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Related Collections Hematopoiesis and Stem Cells Signal Transduction Social Bookmarking                   What's this?  Previous Article  |  Table of Contents  |  Next Article  Blood, 15 April 2001, Vol. 97, No. 8, pp. 2230-2237 HEMATOPOIESIS A common epitope is shared by activated signal transducer and activator of transcription-5 (STAT5) and the phosphorylated erythropoietin receptor: implications for the docking model of STAT activation Dwayne L. Barber, Bryan K. Beattie, Jacqueline M. Mason, Melody H.-H. Nguyen, Monique Yoakim, Benjamin G. Neel, Alan D. D'Andrea, and David A. Frank From the Division of Cellular and Molecular Biology, Ontario Cancer Institute; Department of Laboratory Medicine and Pathobiology, Toronto General Hospital; Departments of Medical Biophysics and Laboratory Medicine and Pathobiology, University of Toronto; Toronto, ON; Cancer Biology Program and Division of Hematology-Oncology, Department of Medicine, Beth Israel Deaconess Medical Center; and Departments of Pediatric Oncology and Adult Oncology, Dana-Farber Cancer Institute; Boston, MA. Erythropoietin (EPO) specifically activates the Janus kinase JAK2 and the transcription factor signal transducer and activator of transcription-5 (STAT5). All members of the STAT family are tyrosine phosphorylated in response to cytokine stimulation at a conserved carboxy-terminal tyrosine, Y694, in the case of STAT5. To determine structural features important for STAT signaling, we generated an activation-specific STAT5 antibody using a phosphopeptide containing amino acids 687 to 698 of STAT5 as antigen. This antibody specifically recognizes tyrosine- phosphorylated STAT5 but not nonphosphorylated STAT5. In immunoprecipitation reactions from cell lines and primary erythroblasts, 2 distinct polyclonal activation-specific STAT5 antibodies selectively immunoprecipitate the tyrosine phosphorylated EPO receptor (EPO-R) in addition to STAT5 under native and denaturing conditions. We propose that the activation-specific STAT5 antibody recognizes the 2 substrates to which the STAT5 SH2 domain interacts, namely, the tyrosine- phosphorylated EPO-R and STAT5 itself. Several studies have implicated EPO-R Y343, Y401, Y431, and Y479 in the recruitment of STAT5. Using a series of EPO-R tyrosine mutants expressed in Ba/F3 cells, we have shown that the activation-specific STAT5 antibody immunoprecipitates an EPO-R containing only 2 tyrosines at positions 343 and 401, confirming the importance of these tyrosines in STAT5 recruitment. These data uncover a novel aspect of STAT SH2 domain recognition and demonstrate the utility of activation-specific antibodies for examining the specificity of STAT-cytokine receptor interactions. © 2001 by The American Society of Hematology.   CiteULike    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this? This article has been cited by other articles: O. Ravid, I. Shams, N. Ben Califa, E. Nevo, A. Avivi, and D. Neumann An extracellular region of the erythropoietin receptor of the subterranean blind mole rat Spalax enhances receptor maturation PNAS, September 4, 2007; 104(36): 14360 - 14365. [Abstract] [Full Text] [PDF] P. Sathyanarayana, M. P. Menon, O. Bogacheva, O. Bogachev, K. Niss, W. S. 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	Copyright © 2001 by American Society of Hematology         Online ISSN: 1528-0020