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

Blood. 2001 Apr 15;97(8):2230-7. doi: 10.1182/blood.v97.8.2230.

Abstract

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.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Antibody Specificity
  • Blotting, Western
  • Cells, Cultured
  • DNA-Binding Proteins / chemistry
  • DNA-Binding Proteins / immunology*
  • Epitopes / chemistry
  • Epitopes / immunology*
  • Erythropoietin / pharmacology*
  • Mice
  • Mice, Inbred C57BL
  • Milk Proteins*
  • Models, Biological
  • Peptide Fragments / immunology
  • Phosphorylation
  • Phosphotyrosine / chemistry
  • Precipitin Tests
  • Protein Denaturation
  • Protein Processing, Post-Translational
  • Rabbits
  • Receptors, Erythropoietin / chemistry
  • Receptors, Erythropoietin / immunology*
  • STAT5 Transcription Factor
  • Sheep
  • Trans-Activators / chemistry
  • Trans-Activators / immunology*
  • src Homology Domains

Substances

  • DNA-Binding Proteins
  • Epitopes
  • Milk Proteins
  • Peptide Fragments
  • Receptors, Erythropoietin
  • STAT5 Transcription Factor
  • Trans-Activators
  • Erythropoietin
  • Phosphotyrosine