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GROWTH, DIFFERENTIATION, AND APOPTOSIS

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

¹Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, Virginia; ²Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, New York; ³Genomics Institute of the Novartis Research Foundation, San Diego, California; ⁴Department of Chemistry, University of Virginia, Charlottesville; ⁵Department of Molecular Biology and Immunology, University of Southern California, Los Angeles, California; and ⁶Department of Pathology, University of Virginia, Charlottesville, Virginia

Submitted 11 April 2006 ; accepted in final form 17 December 2006

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-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.

immobilized metal affinity chromatography; mass spectrometry; UBF; TBP; RNA polymerase I