Erythropoietin Induces Activation of Stat5 through Association with Specific Tyrosines on the Receptor That Are Not Required for a Mitogenic Response

Abstract

The cytoplasmic domain of the erythropoietin receptor (EpoR) contains a membrane-distal region that is dispensable for mitogenesis but is required for the recruitment and tyrosine phosphorylation of a variety of signaling proteins. The membrane-proximal region of 96 amino acids is necessary and sufficient for mitogenesis as well as Jak2 activation, induction of c-fos, c-myc, cis, the T-cell receptor ␥ locus (TCR-␥), and c-pim-1.The studies presented here demonstrate that this region is also necessary and sufficient for the activation of Erythropoietin (Epo) regulates the proliferation and differentiation of cells of the erythroid lineage (25). Epo functions through its interaction with a single chain, Epo-specific receptor (EpoR), of the cytokine receptor superfamily (4, 9). Members of the cytokine receptor superfamily couple ligand binding to the induction of cellular protein tyrosine phosphorylation through their interaction with members of the Janus protein tyrosine kinase (Jak) family (23). EpoR specifically associates with Jak2 through the receptor cytoplasmic, membrane-proximal domain containing the conserved box 1 and box 2 motifs found in a number of the members of the cytokine receptor superfamily. Mutations or deletions in the membrane-proximal domain which affect Jak association affect all receptor functions that have been examined.Epo, like a number of cytokines, activates a variety of signaling pathways. Previous studies have shown that the cytoplasmic membrane-distal region of the receptor is required for recruitment and tyrosine phosphorylation of SHC and the subsequent activation of the ras pathway (33). The membranedistal region is also required for Epo-induced tyrosine phosphorylation of the p85 subunit of phosphatidylinositol 3-kinase (PI 3-kinase) (8, 32) and phospholipase C-␥1 (40). The membrane-distal region is also required for the association of hematopoietic cell phosphatase (59), which negatively influences the receptor complex (44,58,59) and is hypothesized

Signalling by the oncogenic receptor tyrosine kinase Xmrk leads to activation of STAT5 in Xiphophorus melanoma

“…In BaF Hm cells, the activated Xmrk kinase induced the expression of the genes cis, osm and pim-1. Since these genes were identi®ed as STAT5 target genes in Ba/F3 cells (Mui et al, 1996;Quelle et al, 1996;Wang et al, 1996) their induction clearly demonstrates the capability of Xmrk to fully activate STAT5. The detection of pim-1 expression in Xiphophorus melanoma points to an analogous situation as in the BaF Hm cells.…”

“…Inhibition of Xmrk by the tyrosine kinase inhibitor AG555 resulted in a loss of constitutive STAT5 tyrosine phosphorylation in ®sh melanoma cells, indicating that this phosphorylation is depent on an active Xmrk kinase either in a direct or indirect way. For cytokine receptors (like the prolactin, IL-3, GM-CSF and Epo receptor), which do not possess a tyrosine kinase domain, members of the family of janus kinases (JAKs) are essential for STAT tyrosine phosphorylation and activation Mui et al, 1995;Quelle et al, 1996). However, although RTKs are able to activate JAKs, they do not require them for the induction of the STAT pathway.…”

“…Although a variety of studies attempted to de®ne the role of STAT5 in cell growth and di erentiation, its requirement for such processes is still a subject of dispute. Some data describe an implication of STAT5 in the control of proliferation (Damen et al, 1995;Friedmann et al, 1996;Mellitzer et al, 1996), whereas others show that receptor mutants lacking the ability to activate STAT5 are still able to transduce mitogenic signals (Quelle et al, 1996;Fujii et al, 1995). Moreover, while a dominant negative STAT5 protein was found to inhibit IL-3 driven DNA synthesis in Ba/F3 cells (Mui et al, 1996), the expression of another dominant negative STAT5 had no e ect on IL3-induced proliferation in myeloid cells .…”

IL-3 dependent regulation of Bcl-xL gene expression by STAT5 in a bone marrow derived cell line

“…Conicting results were obtained on the role of STAT5 in the proliferation of hematopoietic cell lines. The use of cytokine receptor mutants unable to activate STAT5, or the use of dominant negative form of STAT5, indicated that STAT5 activation was required for Epo, IL-2-or IL-3-induced cell proliferation, while other studies suggested that STAT5 was not essential for this e ect (Friedmann et al, 1996;Fujii et al, 1995;Gobert et al, 1996;Mui et al, 1996;Quelle et al, 1996). Studies with dominant negative STAT5 and receptor mutants also demonstrated a role of STAT5 in erythroid di erentiation Iwatsuki et al, 1997).…”

Induction of erythroid differentiation by inhibition of Ras/ERK pathway in a Friend murine leukemia cell line

“…Stimulation of EPOR by EPO induces tyrosine phosphorylation of EPOR as well as numerous cytoplasmic proteins, including a receptor-associated protein tyrosine kinase Jak2 (Witthuhn et al, 1993b), phosphatidylinositol-3-kinase (PI3K) (Damen et al, 1993), protein tyrosine phosphatases SHP-1 (Klingmuller et al, 1995) and SHP-2 (Klingmuller 1997;Sharlow et al, 1997), and adapter proteins SHC and Grb2 (Miura et al, 1994;Damen et al, 1993a;He et al, 1995). Transient activation of Jak/ STAT pathway as well as Ras/MAP kinase (MAPK) pathways after EPO-stimulation has been documented (Miura et al, 1994;Sawyer and Penta, 1996;Quelle et al, 1996;Torti et al, 1992;Todokoro et al, 1994;Tilbrook et al, 1996;Gobert et al, 1995;Nagata et al, 1997). Among various EPO-dependent cell lines, good correlation has been observed between the basal activity of these signaling pathways and the proliferative potential of the cells (Gobert et al, 1995;Tilbrook et al, 1996;Miura et al, 1994;Carroll and May, 1994;Muszynski et al, 1998).…”