EpoR appear redundant. None of these signal transduction proteins are unique to the EpoR and most are activated by a large number of other cytokine receptors as well as BCR-ABL and other types of growth factor receptors.…”

Dominant action of mutated erythropoietin receptors on differentiation in vitro and erythroleukemia development in vivo

“…Upon hormone binding the epo receptor dimerizes, Janus kinase 2 (JAK2) is activated and tyrosine residues in the cytoplasmic domain of the receptor phosphorylated (Matthews et al, 1996;Watowich et al, 1992;Witthuhn et al, 1993). These phosphorylated tyrosines act as docking sites for signaling proteins including Signal Transducer and Activator of Transcription 5 (STAT 5), the p85 subunit of phosphatidylinositol 3-kinase (PI3-kinase) and phosphatases SHP-1 and SHP-2 (Damen et al, 1995a,b;Gobert et al, 1996;Klingmuller et al, 1995Klingmuller et al, , 1996Klingmuller et al, , 1997Penta and Sawyer, 1995;Quelle et al, 1996;Sharlow et al, 1997;Tauchi et al, 1995Tauchi et al, , 1996Yi et al, 1995). Although Shc and Grb2 bind to the epo receptor and stimulate the Ras/Raf/MAP-kinase pathway (Barber et al, 1997;Bittorf et al, 1994;Carroll et al, 1991;Cutler et al, 1993;Gobert et al, 1995;He et al, 1995;Miura et al, 1994b;Tilbrook et al, 1996a;Torti et al, 1992), their precise binding locations on the receptor have not been identi®ed.…”