Sustrate Identification for Ser/Th Protein Phosphatase
EMSL Project ID
6699
Abstract
I am interested in the structure, regulation and function of the Ser/Thr protein phosphatase, PP5. We purified this enzyme several years ago based on lipid-activated phosphatase activity. Several groups had cloned a cDNA for this enzyme, but at that time there was little insight into its signaling function. More recent observations have implicated PP5 in several signaling pathways, including regulation of p38 and Jun kinase by the stress and hormone activated MAPKKK, Apoptosis Signaling Kinase 1 (ASK 1) and p53-dependent cell cycle regulation. Also, PP5 specifically binds Hsp90 and may regulate proteins dependent on Hsp90 for proper folding and activation such as glucocorticoid receptors and heme regulated-eIF2 alpha kinase. Thus far, only ASK 1 has been shown to be a substrate for PP5. We wish to identify substrates for PP5 using a proteomics approach by analyzing phosphoproteins isolated from cells expressing wild type PP5 or a point mutant that is roughly 500-fold less sensitive to the cell-permeable phosphatase inhibitor okadaic acid (generated in collaboration with David Armstrong, NIEHS). I would like to try your high throughput approach using chromatography based on isotope affinity tags to select for phosphoproteins and distinguish between proteins from control and experimental samples, combined with on-line mass spectrometry to obtain sequence for identification, as a strategy for this study. We are making two sets of stable cell lines expressing various forms of tet-inducible PP5 for these studies, using A549 human lung carcinoma cells and PC12 cells, both of which are made from commercially available cell lines. These samples can be shipped to me once all IRB protocols have been satisfied. I understand this is a challenging project and no one has the "magic bullet" for guaranteed success with global cellular phosphoprotein analysis, but even so I should be able to develop sufficient experience to enable me to attack the problem with alternative strategies requiring many of the same skills.
Project Details
Project type
Exploratory Research
Start Date
2004-02-14
End Date
2007-02-18
Status
Closed
Released Data Link
Team
Principal Investigator