Regulation of Erythropoietin-induced STAT Serine Phosphorylation by Distinct Mitogen-activated Protein Kinases

Abstract

Erythropoietin (EPO)1 is an essential growth factor that promotes survival, proliferation, and differentiation of erythroid progenitor cells (1). These effects are conveyed through engagement of the EPO receptor (EPO-R) (2), a member of the superfamily of cytokine receptors. Erythropoietin induces dimerization of its receptor, resulting in trans-phosphorylation of receptor-associated Janus Kinase 2 (JAK2) molecules (3, 4). Activated JAK2 phosphorylates some or all of the eight tyrosine residues in the intracellular domain of the EPO-R. Phosphorylation of these intracellular tyrosines in turn recruits other intracellular proteins that bind to the EPO-R via their Src homology 2 (SH2) domains (reviewed in Ref. 5). The critical importance of EPO (6), , and JAK2 (9, 10) for efficient erythopoiesis have been demonstrated by targeted deletion in mice.The EPO-R recruits several distinct SH2-containing proteins, including signal transducer and activator of transcription-1 (STAT1) (11, 12), STAT3 (12), and STAT5 proteins (13-18). The EPO-R also associates with a diverse array of SH2-domain containing proteins, including Ship1 inositol phosphatase (19), Shp1 (20), Shp2 (21), phospholipase C␥1/C␥2 (22, 23), phosphatidylinositol 3Ј-kinase (24), as well as many non-catalytic adapter molecules (reviewed in Ref. 5). Binding of some of these molecules leads to their JAK2-dependent tyrosine phosphorylation.STAT proteins are an important class of signaling molecules that are recruited to the erythropoietin receptor. These latent transcription factors are activated at the receptor and transduce signals to the nucleus. While seven STAT molecules have been identified, only STAT5A and STAT5B are consistently activated by EPO in all cell types (13-18), whereas STAT1 and STAT3 are activated in erythroid cell types or by Friend virus transformation (11,12,25). STAT5A/B knockout mice have defects in prolactin (26), growth hormone (26), and interleukin-2 signaling (27), display anemia during fetal development (28, 29) and decreased formation of CD71 lo Ter119 hi cells in STAT5A/B Ϫ/Ϫ adult mice (30). No erythroid abnormalities were been described in the original reports of STAT1 Ϫ/Ϫ mice (31, 32), however, a more recent report suggested differences in clonogenic potential of burst forming unit-erythroid cells (33). STAT3 Ϫ/Ϫ mice are embryonic lethal, precluding analysis of their role in erythropoiesis by conventional gene targeting approaches (34).While tyrosine phosphorylation is required for cytokine-induced STAT dimerization, nuclear translocation, and DNA binding, full transcriptional activity of the homodimer is manifested only when a serine residue in the transcription activation domain is also phosphorylated (35). While the identity of the serine kinases is unclear, these putative proline-directed phosphorylation sites may be targetted by the mitogen-activated protein kinases (MAPKs).The MAPKs comprise a family of serine/threonine kinases that are activated by extracellular signals. There are at least three distinct types of MAPKs:...

Arsenic inhibition of the JAK-STAT pathway

“…Whole-cell lysates from treated HepG2 cells were analysed for STAT3 tyrosine phosphorylation with antibody recognizing STAT3 pY705 (Cell Signaling) or STAT3 serine phosphorylation with antibody recognizing STAT3 pS727 (Cell Signaling). STAT3 protein expression was measured as control phosphorylation of STAT3 on conserved residue (S727), on the other hand, is reportedly involved in the regulation of STAT3 activity in gene transcription and tyrosine phosphorylation (Lim and Cao, 1999;Haq et al, 2002). We then examined the effect of arsenite on the phosphorylation of STAT3 tyrosine (Y705) and serine (S727) by immunoblotting.…”

Epo-induced erythroid maturation is dependent on Plcγ1 signaling

“…Binding of Epo to its receptor activates the catalytic activity of several mitogen-activated protein kinases, including extracellular-regulated kinases 1/2, which participates in mitogenesis. 22 To determine whether activation of Plcγ1 is affected by MAPK pathway, we treated different cell lines with the Mek inhibitor U0126. Similarly, inhibition of Mek did not result in aberrant Plcγ1 phosphorylation ( Figure 1f).…”

Neuropeptide Y activates phosphorylation of ERK and STAT3 in stromal vascular cells from brown adipose tissue, but fails to affect thermogenic function of brown adipocytes

“…The difference in cellular response to NPY in adipocyte precursor cells between the white and brown adipose tissues may be simply due to the different characteristics of these cells which expressing different genes. previously [15,17,22,24,39].…”

“…As shown in Figure 4, treatment of cells with NPY (30 nM and 100 n) induced the phosphorylation, indicating that functional NPY receptor was present in SVC. We further examined STAT3 phosphorylation at Ser727 and Tyr705, as the serine residue of STAT3 is known to be a downstream target of the ERK pathway [15,17,22,24,39]. NPY treatment at the concentration of 30 nM or higher induced the Ser phosphorylation, but not the Tyr phosphorylation (Fig.…”