MASS SPECTRAL ANALYSIS OF TRANSCRIPTION FACTOR LSF IN VITRO PHOSPHORYLATION

转录因子 LSF 体外磷酸化的质谱分析

• 批准号: 7369210
• 负责人: ULLA M HANSEN
• 金额: $ 0.13万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-07-01 至 2007-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7369210
• 关键词: MASS; SPECTRAL; ANALYSIS; TRANSCRIPTION; FACTOR

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.

该子项目是利用 NIH/NCRR 资助的中心拨款提供的资源的众多研究子项目之一。子项目和研究者 (PI) 可能已从另一个 NIH 来源获得主要资金，因此可以在其他 CRISP 条目中得到体现。列出的机构是中心的机构，不一定是研究者的机构。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。激酶 Akt 和 GSK3？有助于调节细胞决策（例如生长、生存和死亡）的细胞信号传导途径。具体来说，Akt 促进生存和生长，而 GSK3？促进细胞死亡。这两种激酶的底物之一是转录因子 LSF（晚期 SV40 因子）。 LSF 与这些激酶一样，已被证明参与调节细胞存活、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，我们进行了研究来定位 Akt 和 GSK3？ LSF 磷酸化。我们定位 LSF 磷酸化的方法利用重组纯化 LSF 的体外磷酸化以及质谱分析。体外磷酸化的 LSF 经过酶消化，产生大小适合 MS 分析的肽。然后通过基质辅助激光解吸/电离飞行时间 (MALDI-TOF) 质谱分析消化物，以鉴定与磷酸化一致的质量增加（80 Da 增量）的肽。对于那些含有比添加的磷酸基团数量更多的潜在磷酸化位点（Ser、Thr）的肽，可以使用串联质谱（MS/MS）分析来定位精确的磷酸化位置。在本研究的早期，我们使用 MALDI MS 鉴定了两种 LSF 肽，这些肽在与 Akt 孵育后被磷酸化。这些肽的丰度较低，无法通过直接 MS/MS 分析来定位磷酸化位点。为了富集磷酸肽，我们在 MS 分析之前利用固定金属离子色谱 (IMAC) 进行甲酯化。然而，通过 MALDI MS 或负离子 nanoESI 前体离子扫描实验，没有从该制剂中鉴定出磷酸肽。另一种方法是将 32P 掺入体外激酶反应中，并从 2D TLC 肽图中分离标记的肽。从 TLC 板中提取磷酸化肽并储存直至检测不到 32P 信号。随后重复实验的 MALDI MS 结果表明肽的丰度太低而无法检测到。我们跟踪了合成肽的体外磷酸化，该肽对应于从 Ser289 到 Ser302 的 LSF 序列（添加了 C 末端 Cys，并在位置 296 处添加了磷酸化 Ser，S289PSPGFNpSSHSSFS302C）。该肽在体外用 GSK-3 磷酸化，然后进行还原、用碘乙酰胺烷基化和 C18 反相纯化。 NanoESI MSMS 分析鉴定出 Ser291 处的磷酸化。此外，我们发现了 Ser289 磷酸化的证据，然而，潜在磷酸化 b2(1+) 离子的质量与 y2(1+) 离子的质量重叠。随后用碘乙酸而不是碘乙酰胺对肽进行烷基化，从而改变了 y2 离子质量，从而证实了 Ser289 处的磷酸化。该数据被用来在 LSF 中生成 Ser 到 Ala 的突变，以确定这种磷酸化的功能意义。此外，我们可用于测定低水平磷酸化的方法的改进正在改善 MS 结果。