MASS SPECTRAL ANALYSIS OF TRANSCRIPTION FACTOR LSF IN VITRO PHOSPHORYLATION

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。所列机构为中心，不一定是研究者所在机构。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。受调节的磷酸化由特定蛋白激酶的酶活性启动。激酶Akt和GSK 3？有助于调节细胞决策（如生长、存活和死亡）的细胞信号通路。具体来说，Akt促进生存和生长，而GSK 3？促进细胞死亡。这两种激酶的一种底物是转录因子LSF（晚期SV 40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活动是如何调节的，我们进行了研究，以定位Akt和GSK 3？LSF的磷酸化。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。对体外磷酸化LSF进行酶消化，以产生大小适合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）质谱法分析消化物，以鉴定具有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的数量更多的潜在磷酸化位点（Ser，Thr）的肽，串联质谱（MS/MS）分析用于定位磷酸化的精确位置。在本研究的早期，我们使用MALDI MS鉴定了两种LSF肽，在与Akt孵育后，这两种LSF肽被磷酸化。这些肽的低丰度排除了通过直接MS/MS分析定位磷酸化位点。为了丰富的磷酸肽，我们利用固定化金属离子色谱（IMAC）与甲酯化之前的MS分析。然而，没有磷酸化肽，确定从该制剂无论是MALDI MS或负离子nanoESI前体离子扫描实验。另一种方法是将32 P掺入体外激酶反应中，并从2D TLC肽图中分离标记的肽。从TLC板中提取磷酸化肽并储存直至检测不到32 P信号。重复实验的后续MALDI MS结果表明，肽的丰度太低而无法检测。我们跟踪了合成肽的体外磷酸化，所述合成肽对应于LSF从Ser 289到Ser 302的序列（在位置296处添加C末端Cys和磷酸化Ser，S289 PSPGFNpSSHSSFS 302 C）。该肽在体外用GSK-3磷酸化，然后还原，用碘乙酰胺烷基化和C18反相纯化。NanoESI MSMS分析鉴定了Ser 291处的磷酸化。此外，我们发现了Ser 289磷酸化的证据，然而，潜在磷酸化的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，使γ 2离子质量发生变化，从而确认丝氨酸289处的磷酸化。该数据被用于产生LSF中的Ser至Ala突变，以确定该磷酸化的功能意义。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.