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.
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.
Barber
D L
DL
Division of Cellular and Molecular Biology, Ontario Cancer Institute, Toronto, Canada. dbarber@oci.utoronto.ca
Beattie
B K
BK
Mason
J M
JM
Nguyen
M H
MH
Yoakim
M
M
Neel
B G
BG
D'Andrea
A D
AD
Frank
D A
DA
D000818:Animals; D000918:Antibody Specificity; D015153:Blotting, Western; D002478:Cells, Cultured; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000276:immunology*; D000939:Epitopes / Q000737:chemistry / Q000276:immunology*; D004921:Erythropoietin / Q000494:pharmacology*; D051379:Mice; D008810:Mice, Inbred C57BL; D008894:Milk Proteins; D008954:Models, Biological; D010446:Peptide Fragments / Q000276:immunology; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000737:chemistry; D011233:Precipitin Tests; D011489:Protein Denaturation; D011499:Protein Processing, Post-Translational; D011817:Rabbits; D017467:Receptors, Erythropoietin / Q000737:chemistry / Q000276:immunology*; D050799:STAT5 Transcription Factor; D012756:Sheep; D015534:Trans-Activators / Q000737:chemistry / Q000276:immunology*; D018909:src Homology Domains
D004268:DNA-Binding Proteins; D000939:Epitopes; D008894:Milk Proteins; D010446:Peptide Fragments; D017467:Receptors, Erythropoietin; D050799:STAT5 Transcription Factor; D015534:Trans-Activators; D004921:Erythropoietin; D019000:Phosphotyrosine