Submitted on April 11, 2006
Accepted on December 17, 2006
Mass Spectrometric Identification of Phosphorylation Sites of the rRNA Transcription Factor Upstream Binding Factor (UBF)
C. Huie Lin1, Mark D Platt2, Scott B Ficarro3, Mark H Hoofnagle1, Jeffrey Shabanowitz4, Lucio Comai5, Donald F Hunt4, and Gary K Owens1*
1 Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, Virginia, United States
2 Rensselaer Polytechnic Institute, Center for Biotechnology and Interdisciplinary Studies, Troy, New York, United States
3 Genomics Research Institute, Novartis, San Diego, California, United States
4 Chemistry, University of Virginia, Charlottesville, Virginia, United States
5 Molecular Microbiology & Immunology, University of Southern California, Los Angeles, California, United States
* To whom correspondence should be addressed. E-mail: gko{at}virginia.edu.
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. However, despite the fact that it has been known for over a decade 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. Results of the present studies employed sophisticated nano-flow high-pressure liquid-chromatography-microelectrospray-ionization tandem mass spectrometry (nHPLC-µESI-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.
