title: Identification of isoform-specific dynamics in phosphorylation-dependent STAT5 dimerization by quantitative mass spectrometry and mathematical modeling.
abstract: STAT5A and STAT5B are important transcription factors that dimerize and transduce activation signals of cytokine receptors directly to the nucleus. A typical cytokine that mediates STAT5 activation is erythropoietin (Epo). Differential functions of STAT5A and STAT5B have been reported. However, the extent to which phosphorylated STAT5A and STAT5B (pSTAT5A, pSTAT5B) form homo- or heterodimers is not understood, nor is how this might influence the signal transmission to the nucleus. To study this, we designed a concept to investigate the isoform-specific dimerization behavior of pSTAT5A and pSTAT5B that comprises isoform-specific immunoprecipitation (IP), measurement of the degree of phosphorylation, and isoform ratio determination between STAT5A and STAT5B. For the main analytical method, we employed quantitative label-free and -based mass spectrometry. For the cellular model system, we used Epo receptor (EpoR)-expressing BaF3 cells (BaF3-EpoR) stimulated with Epo. Three hypotheses of dimer formation between pSTAT5A and pSTAT5B were used to explain the analytical results by a static mathematical model: formation of (i) homodimers only, (ii) heterodimers only, and (iii) random formation of homo- and heterodimers. The best agreement between experimental data and model simulations was found for the last case. Dynamics of cytoplasmic STAT5 dimerization could be explained by distinct nuclear import rates and individual nuclear retention for homo- and heterodimers of phosphorylated STAT5.
authors.affiliation: Molecular Structure Analysis, ‡Systems Biology of Signal Transduction, German Cancer Research Center (DKFZ) , Im Neuenheimer Feld 280, 69120 Heidelberg, Germany.
authors.forename: Martin E
authors.initials: ME
authors.lastname: Boehm
authors.forename: Lorenz
authors.initials: L
authors.lastname: Adlung
authors.forename: Marcel
authors.initials: M
authors.lastname: Schilling
authors.forename: Susanne
authors.initials: S
authors.lastname: Roth
authors.forename: Ursula
authors.initials: U
authors.lastname: Klingmüller
authors.forename: Wolf D
authors.initials: WD
authors.lastname: Lehmann
mesh_terms: D000465:Algorithms; D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D002853:Chromatography, Liquid; D003593:Cytoplasm / Q000378:metabolism; D004921:Erythropoietin / Q000494:pharmacology; D015151:Immunoblotting; D007700:Kinetics; D013058:Mass Spectrometry / Q000379:methods*; D051379:Mice; D008962:Models, Theoretical; D008969:Molecular Sequence Data; D010766:Phosphorylation; D055503:Protein Multimerization; D021381:Protein Transport / Q000187:drug effects; D017467:Receptors, Erythropoietin / Q000235:genetics / Q000378:metabolism; D050799:STAT5 Transcription Factor / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
chemical_list: D017467:Receptors, Erythropoietin; D050799:STAT5 Transcription Factor; C494093:Stat5a protein, mouse; C494096:Stat5b protein, mouse; D004921:Erythropoietin
keywords: Epo stimulation; STAT5; dimerization; mathematical modeling; phosphorylation; relative isoform quantification