title: Analysis of phosphorylation-dependent protein-protein interactions of histone h3.
abstract: Multiple posttranslational modifications (PTMs) of histone proteins including site-specific phosphorylation of serine and threonine residues govern the accessibility of chromatin. According to the histone code theory, PTMs recruit regulatory proteins or block their access to chromatin. Here, we report a general strategy for simultaneous analysis of both of these effects based on a SILAC MS scheme. We applied this approach for studying the biochemical role of phosphorylated S10 of histone H3. Differential pull-down experiments with H3-tails synthesized from l- and d-amino acids uncovered that histone acetyltransferase 1 (HAT1) and retinoblastoma-binding protein 7 (RBBP7) are part of the protein network, which interacts with the unmodified H3-tail. An additional H3-derived bait containing the nonhydrolyzable phospho-serine mimic phosphonomethylen-alanine (Pma) at S10 recruited several isoforms of the 14-3-3 family and blocked the recruitment of HAT1 and RBBP7 to the unmodified H3-tail. Our observations provide new insights into the many functions of H3S10 phosphorylation. In addition, the outlined methodology is generally applicable for studying specific binding partners of unmodified histone tails.
authors.affiliation: Laboratory of Protein Chemistry and §Mass Spectrometry, Leibniz-Institut für Molekulare Pharmakologie , Robert-Rössle-Strasse 10, 13125 Berlin, Germany.
authors.forename: Rebecca
authors.initials: R
authors.lastname: Klingberg
authors.forename: Jan Oliver
authors.initials: JO
authors.lastname: Jost
authors.forename: Michael
authors.initials: M
authors.lastname: Schümann
authors.forename: Kathy Ann
authors.initials: KA
authors.lastname: Gelato
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Fischle
authors.forename: Eberhard
authors.initials: E
authors.lastname: Krause
authors.forename: Dirk
authors.initials: D
authors.lastname: Schwarzer
mesh_terms: D000596:Amino Acids / Q000737:chemistry; D018929:Cell Culture Techniques; D005454:Fluorescence Polarization; D006367:HeLa Cells; D051548:Histone Acetyltransferases / Q000235:genetics / Q000378:metabolism*; D006657:Histones / Q000378:metabolism*; D006801:Humans; D013058:Mass Spectrometry; D010766:Phosphorylation; D011485:Protein Binding; D020033:Protein Isoforms; D011499:Protein Processing, Post-Translational; D040901:Proteomics / Q000379:methods*; D011994:Recombinant Proteins; D056524:Retinoblastoma-Binding Protein 7 / Q000235:genetics / Q000378:metabolism*
chemical_list: D000596:Amino Acids; D006657:Histones; D020033:Protein Isoforms; D011994:Recombinant Proteins; D056524:Retinoblastoma-Binding Protein 7; D051548:Histone Acetyltransferases; C110880:histone acetyltransferase type B complex