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

Abstract

Lin CH, Platt MD, Ficarro SB, Hoofnagle MH, Shabanowitz J, Comai L, Hunt DF, Owens GK. Mass spectrometric identification of phosphorylation sites of rRNA transcription factor upstream binding factor. Am J Physiol Cell Physiol 292: C1617-C1624, 2007. First published December 20, 2006; doi:10.1152/ajpcell.00176.2006.-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 nanoflow HPLC-microelectrospray-ionization tandem mass spectrometry (nHPLC-ESI-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 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 TBPassociated factors (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 transcription. However, relatively little is known regarding the mechanisms by which UBF phosphorylation regulates transcriptional activity. Previous studies in our laboratory (17...

Dysregulation of upstream binding factor-1 acetylation at K352 is linked to impaired ribosomal DNA transcription in Huntington's disease

“…17 It is noteworthy that reduced HAT activity of CBP is thought to play a role in the toxic, transcriptional, and repressive effects of mutated proteins with expanded polyglutamine repeats such as Htt and atrophin. 19–22 These findings, along with the established ability of UBF1-mediated rDNA transcription to be activated by acetyl transferases, 23 raises the possibility that the neurodegenerative process in HD might be associated with changes in adaptive rDNA transcription caused by altered CBP acetyl transferase that affects the balance of acetylated and deacetylated UBF1. Indeed, we found that the acetylation levels of UBF1 are significantly lowered due to dysregulation of CBP function in both HD cells and HD mice.…”

Ribosome accumulation during early phase resistance training in humans

“…From a mechanistic perspective, UBF is an important transcription factor for rDNA transcription as it, in its active state recruits a secondary transcription factor (SL1) to the rDNA promoter and enables transcription by RNA Pol I. 18 Activation of UBF is controlled by the mechanosensitive mTOR pathway, and rapamycin, a specific mTOR inhibitor, blocks UBF from recruiting SL1 and subsequent rRNA transcription. 19,20 Evidence from human exercise studies confirms training-induced activation of UBF through phosphorylation.…”

“…Ribosomal accumulation is believed to be determined by the rates of pre‐rRNA transcription by RNA polymerase I (Pol I), which in turn is regulated by coordinated assembly of a complex of transcription factors at the rDNA promoter 16 . Specifically, activation of the of the upstream binding factor (UBF) through phosphorylation is needed to initiate transcription 17,18 . Such activation is at least partly controlled by the mechanosensitive mTOR pathway, with its inhibition being associated with blocked UBF phosphorylation and subsequent rRNA transcription 19,20 .…”

ESET methylates UBF at K232/254 and regulates nucleolar heterochromatin plasticity and rDNA transcription

“…Post-translational modifications such as acetylation and phosphorylation of UBF control the transcription of rDNA ( 11 , 12 ). cAMP response element-binding protein (CREB)-binding protein (CBP), a histone acetyltransferase (HAT) and transcriptional coactivator, contributes to UBF-mediated transcription in the nucleolus.…”