Mass spectrometric identification of phosphorylation sites of rRNA transcription factor upstream binding factor.
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.
Lin
C Huie
CH
Department of Molecular Physiology and Biological Physics, University of Virginia, Box 800736, 1300 Jefferson Park Ave., Charlottesville, VA 22908, USA.
Platt
Mark D
MD
Ficarro
Scott B
SB
Hoofnagle
Mark H
MH
Shabanowitz
Jeffrey
J
Comai
Lucio
L
Hunt
Donald F
DF
Owens
Gary K
GK
D000465:Algorithms; D000595:Amino Acid Sequence; D000818:Animals; D002478:Cells, Cultured; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D030562:Databases, Protein; D008969:Molecular Sequence Data; D009154:Mutation; D032389:Myocytes, Smooth Muscle / Q000378:metabolism; D036103:Nanotechnology; D010449:Peptide Mapping / Q000379:methods*; D010766:Phosphorylation; D035463:Pol1 Transcription Initiation Complex Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D012318:RNA Polymerase I / Q000378:metabolism; D012335:RNA, Ribosomal / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D012694:Serine / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization; D053719:Tandem Mass Spectrometry; D014158:Transcription, Genetic; D014162:Transfection
D035463:Pol1 Transcription Initiation Complex Proteins; D012335:RNA, Ribosomal; D011994:Recombinant Proteins; C063844:transcription factor UBF; C063660:transcription initiation factor TIF-IB; D012694:Serine; D012318:RNA Polymerase I