Mass Spectrometric Identification of Phosphorylation Sites of the rRNA Transcription Factor
Upstream Binding Factor (UBF)
Running Title: Identification of UBF Phosphorylation Sites
C. Huie Lin1, Mark D. Platt2, Scott B. Ficarro3, Mark H. Hoofnagle1, Jeffrey Shabanowitz4,
Lucio Comai5, Donald F. Hunt4,6, Gary K. Owens1
Address correspondence to:
Gary K Owens,
Department of Molecular Physiology and Biological Physics,
University of Virginia, Box 800736;
1300 Jefferson Park Ave;
Charlottesville, Virginia 22908,
1Dept Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, VA
2Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY
3Genomics Institute of the Novartis Research Foundation, San Diego, CA
4Dept of Chemistry, University of Virginia, Charlottesville, VA
5Dept of Molecular Biology and Immunology, University of Southern California, Los Angeles, CA
6Dept of Pathology, University of Virginia, Charlottesville, VA
Abbreviations: UBF=Upstream Binding Factor, rRNA=ribosomal ribonucleic acid, MS/MS=tandem mass spectrometry,
SL1=Selectivity Factor 1, Pol I= RNA Polymerase I
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Articles in PresS. Am J Physiol Cell Physiol (December 20, 2006). doi:10.1152/ajpcell.00176.2006
Copyright © 2006 by the American Physiological Society.
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.
Activation of rRNA transcription is required for sustained growth of all cells. In vitro, eukaryotic rRNA transcription
can be reconstituted by the addition of three factors: RNA Polymerase I (Pol I), Selectivity Factor 1 (SL1, consisting
of the TATA Binding Protein, TBP, and three Pol I specific TBP associated factors, TAFIs 48, 63, and 110) and the
Upstream Binding Factor (UBF). It has been well recognized for nearly a decade that UBF is a necessary
component for the activation of efficient rRNA transcription, and that phosphorylation of UBF dramatically enhances
rRNA transcription in vitro. Furthermore, UBF phosphorylation is upregulated in states of cellular growth, consistent
with a model whereby phosphorylation of UBF is a key mechanism linking cellular growth to activation of rRNA
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transcription. However, relatively little is known regarding the mechanisms by which UBF phosphorylation regulates
transcriptional activity. Previous studies in our lab(17) and others(33;40) demonstrated that growth-induced
phosphorylation of UBF resulted in increased binding between SL1 (or TBP) and UBF. UBF and SL1 (known as
TIFIB in mouse) are the first factors that bind rDNA to initiate formation of a stable transcription complex in vitro(30).
However, unlike UBF, SL1 alone does not bind efficiently to the rDNA promoter which lacks a TATA box. However,
addition of UBF substantially extends the in vitro DNase footprint generated by SL1 alone, suggesting cooperative
interactions between these factors(1;18). Thus, UBF phosphorylation may control the kinetics and stability of
transcription initiation complex assembly via recruitment of SL1 to the rDNA promoter. Taken together, results
suggest that growth factor induced increases in rRNA transcription are mediated in part by phosphorylation of UBF
and subsequent enhanced recruitment of SL1/TBP to rDNA promoters.
Recently, two kinase families have been implicated in UBF phosphorylation. Drakas et al. demonstrated that
PI3 kinase phosphorylates UBF as a result of insulin-like growth factor signaling (IGF-1). This is of particular
functional significance as IGF signaling has been shown to play a role in cellular growth and hypertrophy(7).
Likewise, ERK1/2 plays a well-characterized role in phosphoregulation of cell growth and was recently implicated in
UBF phosphorylation by Stefanovsky et al.(32). However, the role of this phosphorylation in UBF-mediated rDNA
transcription initiation complex has not been completely elucidated.
UBF phosphorylation may also mediate sub-nuclear localization of UBF. Under conditions of cellular
quiescence or serum starvation, UBF is localized diffusely throughout the nucleus. In contrast, agonist or serum
stimulation of the cells induced rapid nucleolar localization of UBF (13;23), suggesting that spatial localization of
components of the transcription initiation complex contribute to regulation of rRNA transcription(3;8).
A major limitation in the field is that there are no reported studies that have directly mapped which of the 70
serine residues in the primary sequence of UBF modulate its transcriptional activity and by what mechanism they do
so. Our laboratory(17) and others(23) have shown that UBF is phosphorylated exclusively on multiple serine
residues in vivo and is extremely complex. For example, results of 2D phosphotryptic mapping studies in our lab
provided compelling evidence that growth factor stimulation of cells increased the stoichiometry of phosphorylation
on at least 11 different sites as compared to quiescent cells in positive protein balance in a defined serum free
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medium, but did not induce new sites of phosphorylation (11). Studies by Voit et al. showing that mutation of serine
388(34) or 484(37) abrogated transcriptional activity of UBF in vitro represented a major advance for the field.
However, there is no direct evidence that these sites are phosphorylated on full-length UBF and many additional sites
remain to be identified and functionally characterized. In summary, despite it being widely acknowledged for nearly a
decade that regulation of UBF phosphorylation is a key rate limiting step in control of rRNA transcription and
sustained cell growth, very little is known as to how this critical process is regulated.
Here, we report the first direct mapping of phosphorylation sites on full-length UBF1 (see Table) obtained using
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 and demonstrate that at least one of these sites is necessary for transcriptional activation via SL1
nucleation to the rDNA transcription initiation site.
Materials and Methods
Cell culture, Co-transfection assays, and In vitro transcription
Culture of rat aortic VSMCs has been described previously(13). For transient transfection experiments, VSMCs
were plated at a density of 104 cells/cm2. After 24 hours, cells were transfected in triplicate with FuGENE6 (Roche)
following manufacturer's protocol. Transfection was with equal (1:1) ratio of pGRIL rDNA-IRES luciferase construct
to plasmid of interest (pcDNA3 or pcDNA3 carrying wildtype or mutant rUBF1 cDNA (pcDUBF, pcDTR2, pcD389A,
pcD389E, or pcD584A). One day after transfection cells had reached confluence were rendered quiescent(11) in a
defined serum free medium for 24 hours, after which they were refed with serum-containing media for 9 additional
hours. Cells were then lysed with 150µl 1x Passive Lysis Buffer (Promega) for one half hour at room temperature.
Luciferase reporter activity was assayed on Monolight 2010 luminometer using 20µl cell lysate and 100µl Luciferase
Assay Substrate (Promega) with 10 second measurements. Values were normalized using protein concentration
assayed by Coomassie Plus Protein Assay Reagent (Pierce) according to manufacturer directions.
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In vitro transcription assays were performed as previously described(40).
See Supplemental Materials.
Purification and In vitro phosphorylation of UBF
Full-length FLAG - rUBF1 or mutant FLAG - rUBF1 was prepared by infecting a SF9 culture at 5 × 105 cells/ml
with a UBF expression baculovirus, MOI 1.0-10 (constructed using the Baculogold co-transfection kit from
Pharmingen exactly as described in product materials). Cells were incubated as a suspension in a spinner flask at
27°Cfor 44-48 h. At the termination of the incubation, the cells werepelleted by centrifugation, washed once in PBS
(Invitrogen) and snap frozen in liquid N2. Cells were lysed in hypotonic Lysis buffer (10mM Tris-HCl, pH7.9, 10mM
KCl, 1.5mM MgCl2) by sonication (50% duty cycle, 600W X 40% power). After sonication, nuclear debris was
pelleted by centrifugation, 12krpm X 10 min. at 4ºC. Nuclear debris was then extracted by Extraction buffer (50mM
Tris HCl, pH 7.9, 600mM NaCl, 5mM MgCl2, 25% glycerol, 0.5mM EDTA); extract was then cleared by
centrifugation, 12krpm X 10 min. at 4ºC. Lysate and extract were combined, adjusted to pH 7.9 using 1M Tris base,
added to anti-FLAG M2 agarose beads, and rotated at 4ºC for 2 hours. Beads were extensively washed in >10
volumes of Tris buffered saline pH 7.9, and FLAG - rUBF1 (or mutant) eluted in 100mM Glycine pH 2.5. Fractions
were immediately neutralized to 50mM Tris by addition of 1M Tris Base and dialyzed against 50mM Tris pH7.9,
150mM KCl, 10% glycerol. 50µg of purified FLAG -rUBF1 was phosphorylated by 2 µg of Angiotensin II-stimulated
VSMC nuclear extract in vitro for one hour at 37ºC in the presence of Phosphatase Inhibitor Cocktail (Sigma).
Samples were then snap frozen in liquid N2 and stored at -80ºC until ready for proteolytic digestion and MS/MS
analysis. Final concentration of UBF was approximately 100ug/ml. All buffers contained Complete protease inhibitor
cocktail (Roche), 0.5mM DTT, and 1mM MBS unless otherwise stated.
After transient transfection (described above), cells were allowed to recover for 48 hours at 37ºC. Cells were
washed 3 times with PBS, fixed in 2% paraformaldehyde for 15 min, and permeabilized with 0.25% Triton X-100 for
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