title: Quantitative Analysis of Dynamic Protein Interactions during Transcription Reveals a Role for Casein Kinase II in Polymerase-associated Factor (PAF) Complex Phosphorylation and Regulation of Histone H2B Monoubiquitylation.
abstract: Using affinity purification MS approaches, we have identified a novel role for casein kinase II (CKII) in the modification of the polymerase associated factor complex (PAF-C). Our data indicate that the facilitates chromatin transcription complex (FACT) interacts with CKII and may facilitate PAF complex phosphorylation. Posttranslational modification analysis of affinity-isolated PAF-C shows extensive CKII phosphorylation of all five subunits of PAF-C, although CKII subunits were not detected as interacting partners. Consistent with this, recombinant CKII or FACT-associated CKII isolated from cells can phosphorylate PAF-C in vitro, whereas no intrinsic kinase activity was detected in PAF-C samples. Significantly, PAF-C purifications combined with stable isotope labeling in cells (SILAC) quantitation for PAF-C phosphorylation from wild-type and CKII temperature-sensitive strains (cka1Δ cka2-8) showed that PAF-C phosphorylation at consensus CKII sites is significantly reduced in cka1Δ cka2-8 strains. Consistent with a role of CKII in FACT and PAF-C function, we show that decreased CKII function in vivo results in decreased levels of histone H2B lysine 123 monoubiquitylation, a modification dependent on FACT and PAF-C. Taken together, our results define a coordinated role of CKII and FACT in the regulation of RNA polymerase II transcription through chromatin via phosphorylation of PAF-C.
authors.affiliation: From the Department of Biology, DePauw University, Greencastle, Indiana 46135, the Department of Biochemistry and Molecular Biology and.
authors.forename: Lynn Glowczewski
authors.initials: LG
authors.lastname: Bedard
authors.affiliation: the Department of Biochemistry and Biophysics.
authors.forename: Raghuvar
authors.initials: R
authors.lastname: Dronamraju
authors.affiliation: the Department of Biochemistry and Biophysics, Lineberger Comprehensive Cancer Center, and.
authors.forename: Jenny L
authors.initials: JL
authors.lastname: Kerschner
authors.affiliation: the Department of Biochemistry and Molecular Biology and.
authors.forename: Gerald O
authors.initials: GO
authors.lastname: Hunter
authors.affiliation: the Department of Biochemistry and Molecular Biology and.
authors.forename: Elizabeth DeVlieger
authors.initials: ED
authors.lastname: Axley
authors.affiliation: From the Department of Biology, DePauw University, Greencastle, Indiana 46135, the Department of Biochemistry and Molecular Biology and.
authors.forename: Asha K
authors.initials: AK
authors.lastname: Boyd
authors.affiliation: the Department of Biochemistry and Biophysics, Lineberger Comprehensive Cancer Center, and Curriculum in Genetics and Molecular Biology, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599.
authors.forename: Brian D
authors.initials: BD
authors.lastname: Strahl
authors.affiliation: the Department of Biochemistry and Molecular Biology and Center for Computational Biology and Bioinformatics, Indiana University School of Medicine, Indianapolis, Indiana 46202, and almosley@iu.edu.
authors.forename: Amber L
authors.initials: AL
authors.lastname: Mosley
mesh_terms: D047390:Casein Kinase II / Q000235:genetics / Q000378:metabolism*; D002843:Chromatin / Q000235:genetics / Q000378:metabolism; D006657:Histones / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation / Q000502:physiology; D012319:RNA Polymerase II / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D014158:Transcription, Genetic / Q000502:physiology*; D054875:Ubiquitination / Q000502:physiology*
chemical_list: D002843:Chromatin; D006657:Histones; D029701:Saccharomyces cerevisiae Proteins; D047390:Casein Kinase II; D012319:RNA Polymerase II
keywords: FACT; PAF-C; SILAC; casein kinase II; chromatin; histone modification; phosphorylation; quantitative proteomics; transcription; ubiquitylation (ubiquitination)