Abstract
The production of red blood cells is tightly regulated by erythropoietin (Epo). The phosphoinositide 3-kinase (PI 3-kinase) pathway was previously shown to be activated in response to Epo. We studied the role of this pathway in the control of Epo-induced survival and proliferation of primary human erythroid progenitors. We show that phosphoinositide 3 (PI 3)-kinase associates with 4 tyrosine-phosphorylated proteins in primary human erythroid progenitors, namely insulin receptor substrate-2 (IRS2), Src homology 2 domain-containing inositol 5'-phosphatase (SHIP), Grb2-associated binder-1 (Gab1), and the Epo receptor (EpoR). Using different in vitro systems, we demonstrate that 3 alternative pathways independently lead to Epo-induced activation of PI 3-kinase and phosphorylation of its downstream effectors, Akt, FKHRL1, and P70S6 kinase: through direct association of PI 3-kinase with the last tyrosine residue (Tyr479) of the Epo receptor (EpoR), through recruitment and phosphorylation of Gab proteins via either Tyr343 or Tyr401 of the EpoR, or through phosphorylation of IRS2 adaptor protein. The mitogen-activated protein (MAP) kinase pathway was also activated by Epo in erythroid progenitors, but we found that this process is independent of PI 3-kinase activation. In erythroid progenitors, the functional role of PI 3-kinase was both to prevent apoptosis and to stimulate cell proliferation in response to Epo stimulation. Finally, our results show that PI 3-kinase-mediated proliferation of erythroid progenitors in response to Epo occurs mainly through modulation of the E3 ligase SCF(SKP2), which, in turn, down-regulates p27(Kip1) cyclin-dependent kinase (CDK) inhibitor via proteasome degradation.
Highlights
The production of red blood cells is tightly regulated by the cytokine erythropoietin (Epo), which supports the survival and proliferation of erythroid progenitors.[1]
Our results indicate that the mitogen-activated protein kinase (MAPK) pathway is activated by Epo independently of phosphoinositide 3 (PI 3)-kinase activation
Erythroid progenitors isolated from human cord blood were deprived of growth factor and serum in culture medium complemented with bovine serum albumin (BSA) and transferrin, stimulated for 10 minutes with a saturating concentration of Epo (10 U/mL)
Summary
The production of red blood cells is tightly regulated by the cytokine erythropoietin (Epo), which supports the survival and proliferation of erythroid progenitors.[1]. The class IA family of PI 3-kinases consists of a regulatory subunit (p85) and a catalytic subunit (p110).[5] PI 3-kinase plays a central role in controlling cell survival and cell cycle progression in different systems.[6] In the erythroid lineage, PI 3-kinase is required for the protection of erythroid cells from apoptosis[7] and is involved in Epo-induced mitogenic responses.[8] We and others have previously shown that PI 3-kinase is associated through its SH2 domains with the activated EpoR.[9,10,11] The last tyrosine residue (Tyr479) in the EpoR cytoplasmic domain was further shown to be a binding site for the p85 subunit of PI 3-kinase.[12,13] This cytosolic tyrosine residue was identified as the origin of a major signal transduction pathway leading to proliferation and differentiation of primary erythroid progenitors.[14] EpoR devoid of this tyrosine still activates PI 3-kinase, suggesting that alternative mechanisms for EpoRinduced PI 3-kinase activation exist.[12,15] analyses of receptor forms with cytosolic truncations and deletions delineated the extended EpoR box 2 as a candidate subdomain for p85 binding.[16] More recently, it was shown that the distal region and tyrosine residues of the EpoR were nonessential for in vivo erythropoiesis.[17] the molecular mechanisms of PI 3-kinase activation and their role in EpoR-mediated signaling are still unclear
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