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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fifteen minutes. Primary antibodies, anti-FLAG M2 1:500 (Kodak) and anti-fibrillarin 1:100 ("ANA-N", Sigma) were
applied with 0.1% Triton X-100, 5% Goat serum, and 1% horse serum in PBS for 2 hours at room temperature. Cells
were then washed 3 times with PBS and stained with 300nM DAPI and secondary antibodies 1:2000 Alexa Fluor 488
anti-mouse IgG, 1:2000 Alexa Fluor 533 anti-human IgG, 1:2000 (Molecular Probes). Before visualizing, cells were
washed three times with PBS. Images were acquired on an Olympus inverted fluorescence microscope and a cooled
digital camera (Roper Scientific).
Mass Spectrometric Identification of Phosphorylation Sites
100ul (~100pmol) of purified, phosphorylated UBF1 was reduced, alkylated, and digested with trypsin(14) and
aliquots of the digest corresponding to 12.5pmol of UBF1 were subjected to sample clean-up and IMAC enrichment
as previously described(10), with minor modifications. As a single, highly-purified protein was being analyzed, no
attempt was made to reduce the binding of non-phosphorylated peptides to the IMAC material by utilizing the Fisher
esterification. Rather, an aliquot was loaded directly onto a 360µm o.d. × 200µm i.d. fused-silica clean-up column
containing a LiChrosorb frit and packed with 8cm of 5-20µm C-18 particles. The peptide-loaded clean-up column
was rinsed extensively with 0.1% (v/v) HOAc and connected to a 360µm o.d. × 100µm i.d. fused-silica column,
packed with 8cm POROS 20 MC IMAC packing material and activated with 200µL 100mM FeCl3. Peptides were
eluted from the clean-up column to the IMAC column with a solution of 40% (v/v) acetonitrile, and 1% (v/v) HOAc.
The IMAC column was disconnected from the clean-up column and rinsed with a solution containing 100mM NaCl in
25% (v/v) MeCN and 1% (v/v) HOAc to remove non-specifically bound peptides. The IMAC column was re-
equilibrated with a solution of 0.1% (v/v) HOAc, and the phosphopeptides were eluted to an HPLC analytical
column/pre-column assembly containing an integrated laser-pulled electrospray ionization emitter tip(21). Samples
were analyzed by nanoflow HPLC-microelectrospray ionization on a Finnigan LCQ DECA ion trap mass
spectrometer, operated in data dependent mode. Rapid identification of phosphopeptide candidates from MS/MS
spectra was accomplished with an in-house computer program called Neutral Loss Tool(39) which screens for the
loss of phosphoric acid from the phosphopeptide precursor(6). Candidate MS/MS spectra were searched against the
UBF1 protein sequence using the SEQUEST algorithm with "No Enzyme" specificity(9). Search parameters included
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a differential modification of 80 Da on serine, threonine, and tyrosine, representing the presence or absence of
phosphate, and a static modification of 57 Da on cysteine, representing alkylation with iodoacetamide.
Wang resins, 9-fluorenylmethoxycarbonyl (Fmoc) protected amino acids, and phosphorylated amino acids
(Calbiochem, San Diego, CA) were used to synthesize peptides on an AMS 422 multiple peptide synthesizer (Gilson
Medical Electronics, Middletown, WI). Peptides were characterized by mass spectrometry.
SL1 interaction assay
The UBF-SL1 interaction assay was performed largely as previously described(33). Briefly, 500ng purified
wildtype FLAG - rUBF1 or mutant was incubated with anti-FLAG M2 agarose bead (Sigma) for two hours at 4ºC,
washed extensively in TM++0.1 (50mM TrisHCl pH7.9, 100mM KCl, 12.5mM MgCl2, 10% glycerol, 0.1%NP-40), and
incubated with 20ug of partially purified SL1 overnight. Beads were washed extensively in TM++0.1, rUBF1 bound
SL1 was eluted in BCO (20mM TrisHCl, pH8.0, 10% glycerol, 1M KCl, 0.5mM EDTA, 1% Deoxycholic acid) buffer,
TCA precipitated, resolved on 10% SDS PAGE and analyzed by anti-TAF110 Western.
Effective expression, purification, and phosphorylation of UBF
The development of a reliable and reproducible method for the efficient expression and purification of UBF was
the first major step in our attempts to identify phosphorylation sites within the protein. Indeed, efficient production of
full-length recombinant UBF continues to represent a major challenge for the field (L Comai, L Rothblum, CH Lin,
unpublished observations), and a number of studies continue to use truncated forms of the protein for biochemical
studies (32). Baculovirus-infected, eukaryotic SF9 cells were chosen due to the fact that: 1) the expression of
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eukaryotic proteins in bacteria often results in incorrectly folded, insoluble molecules (20); 2) there is no known limit
for the size of the heterologous gene insertion in baculovirus (16); and 3) the expression of the gene occurs after the
virus has been packaged, so that the expressed gene should not affect viral production (11;16;31). The incorporation
of the FLAG tag was also an important advance, as the tagging did not interfere with the function of UBF (as
assessed in in vitro rRNA transcription assays), but it did allow for purification of the protein to apparent homogeneity
(Supplemental Figure 1a). In addition, this technique was readily applied to the production of phosphorylation site
mutants and was also used in the over-expression of full length UBF in mammalian cells.
Purified FLAG-rUBF was then treated with nuclear extract from angiotensin II stimulated VSMC which we have
previously shown leads to rapid phosphorylation of the protein (Supplemental Figure 1b) and binding to TBP(17) and
activation of its ability to stimulate rRNA transcription in vitro (Kihm and Owens, unpublished results). Alternatively,
purified recombinant UBF phosphorylated by a Large T antigen associated kinase was used.
Sequence coverage of FLAG-rUBF using multiple proteolytic enzymes
As there are 70 serine residues in UBF, multiple enzymes were used to digest the protein prior to nHPLC-µESI
MS in an effort to ensure visualization of all potential phosphorylation sites. Supplemental Figure 2 shows that
reduction, alkylation, and parallel digestion of FLAG-rUBF with Asp-N, Glu-C, and trypsin resulted in 35.7%, 53.4%,
and 64.4% sequence coverage respectively. Combining the coverages obtained using each individual enzyme
provided over 80% sequence coverage, allowing direct visualization of 46 of the 47 serine residues present in the
main body of the protein. Importantly, the serine residue not visualized in this analysis is converted to an alanine
residue in the human sequence of UBF1. One phosphopeptide was identified in this reversed phase analysis: the
serine 584 species, FSQELLpSNGELNHLPLK.
IMAC enrichment for the identification of phosphorylation sites
To selectively enrich the sample for phosphorylated peptides, immobilized metal affinity chromatography
(IMAC) using Fe+3 was used (10;25;29). This approach separates phosphopeptides from their non-phosphorylated
counterparts and permits subsequent on-line (22;38) or off-line (26) mass spectrometric analysis. In this study, off-
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line IMAC enrichment used in conjunction with nHPLC-µESI MS permitted the identification of nine specific sites of
phosphorylation in UBF.
Ten Different UBF Serine Phosphorylation Sites Were Identified by LC-MS/MS on an Ion Trap Mass Spectrometer
Ten different phosphorylation sites in rUBF1 were identified in toto including serines 273, 336, 364, 3891, 412,
433, 484, 546, 584, and 638 (Table, Figure 1, and Supplemental Figure 3). All assigned sequences were confirmed
by comparing the fragmentation pattern of the experimental spectrum to that of a synthetic phosphopeptide (example
shown in Figure 1b). Importantly, all phosphorylations occurred on serine residues, in agreement with previously
conducted phosphoamino acid analysis (13).
Mutation of serine 389 decreased UBF induced rRNA transcriptional activation in vitro and in vivo whereas mutation
of serine 584 alanine had no effect
Given the large technical burden of producing purified, recombinant full-length UBF, we chose to focus on two
phosphorylation sites for further functional characterization. Since our interest has been in determining the
phosphorylation events that lead to UBF-SL1 interaction, we selected sites that may contribute to controlling this
interaction. Recent structural studies have revealed conserved amino acid residues in NC2 (Negative Cofactor 2)
that interacts directly with TBP(15) bound to the TATA sequence, and this motif is found on UBF at several sites
(Supplemental Figure 4), including one adjacent to serine 389. This suggested that UBF-TBP interaction may be
regulated by phosphorylation at serine 389.
Next, we chose to focus on serine 584 as it was identified without requiring IMAC enrichment, and was
therefore one of the first sites identified.
To test the function of the serine 389 and 584 phosphorylation sites on rRNA transcription, serine to alanine
substitution mutations of rUBF1 were tested in an in vitro reconstitution rRNA transcription assay. In addition, to
determine if the phosphorylation site mutations altered rRNA transcription in intact cells, a series of co-transfection
studies were performed in cultured VSMCs with wildtype or mutant UBF expression plasmids and a unique rDNA
promoter luciferase reporter plasmid, pGRIL (see Supplemental Methods).
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Results showed that wild-type rUBF1 activated rRNA transcription by nearly two-fold in an in vitro reconstitution
assay (Figure 2A) and nearly three-fold in in vivo transient co-transfection assays (Figure 2B). In contrast, mutation of
serine 389 to alanine virtually abolished UBF-activated transcription in both systems. To simulate the negative charge
of a phosphate group at residue 389, this site was also mutated from a serine to a glutamate residue, and mutation of
serine 389 to glutamate fully restored activity in vitro (data not shown) and restored ~80% activity in vivo (Figure 2B).
In contrast to effects of mutation of serine 389, mutation of serine 584 to an alanine residue did not seem to affect
transcription in vitro but decreased transcription in vivo by 75%. Interestingly, transient transfection of pcDNA3
encoding for a truncation mutation (TR) in which the C-terminus was removed downstream of amino acid 674
repressed transcription from the rDNA-IRES-Luciferase construct in vivo below the activity of samples containing the
empty vector (pcDNA3) control suggesting this construct functions as a dominant negative.
Mutation of serine 389 to alanine abrogated UBF-SL1 interaction in vitro, whereas a serine 389 to glutamate mutation
partially restored interaction.
UBF phosphorylation has been shown to modulate UBF-SL1 interactions(17;33). To determine if
phosphorylation of serine 389 is required for UBF-SL1 interaction, we performed in vitro interaction assays; results
showed that wildtype rUBF1 bound to SL1 with an efficacy equivalent to 10-20% of input SL1 (Figure 3, Lanes 4, 1,
and 2, respectively). Mutation of serine 389 to alanine abolished this UBF-SL1 interaction (Figure 3, Lane 5) as did
removal of the acidic tail (TR) (Figure 3, Lane 7), while mutation of serine 389 to glutamate partially restored UBF-
SL1 interaction (Figure 3, Lane 6).
Phosphorylation of serine 389 and 584 sites are not necessary for nucleolar localization of UBF
We and others(13;23) previously demonstrated that UBF is distributed throughout the nucleus in quiescent cells
and is localized specifically to the nucleolus within 15 minutes of growth factor stimulation. Due to the rapid kinetics
of this event, we hypothesized that localization of UBF may be due to phosphorylation of serine 389 or 584. Under
our transfection culture conditions (cells were asynchronous and in logarithmic growth phase), we found that wildtype
FLAG-UBF (Green) in most cells was localized to the nucleoli (Red). Consistent with previous reports, removal of the
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acidic tail (UBF 674C, Green) abrogated nucleolar localization of UBF(19) (Figure 4B) such that the protein was
distributed diffusely throughout the nucleus (Blue). In contrast, mutation of serine 389 and 584 to alanine had no
affect on nucleolar localization of UBF. Further, mutation of serine 389 to glutamate also did not significantly affect
subnuclear localization of UBF.
The importance of UBF phosphorylation in activation of rRNA transcription was first demonstrated nearly a
decade ago by Voit and co-workers(35;36). Since then, several effects of growth stimulation on UBF have been
defined. First, UBF is hyperphosphorylated in response to growth stimulation(13;23;36). Second, UBF is localized
specifically to the nucleolus within fifteen minutes of stimulation(13). Third, UBF affinity for SL1/TBP is increased in
response to growth stimulation(17;40). Fourth, UBF activation of rRNA transcription is increased by growth
stimulation(34-37). As such, observations in the present studies showing direct evidence of phosphorylation of ten
unique serine sites within UBF, and that mutation of serine 389 resulted in significant loss in the transcriptional
activity of UBF in vitro and in vivo represent a major advance for the field. These findings will allow further functional
analysis of UBF phosphorylation in the control of rRNA transcription in intact cells.
To that end, we are presently designing anti-phospho UBF antibodies to directly probe the functional
significance of these sites in growth-stimulated cells as present methods precluded the identification of sites
differentially phosphorylated in UBF from growth-stimulated versus unstimulated cells. Of note, we have previously
demonstrated that in cells rendered quiescent and maintained in neutral protein balance, the pattern of UBF
phosphorylation is similar to that in stimulated cells, and differs only in stoichiometry of phosphorylation at each
site(17). Indeed, while MS/MS is an exceedingly powerful tool, the technique cannot rule out phosphorylation at a
given site if there is an absence of signal. Therefore, it would not have been possible to unequivocally use the
technique to delineate differential phosphorylation between stimulated and unstimulated cells.
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Likewise we cannot completely exclude the possibility of phosphorylation on threonine or tyrosine residues,
although we (present manuscript)(13;17) and others(23;34;37) have reported phosphorylation of only serine residues
on UBF. Stefanovsky and co-workers have recently demonstrated that UBF peptides mimicking HMG boxes 1 and 2
are phosphorylated in vitro on Threonine 117 and Threonine 201, respectively(32). Investigators were able to
demonstrate in a co-transfection assay that both threonine residues were important in activating rRNA transcription.
However, it remains to be determined whether or not the full-length protein is phosphorylated in vivo on these sites
as well. Indeed, in the present studies, we have exhaustively attempted to identify phosphorylation on threonine and
tyrosine residues on full-length UBF, but were not successful. As previously noted, the technical limitations of
tandem mass spectrometry precluded our ability to completely rule-out phosphorylation on non-serine residues.
Of the serine phosphorylation sites identified in present studies, the two tested in co-transfection studies both
caused disruption of rRNA transcription from our chimeric reporter construct. While not a novel model of rRNA
transcription(12), results from the in vivo transfection assay may have uncovered unique roles for different functional
regions of UBF. Specifically, mutation of serine 584 to alanine decreased transcription from the reporter construct,
but had little effect in vitro. Several explanations may account for this effect, at least three of which may be related to
the role of phosphorylation of serine 584. First, whereas the in vitro reconstitution assay does not include chromatin
or tertiary DNA-protein structures, the reporter gene likely forms tertiary and chromatin structures in a manner that
may be physiologically relevant(28). Indeed, we have found that UBF preferentially associates with histone H3
methylated-lysine 9 (Lin, Cheung, Allis, and Owens, unpublished observations), suggesting a direct interaction
between UBF and specific rDNA chromatin structures. Second, whereas activity in the reconstitution assay is
primarily dependent upon total concentrations of each factor in solution, the transfection assay is also dependent
upon intranuclear spatial localization of factors. Third, since the reconstitution assay consists of only factors
necessary for transcription, results of the transfection assay may suggest the effect of co-activators or repressors
(eg. Rb(2) or p130(5)). It may be that phosphorylation of serine 584 plays an important role in one or more of these
phenomena; further study of this phosphorylation site may provide insight into as of yet undiscovered roles for UBF
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Importantly, one of the most well-characterized roles of UBF phosphorylation, modulation of the interaction with
SL-1, was perturbed by mutation of serine 389. The fact that mutation of serine 389 virtually abolished UBF-SL1
interaction was somewhat surprising as previous work by Tuan et al.(33) demonstrated that a fragment containing
only the amino acid residues of UBF between 491 and 764 was sufficient for UBF-SL1 interaction in an in vitro assay.
Furthermore, removal of the C-terminal residues between 670 and 764 of UBF completely abrogated UBF-SL1
interaction. Whereas these data suggest that the acidic C-terminus of UBF is most likely the portion of the protein
that directly interacts with the SL1 complex, recent structural studies have revealed a great deal regarding the nature
of TBP and cofactor interactions. The Negative Cofactor (NC2) has recently been co-crystallized with TBP bound to
a TATA DNA fragment. Based on the crystal structure, investigators identified several conserved amino acid
residues in the NC2 beta chain (helix 5) that interact directly with TBP via its C-terminal domain(15). Interestingly,
the motif formed by these residues can be found in several regions in UBF (Supplemental Figure 4). More
importantly, several UBF phosphorylation sites are located flanking these motifs, including Serine 389, consistent
with phosphoregulation of these putative TBP-interaction motifs. Consistent with this hypothesis, the 40 amino acid
region surrounding Serine 389 contains one theoretical alpha helix based on both the Chou-Fasman(4) and Garnier-
Robson(27) methods (Supplemental Figure 4) that terminates at Serine 389. However, Glutamate substitution of
Serine 389 extends the alpha helix through residue 389 to 391A or 393K (Chou-Fasman or Garnier-Robson
methods, respectively). Additional studies will be required to further delineate the kinetic and spatial relationship
between UBF phosphorylation and SL1 recruitment in the context of transcription initiation.
Indeed the kinetics of UBF nucleolar localization suggest involvement of a rapid post-translational modification
event such as phosphorylation(13). In fact, recent fluorescence recovery after photobleaching (FRAP) and
fluorescence loss in photobleaching (FLIP) experiments in vivo have demonstrated that UBF moves rapidly in and
out of the nucleolus(3;8), such that sustained nucleoplasmic or nucleolar localization is likely due to a shift in
equilibrium by some post-translational modification. However, alanine substitution mutations for serine 389 and 584
did not alter nucleolar localization. Since we have mapped ten phosphorylation sites so far, and we only present data
for two sites here, we certainly cannot exclude the possibility that phosphorylation of a single site controls localization
of UBF to the nucleolus. Alternatively, it may be that UBF must be phosphorylated on a certain number of sites to
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reach a threshold negative charge as opposed to phosphorylation at a specific site in order to be localized to the
nucleolus. For example, nuclear import of NFAT requires complete dephosphorylation of 13 sites to expose a
nuclear localization signal and mask a nuclear export signal(24). However, to adequately test this hypothesis in UBF
would require an exhaustive panel of combinatorial mutations of the ten sites we have mapped.
While present studies are the first direct identification of UBF phosphorylation sites, two key studies by Voit and
co-workers significantly advanced the field by presenting the first evidence suggesting specific differential UBF
phosphorylation at serine 484(37) and serine 388(34) (identical to the site we have called serine 389). These studies
employed a combination of 2D phosphotryptic mapping and site-directed mutagenesis to indirectly identify
phosphorylation at these sites. In contrast, the present studies report the first unequivocal direct identification of UBF
phosphorylation sites by tandem mass spectrometry, and therefore represent a required validation of phosphorylation
of UBF on sites also identified by Voit and co-workers (serines 388/389 and 484) as well as eight additional novel
phosphorylation sites. These findings represent a major advance for the field and will provide the basis for extensive
further studies to elucidate the mechanisms that control growth induced activation of UBF and increased rRNA
Authors gratefully thank Mary McCanna, Diane Raines, and Sonia Navarro for their excellent technical
assistance. Authors also thank Benjamin Garcia and Jie Qian for their assistance in obtaining tandem mass spectra
for the synthetic peptides. This work was supported by NIH grants P01 HL19242 (to GKO), and NIH GM37537 (to
D.F.H.). CHL was supported by NIH training grants T32 GM 007267 and 5T32 HL07284. HeLa cell pellets for
preparation of SL1 were obtained from National Cell Culture Center (NCCC).
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15 Download full-text
Table. Summary of UBF phosphorylation sites identified by tandem mass spectrometry
Figure 1. MS/MS spectrum of a tryptic phosphorylated peptide derived from rUBF1.
Predicted masses for the ions of type b and y are shown above and below the sequence, respectively. The b(2+)
and y(2+) masses are average values while the singly charged b and y ion masses are monoisotopic. Ions observed
in the spectrum are underlined and those that lose phosphoric acid are presented in bold type. The label, ∆, denotes
loss of phosphoric acid from the corresponding ion of type b or y. A) Identification of phosphorylation at serine 389.
B) The MS/MS spectrum of the synthetic phosphopeptide VLGEEKMLNINKKQTTpSPASK. C) Identification of
phosphorylation at serine 584.
Figure 2. Effect of UBF Phosphorylation Site Mutation on rRNA Transcription.
A. Representative S1 Nuclease assay of in vitro transcription reaction. Increasing amounts of purified
recombinant wt UBF1 (Lanes 2-3), S389A mut (Lanes 4-5), or S584A mut (Lanes 6-7) were added to in vitro rRNA
transcription reactions. Lane 1 serves as a negative control in which no UBF was added to the reaction. B. In vivo
reporter assay of rRNA transcription. VSMCs were co-transfected with the mammalian expression vector pcDNA3
(Invitrogen) encoding WT UBF1 or phosphorylation site mutant and pGRIL (rDNA IRES Luciferase) reporter construct
in triplicate. Co-transfected cells were lysed and assayed for luciferase activity. Activity was normalized against
lysate protein concentration and plotted as mean (of each triplicate) +/- standard deviation. Results represent at
least three independent experiments. pcDTR2 = pcDNA3 encoding acidic tail truncation mutation (truncated C-
terminal of aa 674).
Figure 3. Effect of phosphorylation site mutation on UBF-SL1 interaction in vitro.
Purified recombinant FLAG-tagged wildtype UBF1 or mutant was immobilized on anti-FLAG M2 agarose beads
and then incubated with partially purified SL1 (Lanes 4-7). Bound SL1 was eluted in BCO buffer and analyzed by
anti-TAF110 western (top panel). After BCO elution, beads were boiled in Laemmli Sample buffer and resolved by
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