Identification of isoform-specific dynamics in phosphorylation-dependent STAT5 dimerization by quantitative mass spectrometry and mathematical modeling

TY - JOUR

T1 - Identification of isoform-specific dynamics in phosphorylation-dependent STAT5 dimerization by quantitative mass spectrometry and mathematical modeling

AU - Boehm, Martin E

AU - Adlung, Lorenz

AU - Schilling, Marcel

AU - Roth, Susanne

AU - Klingmüller, Ursula

AU - Lehmann, Wolf D

PY - 2014/12/5

Y1 - 2014/12/5

N2 - 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.

AB - 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.

KW - Algorithms

KW - Amino Acid Sequence

KW - Animals

KW - Cell Line

KW - Cell Nucleus/metabolism

KW - Chromatography, Liquid

KW - Cytoplasm/metabolism

KW - Erythropoietin/pharmacology

KW - Immunoblotting

KW - Kinetics

KW - Mass Spectrometry/methods

KW - Mice

KW - Models, Theoretical

KW - Molecular Sequence Data

KW - Phosphorylation

KW - Protein Multimerization

KW - Protein Transport/drug effects

KW - Receptors, Erythropoietin/genetics

KW - STAT5 Transcription Factor/chemistry

KW - Sequence Homology, Amino Acid

U2 - 10.1021/pr5006923

DO - 10.1021/pr5006923

M3 - SCORING: Journal article

C2 - 25333863

VL - 13

SP - 5685

EP - 5694

JO - J PROTEOME RES

JF - J PROTEOME RES

SN - 1535-3893

IS - 12

ER -