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Quantitative mass spectrometry of TATA binding protein-containing complexes and subunit phosphorylations during the cell cycle.
Pijnappel, Wwm Pim; Kolkman, Annemieke; Baltissen, Marijke Pa; Heck, Albert Jr; Timmers, Ht Marc.
Afiliación
  • Pijnappel WP; Netherlands Proteomics Centre, Department of Physiological Chemistry, University Medical Center Utrecht, Universiteitsweg 100, 3584 CG Utrecht, the Netherlands.
  • Kolkman A; Netherlands Proteomics Centre, Biomolecular Mass Spectrometry and Proteomics Group, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Faculty of Science, Padualaan 8, 3584 CH Utrecht, the Netherlands.
  • Baltissen MP; Netherlands Proteomics Centre, Department of Physiological Chemistry, University Medical Center Utrecht, Universiteitsweg 100, 3584 CG Utrecht, the Netherlands.
  • Heck AJ; Netherlands Proteomics Centre, Biomolecular Mass Spectrometry and Proteomics Group, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Faculty of Science, Padualaan 8, 3584 CH Utrecht, the Netherlands.
  • Timmers HM; Netherlands Proteomics Centre, Department of Physiological Chemistry, University Medical Center Utrecht, Universiteitsweg 100, 3584 CG Utrecht, the Netherlands.
Proteome Sci ; 7: 46, 2009 Dec 24.
Article en En | MEDLINE | ID: mdl-20034391
BACKGROUND: Progression through the cell cycle is accompanied by tightly controlled regulation of transcription. On one hand, a subset of genes is expressed in a cell cycle-dependent manner. On the other hand, a general inhibition of transcription occurs during mitosis. Genetic and genome-wide studies suggest cell cycle regulation at the level of transcription initiation by protein complexes containing the common DNA-binding subunit TATA binding protein (TBP). TBP is a key player in regulating transcription by all three nuclear RNA polymerases. It forms at least four distinct protein complexes with TBP-associated factors (TAFs): SL1, B-TFIID, TFIID, and TFIIIB. Some TAFs are known to remain associated with TBP during the cell cycle. Here we analyze all TAFs and their phosphorylation status during the cell cycle using a quantitative mass spectrometry approach. RESULTS: TBP protein complexes present in human cells at the G2/M and G1/S transitions were analyzed by combining affinity purification with quantitative mass spectrometry using stable isotope labeling with amino acids in cell culture (SILAC). Phosphorylations were mapped and quantified after enrichment of tryptic peptides by titanium dioxide. This revealed that subunit stoichiometries of TBP complexes remained intact, but their relative abundances in nuclear extracts changed during the cell cycle. Several novel phosphorylations were detected on subunits of the TBP complexes TFIID and SL1. G2/M-specific phosphorylations were detected on TAF1, TAF4, TAF7, and TAFI41/TAF1D, and G1/S-specific dephosphorylations were detected on TAF3. Many phosphorylated residues were evolutionary conserved from human to zebrafish and/or drosophila, and were present in conserved regions suggesting important regulatory functions. CONCLUSIONS: This study provides the first quantitative proteomic analysis of human TBP containing protein complexes at the G2/M and G1/S transitions, and identifies new cell cycle-dependent phosphorylations on TAFs present in their protein complex. We speculate that phosphorylation of complex-specific subunits may be involved in regulating the activities of TBP protein complexes during the cell cycle.

Texto completo: 1 Base de datos: MEDLINE Tipo de estudio: Prognostic_studies Idioma: En Revista: Proteome Sci Año: 2009 Tipo del documento: Article País de afiliación: Países Bajos

Texto completo: 1 Base de datos: MEDLINE Tipo de estudio: Prognostic_studies Idioma: En Revista: Proteome Sci Año: 2009 Tipo del documento: Article País de afiliación: Países Bajos