A common epitope is shared by activated signal transducer and activator oftranscription-5 (STAT5) and the phosphorylated erythropoietin receptor: implications for the docking model of STAT activation

Erythropoietin (EPO) specifically activates the Janus kinase JAK2 and the t ranscription factor signal transducer and activator of transcription-5 (STA T5). 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 sig naling, we generated an activation-specific STAT5 antibody using a phosphop eptide containing amino acids 687 to 698 of STAT5 as antigen. This antibody specifically recognizes tyrosine-phosphorylated STAT5 but not nonphosphory lated STAT5. In immunoprecipitation reactions from cell lines and primary e rythroblasts, 2 distinct polyclonal activation-specific STAT5 antibodies se lectively immunoprecipitate the tyrosine phosphorylated EPO receptor (EPO-R ) in addition to STAT5 under native and denaturing conditions. We propose t hat the activation-specific STAT5 antibody recognizes the 2 substrates to w hich the STAT5 SH2 domain interacts, namely, the tyrosine-phosphorylated EP O-R and STAT5 itself. Several studies have implicated EPO-R Y343, Y401, Y43 1, and Y479 in the recruitment of STAT5. Using a series of EPO-R tyrosine m utants 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 STAT 5 recruitment. These data uncover a novel aspect of STAT SH2 domain recogni tion and demonstrate the utility of activation-specific antibodies for exam ining the specificity of STAT-cytokine receptor interactions. (Blood. 2001; 97:2230-2237) (C) 2001 by The American Society of Hematology.