Late stage results from the probe development effort to identify inhibitors of the protein methylesterase PME-1: Inhibition of PME-1-mediated demethylation of PP2a
Name: Late stage results from the probe development effort to identify inhibitors of the protein methylesterase PME-1: Inhibition of PME-1-mediated demethylation of PP2a ..more
BioActive Compounds: 2
Depositor Specified Assays
Data Source (MLPCN Center Name): The Scripps Research Institute Molecular Screening Center (SRIMSC)
Center Affiliation: The Scripps Research Institute, TSRI
Assay Provider: Benjamin Cravatt, TSRI
Network: Molecular Libraries Probe Production Center Network (MLPCN)
Grant Proposal Number: 1 2 R01 CA087660-05 Fast Track
Grant Proposal PI: Benjamin Cravatt, TSRI
External Assay ID: PME-1_INH_CL_GEL_Demethylation
Name: Late stage results from the probe development effort to identify inhibitors of the protein methylesterase PME-1: Inhibition of PME-1-mediated demethylation of PP2a
Reversible protein phosphorylation networks play essential roles in most cellular processes. While over 500 kinases catalyze protein phosphorylation, only two enzymes, PP1 and PP2A, are responsible for >90% of all serine/ threonine phosphatase activity (1). Phosphatases, unlike kinases, achieve substrate specificity through complex subunit assembly and post-translational modifications rather than number. PP2A, for example, typically exists as heterotrimer with diverse subunits that may combinatorially make as many as 70 different holoenzyme assemblies (2). Mutations in several of these PP2A subunits have been identified in human cancers, suggesting that PP2A may act as a tumor suppressor (3). Adding further complexity, several residues of the catalytic subunit of PP2A can be reversibly phosphorylated, and the C-terminal leucine residue can be reversibly methylated (4,5). PME-1 is specifically responsible for demethylation of the carboxyl terminus (4).
Methylesterification is thought to control the binding of different subunits to PP2A, but little is known about physiological significance of this post-translational modification in vivo (5). Recently, PME-1 has been identified as a protector of sustained ERK pathway activity in malignant gliomas 6. In order to further elucidate the role of PP2A methylation in vivo, our lab has generated mice that lack PME-1 (PME-1 (-/-) mice) by targeted gene disruption (7). Unfortunately, PME-1 deletion resulted in perinatal lethality, underscoring the importance of PME-1 but hindering our biological studies. Biochemical elucidation of PME-1 would thus greatly benefit from the development of potent and selective chemical inhibitors.
1. Oliver, C. J.; Shenolikar, S., Physiologic importance of protein phosphatase inhibitors. Front. Biosci. 1998, 3, D961-72.
2. Janssens, V.; Goris, J., Protein phosphatase 2A: a highly regulated family of serine/threonine phosphatases implicated in cell growth and signalling. Biochem. J. 2001, 353 (Pt 3), 417-39.
3. Janssens, V.; Goris, J.; Van Hoof, C., PP2A: the expected tumor suppressor. Curr. Opin. Genet. Dev. 2005, 15 (1), 34-41.
4. Lee, J.; Chen, Y.; Tolstykh, T.; Stock, J., A specific protein carboxyl methylesterase that demethylates phosphoprotein phosphatase 2A in bovine brain. Proc. Natl. Acad. Sci. U. S. A. 1996, 93 (12), 6043-7.
5. Wu, J.; Tolstykh, T.; Lee, J.; Boyd, K.; Stock, J. B.; Broach, J. R., Carboxyl methylation of the phosphoprotein phosphatase 2A catalytic subunit promotes its functional association with regulatory subunits in vivo. Embo J. 2000, 19 (21), 5672-81.
6. Puustinen, P.; Junttila, M. R.; Vanhatupa, S.; Sablina, A. A.; Hector, M. E.; Teittinen, K.; Raheem, O.; Ketola, K.; Lin, S.; Kast, J.; Haapasalo, H.; Hahn, W. C.; Westermarck, J., PME-1 protects extracellular signal-regulated kinase pathway activity from protein phosphatase 2A-mediated inactivation in human malignant glioma. Cancer Res. 2009, 69 (7), 2870-7.
7. Ortega-Gutierrez, S.; Leung, D.; Ficarro, S.; Peters, E. C.; Cravatt, B. F., Targeted disruption of the PME-1 gene causes loss of demethylated PP2A and perinatal lethality in mice. PLoS ONE 2008, 3 (7), e2486.
PME-1, protein phosphatase methylesterase 1, PPME-1, protein phosphatase 2a, PP2a, lysophospholipase, cancer, inhibitor, chemiluminescence, methylation, demethylation, HRP, antibody, SDS-PAGE, Scripps, The Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN
The purpose of this assay is to monitor the demethylation of PP2a by endogenous PME-1 that occurs during inhibitor incubation with cells in culture.
Following incubation of HeLa cells in culture with inhibitor (at 5 and 20 micromolar concentrations) at 37 degrees Celcius, cells are homogenized, and proteins separated by SDS-PAGE. PP2a methylation is visualized by chemi-luminescent dectection using HRP-antibody specific to C-terminal demethylated PP2a. As designed, test compounds that act as PME-1 inhibitors will inhibit demethylation of PP2a, resulting in a decrease in the demethylated PP2a signal.
PubChem Activity Score:
Compounds that inhibit PP2a demethylation >50% are considered active in situ PME-1 inhibitors.
List of Reagents:
HRP-antibody specific to C-terminal demethylated PP2a (Millipore 05-577)
Sodium Chloride (Fisher, part 980597)
1M Tris, pH 8.0 (Invitrogen, part T-3038)
This assay was performed in the laboratory of the Assay Provider with compounds ordered as powders. Details of protocols, compound structures, and results from the original assays can be found in PubChem at the respective AIDs listed below. The results of our probe development efforts can be found at http://mlpcn.florida.scripps.edu/index.php/probes/probe-reports.html.
** Test Concentration.
Data Table (Concise)