title: Kinetic analysis of BCL11B multisite phosphorylation-dephosphorylation and coupled sumoylation in primary thymocytes by multiple reaction monitoring mass spectroscopy.
abstract: 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.
authors.affiliation: Department of Pharmaceutical Sciences, College of Pharmacy, Oregon State University , Corvallis, Oregon 97331, United States.
authors.forename: Walter K
authors.initials: WK
authors.lastname: Vogel
authors.forename: Philip R
authors.initials: PR
authors.lastname: Gafken
authors.forename: Mark
authors.initials: M
authors.lastname: Leid
authors.forename: Theresa M
authors.initials: TM
authors.lastname: Filtz
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D000001:Calcimycin / Q000494:pharmacology; D061207:Calcium Ionophores / Q000494:pharmacology; D002478:Cells, Cultured; D015151:Immunoblotting; D007700:Kinetics; D008239:Lysine / Q000378:metabolism; D013058:Mass Spectrometry / Q000379:methods*; D051379:Mice; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D008969:Molecular Sequence Data; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D012097:Repressor Proteins / Q000378:metabolism*; D012694:Serine / Q000378:metabolism; D058207:Sumoylation / Q000187:drug effects; D013912:Threonine / Q000378:metabolism; D060168:Thymocytes / Q000166:cytology / Q000378:metabolism*; D013997:Time Factors; D025521:Tumor Suppressor Proteins / Q000378:metabolism*
chemical_list: C511143:Bcl11b protein, mouse; D061207:Calcium Ionophores; D012097:Repressor Proteins; D025521:Tumor Suppressor Proteins; D013912:Threonine; D015240:Phorbol 12,13-Dibutyrate; D000001:Calcimycin; D012694:Serine; D020928:Mitogen-Activated Protein Kinases; D008239:Lysine
keywords: BCL11B; MRM; SRM; T-cell receptor; post-translational modification; signal transduction; sumoylation