Mass spectrometric identification of phosphorylation sites of rRNA transcription factor upstream binding factor
Mass spectrometric identification of phosphorylation sites of rRNA transcription factor upstream binding factor
复制标题
DOI：
10.1152/ajpcell.00176.2006
复制
发表时间：
2007-05-01
影响因子：
5.5
通讯作者：
Owens, Gary K.
中科院分区：
文献类型：
--
作者：
Lin, C. Huie;Platt, Mark D.;Owens, Gary K.
rRNA转录是所有细胞生长过程的基本要求，并通过上游结合因子（UBF）的磷酸化来响应生长刺激。尽管众所周知，UBF的磷酸化是其激活所必需的，并且是激活rRNA转录的关键步骤，但到目前为止，还没有UBF磷酸化位点的直接映射。本研究结果采用先进的纳米流高效液相色谱-微电喷雾-电离串联质谱（nHPLC-mu ESI-MS/MS）结合固定化金属亲和色谱（IMAC）和计算机数据库搜索算法，确定了UBF上273、336、3664、389、412、433、484、546、584和638丝氨酸的10个磷酸化位点。然后，我们对其中两个位点，丝氨酸389和584进行了功能分析。丝氨酸-丙氨酸替代突变389 （S389A）在体外和体内均能消除rRNA转录，而丝氨酸突变584 （S584A）在体内减少转录，但在体外没有。相比之下，389 （S389E）的丝氨酸-谷氨酸突变恢复了转录活性。此外，S389A在体外消除了UBF- SL1相互作用，而S389E则部分恢复了UBF- SL1相互作用。综上所述，这些研究的结果表明，生长因子刺激通过UBF丝氨酸389的磷酸化诱导rRNA转录活性的增加，部分原因是促进了RNA聚合酶I的招募中的限速步骤：即SL1的招募。此外，研究提供了关于多个额外的UBF磷酸化位点的关键新数据，这些数据将需要进一步的领域表征。
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-mu 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.