Mass spectrometric identification of phophorylation sites of rRNA transcription factor upstream binding factor

Department of Molecular Physiology and Biological Physics, University of Virginia, Box 800736, 1300 Jefferson Park Ave., Charlottesville, VA 22908, USA.
AJP Cell Physiology (Impact Factor: 3.78). 06/2007; 292(5):C1617-24. DOI: 10.1152/ajpcell.00176.2006
Source: PubMed


rRNA transcription is a fundamental requirement for all cellular growth processes and is activated by the phosphorylation of the upstream binding factor (UBF) in response to growth stimulation. Even though it is well known that phosphorylation of UBF is required for its activation and is a key step in activation of rRNA transcription, as yet, there has been no direct mapping of the UBF phosphorylation sites. The results of the present studies employed sophisticated nano-flow HPLC-microelectrospray-ionization tandem mass spectrometry (nHPLC-muESI-MS/MS) coupled with immobilized metal affinity chromatography (IMAC) and computer database searching algorithms to identify 10 phosphorylation sites on UBF at serines 273, 336, 364, 389, 412, 433, 484, 546, 584, and 638. We then carried out functional analysis of two of these sites, serines 389 and 584. Serine-alanine substitution mutations of 389 (S389A) abrogated rRNA transcription in vitro and in vivo, whereas mutation of serine 584 (S584A) reduced transcription in vivo but not in vitro. In contrast, serine-glutamate mutation of 389 (S389E) restored transcriptional activity. Moreover, S389A abolished UBF-SL1 interaction in vitro, while S389E partially restored UBF-SL1 interaction. Taken together, the results of these studies suggest that growth factor stimulation induces an increase in rRNA transcriptional activity via phosphorylation of UBF at serine 389 in part by facilitating a rate-limiting step in the recruitment of RNA polymerase I: i.e., recruitment of SL1. Moreover, studies provide critical new data regarding multiple additional UBF phosphorylation sites that will require further characterization by the field.

Download full-text


Available from: Chun Huie Lin, Sep 28, 2015
11 Reads
  • Source
    • "Post-translational modifications such as acetylation and phosphorylation of UBF control the transcription of rDNA (11,12). cAMP response element-binding protein (CREB)-binding protein (CBP), a histone acetyltransferase (HAT) and transcriptional coactivator, contributes to UBF-mediated transcription in the nucleolus. "
    [Show abstract] [Hide abstract]
    ABSTRACT: The remodeling of chromatin in the nucleolus is important for the control of ribosomal DNA (rDNA) transcription and ribosome biogenesis. Herein, we found that upstream binding factor (UBF) interacts with ESET, a histone H3K9 methyltransferase and is trimethylated at Lys (K) 232/254 by ESET. UBF trimethylation leads to nucleolar chromatin condensation and decreased rDNA transcriptional activity. UBF mutations at K232/254A and K232/254R restored rDNA transcriptional activity in response to ESET. Both ESET-ΔSET mutant and knockdown of ESET by short hairpin RNA reduced trimethylation of UBF and resulted in the restoration of rDNA transcription. Atomic force microscopy confirmed that UBF trimethylated by ESET modulates the plasticity of nucleolar chromatin. We further demonstrated that UBF trimethylation at K232/254 by ESET deregulates rDNA transcription in a cell model of Huntington's disease. Together, our findings show that a novel epigenetic modification of UBF is linked to impaired rDNA transcription and nucleolar chromatin remodeling, which may play key roles in the pathogenesis of neurodegeneration.
    Nucleic Acids Research 11/2013; 42(3). DOI:10.1093/nar/gkt1041 · 9.11 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Mammalian sperm need to reside in the female reproductive tract for a finite period of time before acquiring fertilizing competence. The biochemical changes associated with this process are collectively known as "capacitation". With the use of the mouse as an experimental model, we have previously demonstrated that capacitation is associated with a cAMP-dependent increase in protein tyrosine phosphorylation. However, little is known about the identity and function of the protein targets of this phosphorylation cascade. In the present work, we have used differential isotopic labeling coupled with immobilized metal affinity chromatography (IMAC)-based phosphopeptide enrichment and analysis on a hybrid linear ion trap/FT-ICR mass spectrometer to measure the changes in protein phosphorylation resulting from the capacitation process. As no kinase activators and/or phosphatase inhibitors were used in the preparation of the sperm samples, phosphorylated residues identified in this study represent in vivo sites of phosphorylation. Also, in contrast to other methods which rely on the incorporation of isotopically labeled amino acids at the protein level (e.g., SILAC), the present technique is based on the Fisher esterification of protein digests, allowing for the comparison of phosphorylation status in the absence of protein synthesis. This approach resulted in the identification of 55 unique, in vivo sites of phosphorylation and permitted the relative extent of phosphorylation, as a consequence of capacitation, to be calculated for 42 different phosphopeptides. This work represents the first effort to determine which specific protein phosphorylation sites change their phosphorylation status in vivo as a result of the mammalian capacitation process.
    Journal of Proteome Research 03/2009; 8(3):1431-40. DOI:10.1021/pr800796j · 4.25 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The influence of erbium (Er3+) ion concentration on the infrared emission at 1.5 μm in TeO2–WO3 and TeO2–CdO–WO3 glasses was studied. The samples were prepared by conventional melt quenching method. The optical properties were studied by measuring the absorption and luminescence spectra at room temperature. The Judd–Ofelt parameters (Ωt), transition probabilities, branching ratios of various transitions and their radiative lifetimes were calculated from the optical absorption spectra. Absorption and emission cross-section spectra and the Stark Levels splitting for the 4I13/2 to 4I15/2 transition of Er3+ centered at 1.5 μm were determined.
    Optics Communications 02/2011; 284(3):863. DOI:10.1016/j.optcom.2010.09.087 · 1.45 Impact Factor
Show more