Identification of Isoform-Specific Dynamics in Phosphorylation-Dependent STAT5 Dimerization by Quantitative Mass Spectrometry and Mathematical Modeling

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.