Kinetic Analysis of BCL11B Multisite Phosphorylation–Dephosphorylation and Coupled Sumoylation in Primary Thymocytes by Multiple Reaction Monitoring Mass Spectroscopy

Transcription factors with multiple
post-translational modifications
(PTMs) are not uncommon, but comprehensive information on site-specific
dynamics and interdependence is comparatively rare. Assessing dynamic
changes in the extent of PTMs has the potential to link multiple sites
both to each other and to biological effects observable on the same
time scale. The transcription factor and tumor suppressor BCL11B is
critical to three checkpoints in T-cell development and is a target
of a T-cell receptor-mediated MAP kinase signaling. Multiple reaction
monitoring (MRM) mass spectroscopy was used to assess changes in relative
phosphorylation on 18 of 23 serine and threonine residues and sumoylation
on one of two lysine resides in BCL11B. We have resolved the composite
phosphorylation–dephosphorylation and sumoylation changes of
BCL11B in response to MAP kinase activation into a complex pattern
of site-specific PTM changes in primary mouse thymocytes. The site-specific
resolution afforded by MRM analyses revealed four kinetic patterns
of phosphorylation and one of sumoylation, including both rapid simultaneous
site-specific increases and decreases at putative MAP kinase proline-directed
phosphorylation sites, following stimulation. These data additionally
revealed a novel spatiotemporal bisphosphorylation motif consisting
of two kinetically divergent proline-directed phosphorylation sites
spaced five residues apart.