MASS SPECTRAL ANALYSIS OF TRANSCRIPTION FACTOR LSF IN VITRO PHOSPHORYLATION

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。蛋白质磷酸化是一种重要的翻译后修饰，参与调节细胞蛋白质的功能活性。受调节的磷酸化是由特定蛋白激酶的酶活性启动的。Akt和GSK3？参与调节细胞生长、存活和死亡等细胞决策的细胞信号通路。具体地说，Akt促进生存和生长，而GSK3？促进细胞死亡。这两种酶的底物之一是转录因子LSF(晚期SV40因子)。LSF和这些激酶一样，已被证明参与调节细胞生存、生长和死亡的信号通路。为了更好地了解LSF活性是如何调控的，我们进行了定位Akt和GSK3的研究？LSF上的磷酸化。我们的方法是利用重组纯化的LSF的体外磷酸化结合质谱分析来定位LSF的磷酸化。在体外，磷酸化的LSF经过酶消化，产生适合MS分析的大小合适的多肽。然后用基质辅助激光解吸/电离飞行时间(MALDI-TOF)质谱仪对消化产物进行分析，以确定与磷酸化一致的质量增加(80-Da增量)的多肽。对于那些含有比添加的磷酸基团更多的潜在磷酸化位点(Ser，Thr)的多肽，采用串联质谱分析(MS/MS)来定位磷酸化的精确位置。在这项研究的早期，我们使用MALDI MS鉴定了两个与Akt孵育后被磷酸化的LSF多肽。由于这些多肽丰度较低，无法直接进行MS/MS分析，因此无法定位S的磷酸化位点。为了丰富磷酸肽，我们使用了固定化金属离子层析(IMAC)和甲酯化，然后进行了MS分析。然而，无论是通过MALDI MS还是负离子纳米ESI前驱体离子扫描实验，都没有从该制剂中鉴定出任何磷酸肽。另一种方法是将~(32)P掺入体外激酶反应中，并从2D TLC肽图中分离标记的多肽。从TLC板上提取磷酸化的多肽并储存，直到检测不到~(32)P信号。随后重复实验的MALDI MS结果表明，这些多肽的丰度太低，无法检测到。我们从Ser289到Ser302，跟踪了一个与LSF序列相对应的合成肽的体外磷酸化(在C-末端增加了一个半胱氨酸，并在第296位增加了磷酸丝氨酸，S289PSPGFNpSSHSSFS302C)。该多肽经GSK-3体外磷酸化、还原、与碘乙酰胺烷基化、C18反相纯化。NanESI MSMS分析鉴定了Ser291处的磷酸化。此外，我们在Ser289发现了磷酸化的证据，然而，潜在的磷酸化的b2(1+)离子的质量与y2(1+)离子的质量重叠。随后，该肽与碘乙酸烷基化，而不是与碘乙酰胺烷基化，使y2离子质量发生移动，从而使Ser289处的磷酸化得以确认。这些数据被用来在LSF中产生Ser to Ala突变，以确定这种磷酸化的功能意义。此外，我们现有的低水平磷酸化测定方法的改进正在导致MS结果的改善。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Protein phosphorylation is an important posttranslational modification involved in regulating the functional activity of cellular proteins. Regulated phosphorylation is initiated by the enzymatic activity of specific protein kinases. The kinases Akt and GSK3? contribute to cellular signaling pathways that regulate cellular decisions such as growth, survival, and death. Specifically, Akt promotes survival and growth, whereas GSK3? promotes cell death. One substrate of both of these kinases is the transcription factor LSF (Late SV40 Factor). LSF, like these kinases, has been shown to be involved in signaling pathways that regulate cell survival, growth, and death. In order to better understand how LSF activity is regulated, we have undertaken studies to locate the sites of Akt and GSK3? phosphorylation on LSF. Our approach to locating LSF phosphorylation utilizes in vitro phosphorylation of recombinant purified LSF together with mass spectrometry analysis. In vitro phosphorylated LSF is subjected to enzymatic digestion to produce peptides that are of appropriate size for MS analysis. Digests are then analyzed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry to identify peptides that have a mass increase (80-Da increments) consistent with phosphorylation. For those peptides that contain more potential phosphorylation sites (Ser, Thr) than the number of added phosphate groups, tandem mass spectrometry (MS/MS) analysis is used to locate the precise position of phosphorylation. Early in this study, we used MALDI MS to identify two LSF peptides by which were phosphorylated following incubation with Akt. The low abundance of these peptides precluded location of the phosphorylation site(s) by direct MS/MS analysis. In order to enrich for phosphopeptides, we utilized immobilized metal ion chromatography (IMAC) with methyl esterification prior to MS analysis. However, no phosphopeptides were identified from this preparation by either MALDI MS or negative-ion nanoESI precursor ion scanning experiments. Another approach was incorporation of 32P into the vitro kinase reaction and isolation of labeled peptides from 2D TLC peptide maps. Phosphorylated peptides were extracted from the TLC plate and stored until the 32P signal was undetectable. Subsequent MALDI MS results from replicate experiments indicated that abundance of the peptides was too low to be detected. We have followed the in vitro phosphorylation of a synthetic peptide that corresponds to the sequence of LSF fromSer289 to Ser302 (with the addition of a C-terminal Cys, and phosphoSer at position 296, S289PSPGFNpSSHSSFS302C). This peptide was phosphorylated in vitro with GSK-3 followed by reduction, alkylation with iodoacetamide and C18 reversed-phase purification. NanoESI MSMS analysis identified phosphorylation at Ser291. In addition, we found evidence for phosphorylation at Ser289, however, the mass of the potential phosphorylated b2(1+) ion overlapped with the mass of the y2(1+) ion. Subsequent alkylation of the peptide with iodoacetic acid rather than iodoacetamide shifted the y2 ion mass, thus allowing the confirmation of phosphorylation at Ser289. This data is being utilized to generate Ser to Ala mutations in LSF for determination of the functional significance of this phosphorylation. In addition, improvements in the approaches we have available for determinations of low-level phosphorylation are leading to improved MS results.