Erythropoietin Induces the Tyrosine Phosphorylation of Insulin Receptor Substrate-2

Abstract

In this report, we demonstrate that insulin receptor substrate-2 (IRS-2) is phosphorylated on tyrosine following treatment of UT-7 cells with erythropoietin. We have investigated the expression of IRS-1 and IRS-2 in several cell lines with erythroid and/or megakaryocytic features, and we observed that IRS-2 was expressed in all cell lines tested. In contrast, we did not detect the expression of IRS-1 in these cells. In response to erythropoietin, IRS-2 was immediately phosphorylated on tyrosine, with maximal phosphorylation between 1 and 5 min. Tyrosine-phosphorylated IRS-2 was associated with phosphatidylinositol 3-kinase and with a 140-kDa protein that comigrated with the phosphatidylinositol- Insulin receptor substrate-1 (IRS-1) 1 is a major substrate of the IGF-1 and insulin receptors (1). IRS-1 is a hydrophilic protein with a theoretical molecular mass of 131 kDa that migrates between 160 and 185 kDa on SDS-polyacrylamide gel electrophoresis partially because of a high serine phosphorylation state (2, 3). IRS-1 contains a pleckstrin homology domain, a PTB domain, and at least 20 potential tyrosine phosphorylation sites including nine YXXM motifs that are consensus binding sites for the SH2 domains of the regulatory subunit (p85) of PI 3-kinase (4). IRS-1 binds to the tyrosine-phosphorylated IGF-1 and insulin receptors through its PTB domain and becomes a docking protein for signaling proteins such as PI 3-kinase, Grb2, PTP1D, and Nck after tyrosine phosphorylation (5). Moreover, a recent report shows that the pleckstrin homology domain could also be involved in the association of IRS-1 with the insulin receptor (6). More recently, a second IRS protein was identified that was designated IRS-2 (7). This protein corresponds to the previously identified 4PS protein (for IL-4-induced phosphotyrosine substrate) (8, 9). IRS-2 exhibits a high structural similarity to IRS-1, with a strong conservation of the PTB and pleckstrin homology domains. Moreover, tyrosine phosphorylation sites shown to bind PI 3-kinase, Grb2, and PTP1D in IRS-1 are also conserved in IRS-2.Not only insulin and IGF-1 receptors, but also several cytokine and interferon receptors can induce tyrosine phosphorylation of IRS-1 or IRS-2 (9 -18). IRS-1 and IRS-2 activation by IL-4 is well documented. Indeed, it has been shown that IRS-1 and IRS-2 bind to a peptidic sequence of the activated IL-4 receptor with a typical NPXY PTB domain-binding motif (19), and IRS-1 or IRS-2 expression appears to be required for IL-4-induced mitogenesis (10). However, other cytokine receptors that induce the tyrosine phosphorylation of IRS-1 and IRS-2 do not possess typical PTB domain-binding motifs, and the mechanism of IRS-1 and IRS-2 activation by these receptors remains unknown.The erythropoietin (Epo) receptor also belongs to the cytokine receptor family (20,21). Epo binding to its receptor activates the receptor-associated JAK2 tyrosine kinase (22) and induces the tyrosine phosphorylation of the Epo receptor (23-26) and other proteins. Several intracellular signa...

Epo regulates erythroid proliferation and differentiation through distinct signaling pathways: implication for erythropoiesis and Friend virus-induced erythroleukemia

“…Moreover, activation of SHIP and p85/PI3K was also detected in proliferating HB60-ED cells. Our results are consistent with published studies that have shown increased phosphorylation of Shc and other SH2 containing protein such as P85/PI3K, SHIP and Grb2 during Epo-induced proliferation of erythroblasts (Damen et al, 1993(Damen et al, , 1995a(Damen et al, , 1996He et al, 1995;Verdier et al, 1997). Interestingly, Epo-induced di erentiation of the parental HB60-5 cells requires very distinct signaling events, namely the tyrosine phosphorylation of STAT-5b.…”

“…Phosphatidylinositol 3-kinase (PI3K) has been previously shown to be phosphorylated in response to Epo and SCF stimulation and has been shown to bind to both receptors (Verdier et al, 1997;Damen et al, 1995a;Sattler et al, 1997;Lev et al, 1992). Using HB60-5 and HB60-ED cells, we also examined the binding of the p85 subunit of PI3K to the Epo-R and c-Kit.…”

“…However, it is clear that Epo possesses anti-apoptotic activity (Koury and Bondurant, 1990), a function that is somehow tightly coupled to its proliferative and di erentiation-inducing e ects on erythroid progenitor cells (Youssou®an et al, 1993;Liboi et al, 1993). Although the exact molecular mechanisms underlying these contrasting cellular responses are not well understood, a number of downstream signaling e ectors have been identi®ed, including Shc/ras, PI3-kinase, SHIP, as well as the JAK2/STAT5 pathways (Lecoq-Lafon et al, 1999;Damen et al, 1993Damen et al, , 1995aHe et al, 1995;Miura et al, 1994;Verdier et al, 1997;ID: 1002Pallard et al, 1995;Wakao et al, 1995). Epoinduced phosphorylation of the Epo-R at Tyrosine 343 promotes proliferation and an increase in STAT-5 activity (Damen et al, 1995b).…”

Cooperation between STAT5 and phosphatidylinositol 3-kinase in the IL-3-dependent survival of a bone marrow derived cell line

“…Distinct mechanisms of PI 3-kinase activation have been reported. PI 3-kinase is activated by recruitment of the p85 subunit to the phosphorylated receptor, by direct binding to the JAK tyrosine kinases or indirectly by binding to IRS-1/IRS-2 or GAB-1 (Migone et al, 1998;Miura et al, 1994;Verdier et al, 1997;Lecoq-Lafon et al, 1999). Previous report indicated also that STAT3 acts as an adapter molecule that couples the PI 3-kinase to the interferon receptor (Pfe er et al, 1997).…”

Proteasomes Regulate the Duration of Erythropoietin Receptor Activation by Controlling Down-regulation of Cell Surface Receptors

“…Whole cell extracts, immunoprecipitations and Western blots were done as previously described (31). Renaissance (NEN) was used for the Western blot development.…”