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FDCP‐1 cells are hematopoietic progenitor cells which require interleukin‐3 for survival and proliferation. FDCP‐1 cells stably transfected with the murine erythropoietin receptor cDNA survive and proliferate in the presence of erythropoietin. Erythropoietin induces the activation of the short forms (80 kDa) of STAT5 in the cells. Erythropoietin‐induced activation of STAT5 was strongly reduced in cells expressing mutated variants of the erythropoietin receptors in which tyrosine residues in their intracellular domain have been eliminated. We determined that the erythropoietin receptor tyrosine residues 343 and 401 are independently necessary for STAT5 activation. The amino acid sequences surrounding these two tyrosine residues are very similar. Peptides comprising either phosphorylated Tyr343 or phosphorylated Tyr401, but not their unphosphorylated counterparts, inhibited the STAT5 activation. We propose that these two tyrosine residues of the erythropoietin receptor constitute docking sites for the STAT5 SH2 domain. The growth stimulus mediated by erythropoietin was decreased in cells expressing erythropoietin receptors lacking both Tyr343 and Tyr401. This suggests that STAT5 activation could be involved in the growth control of FDCP‐1 cells.

Cis is an Src homology 2 domain-containing protein, which binds to the erythropoietin receptor and decreases erythropoietin-stimulated cell proliferation. We show that Cis associates with the second tyrosine residue of the intracellular domain of the erythropoietin receptor (Tyr 401 ). Two forms of Cis with molecular masses of 32 and 37 kDa were detected, and we demonstrate that the 37-kDa protein resulted from post-translational modifications of the 32-kDa form. Anti-ubiquitin antibodies recognized the 37-kDa form of Cis and the proteasome inhibitors N-acetyl-leucyl-leucyl-norleucinal and lactacystin inhibited its degradation, showing that the 37-kDa form of Cis is a ubiquitinated protein, which seems to be rapidly degraded by the proteasome. In erythropoietin-stimulated UT-7 cells, the activation of the erythropoietin receptor and signal transducer and activator of transcription 5 (STAT5) was transient and returned to basal levels after 30 -60 min of erythropoietin stimulation. In contrast, these proteins remained strongly phosphorylated, and STAT5 remained activated for at least 120 min in the presence of proteasome inhibitors. These experiments demonstrate that the proteasomes are involved in the down-regulation of the erythropoietin receptor activation signals. Because the proteasome inhibitors induced the accumulation of both the ubiquitinated form of Cis and the Cis-erythropoietin receptor complexes, our results suggest that the ubiquitinated form of Cis could be involved in the proteasome-mediated inactivation of the erythropoietin receptor.

Two distinct genes encode the closely related signal transducer and activator of transcription proteins STAT5A and STAT5B. The molecular mechanisms of gene regulation by STAT5 and, particularly, the requirement for both STAT5 isoforms are still undetermined. Only a few STAT5 target genes, among them the CIS (cytokine-inducible SH2-containing protein) gene, have been identified. We cloned the human CIS gene and studied the human CIS gene promoter. This promoter contains four STAT binding elements organized in two pairs. By electrophoretic mobility shift assay studies using nuclear extracts of UT7 cells stimulated with erythropoietin, we showed that these four sequences bound to STAT5-containing complexes that exhibited different patterns and affinities: the three upstream STAT binding sequences bound to two distinct STAT5-containing complexes (C0 and C1) and the downstream STAT box bound only to the slower-migrating C1 band. Using nuclear extracts from COS-7 cells transfected with expression vectors for the prolactin receptor, STAT5A, and/or STAT5B, we showed that the C1 complex was composed of a STAT5 tetramer and was dependent on the presence of STAT5A. STAT5B lacked this property and bound with a stronger affinity than did STAT5A to the four STAT sequences as a homodimer (C0 complex). This distinct biochemical difference between STAT5A and STAT5B was confirmed with purified activated STAT5 recombinant proteins. Moreover, we showed that the presence on the same side of the DNA helix of a second STAT sequence increased STAT5 binding and that only half of the palindromic STAT binding sequence was sufficient for the formation of a STAT5 tetramer. Again, STAT5A was essential for this cooperative tetrameric association. This property distinguishes STAT5A from STAT5B and could be essential to explain the transcriptional regulation diversity of STAT5.STAT proteins are latent transcription factors containing a Src homology 2 domain (SH2 domain) that become activated by tyrosine phosphorylation. The binding of the STAT SH2 domains to the phosphorylated cytokine receptors allows their tyrosine phosphorylation by Jak kinases. After dimerization and nuclear translocation, STAT dimers bind to specific DNA sequences, thereby allowing downstream gene regulation.

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 mitogenactivated 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, downregulates p27 Kip1 IntroductionThe production of red blood cells is tightly regulated by the cytokine erythropoietin (Epo), which supports the survival and proliferation of erythroid progenitors. 1 Epo binding to its cognate receptor activates the receptor-associated Janus kinase-2 (Jak2) tyrosine kinase. 2 The Epo receptor (EpoR) is tyrosine phosphorylated 3 and recruits several Src homology-2 (SH2) domain-containing proteins, thereby leading to the activation of different intracellular signaling pathways. 4 One of these pathways involves phosphoinositide 3-kinase (PI 3-kinase). PI 3-kinase products phosphoinositide 3,4 bisphosphate (PI(3,4)P2) and phosphoinositide 3,4,5-trisphosphate (PI(3,4,5)P3) are major intracellular second messengers acting as mediators between the plasma membrane and intracellular signaling molecules. 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 ...

The mechanism of action of prolactin (PRL) was studied in murine lymphoid BAF‐3 cells transfected with either the long form of the PRL receptor (PRL‐R), or a chimeric receptor consisting of the extracellular domain of the PRL‐R and the transmembrane and intracellular domain of the erythropoietin receptor (PRL/EPO‐R). PRL sustained normal and long‐term proliferation of BAF‐3 cells expressing either the PRL‐R or the hybrid PRL/EPO‐R. Upon [125I]PRL cross‐linking, both types of BAF‐3 transfectants were shown to express two [125I]PRL cross‐linked species differing in size by 20 kDa. These cross‐linked complexes, after denaturation, were recognized by antibody against the PRL‐R, indicating that they contain the transfected receptor. PRL induced rapid and transient tyrosine phosphorylation of both the PRL‐R and the PRL/EPO‐R in BAF‐3 transfectants. Furthermore, PRL induced rapid tyrosine phosphorylation of the Janus kinase 2 (JAK2) which was already physically associated with the PRL‐R or the PRL/EPO‐R in the absence of ligand. JAK1 was also associated with PRL‐R and PRL/EPO‐R in the absence of ligand. However, only in BAF‐3 cells expressing the PRL‐R does PRL induce rapid and transient tyrosine phosphorylation of JAK1. These results demonstrate that JAK protein tyrosine kinases couple PRL binding to tyrosine phosphorylation and proliferation.

Stimulation of sensitive cells with erythropoietin results in rapid induction of protein tyrosine phosphorylation. Other than tyrosine phosphorylation of one chain of the erythropoietin receptor, the identities of the remaining tyrosine-phosphorylated proteins are undefined. In this report, we demonstrate that the stimulation of the erythropoietin-sensitive human UT7 cells by erythropoietin rapidly resulted in the appearance of phosphatidylinositol 3-kinase activity in anti-phosphotyrosine immunoprecipitates. Erythropoietin action was rapid, detectable after as early as 1 niin stimulation, transient, returning to control level after 30 min stimulation and was observed using the erythropoietin concentrations able to stimulate the cell proliferation. Anti-(phosphatidylinositol 3-kinase) antibodies specifically immunoprecipitated '251-erythropoietin bound to its receptor, strongly suggesting that phosphatidylinositol 3-kinase associated with a protein complex containing the activated erythropoietin receptor. To confirm this result, phosphatidylinositol 3-kinase was immunoprecipitated from erythropoietin-stimulated cells using mild conditions followed by Western analysis using antiphosphotyrosine antibodies. Five tyrosine phosphorylated proteins were revealed: the cloned chain of the erythropoietin receptor, the regulatory subunit of phosphatidylinositol 3-kinase and three unidentified proteins of 111, 97 and 64 kDa. None of these tyrosine phosphorylated proteins was detected in anti-(phosphatidylinositol 3-kinase) immunoprecipitates from unstimulated cells. Thus, our results show that phosphatidylinositol3-kinase associates with a tyrosine-phosphorylated protein complex containing the activated erythropoietin receptor.Red blood cell production is mainly regulated by the glycoprotein hormone erythropoietin which controls the survival, proliferation and differentiation of the late erythroid progenitors: the so-called colony-forming-units erythroid (CFU-E) (see [I] for a recent review concerning erythropoietin). Erythropoietin action is mediated through erythropoietin binding to a small number of high-affinity membrane receptors (reviewed in [2]). A cDNA encoding an erythropoietinbinding protein of 55 kDa was cloned by an expression strategy from an erythroleukemia cell line [3]. Although erythropoietin receptors appear to be multimeric complexes [4, 51, the cloned chain alone is able to transmit a proliferative signal when transfected into various non-erythroid hematopoietic cells [6,71. The cloned chain of the erythropoietin receptor belongs to the newly described hematopoietic growth

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