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.
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