title: Identification and characterization of protein phosphatase 2C activation by ceramide.
abstract: Ceramide is a bioactive sphingolipid with many associated biological outcomes, yet there is a significant gap in our current understanding of how ceramide mediates these processes. Previously, ceramide has been shown to activate protein phosphatase (PP) 1 and 2A. While continuing this line of work, a late fraction from a Mono-Q column was consistently observed to be activated by ceramide, yet PP1 and PP2A were undetectable in this fraction. Proteomic analysis of this fraction revealed the identity of the phosphatase to be PP2Cγ/PPM1G. This was consistent with our findings that PP2Cγ 1-eluted in a high salt fraction due to its strongly acidic domain, and 2-was insensitive to okadaic acid. Further characterization was performed with PP2Cα, which showed robust activation by C(6)-ceramide. Activation was specific for the erythro conformation of ceramide and the presence of the acyl chain and hydroxyl group at the first carbon. In order to demonstrate more physiological activation of PP2Cα by ceramide, phospho-p38δ was utilized as substrate. Indeed, PP2Cα induced the dephosphorylation of p38δ only in the presence of C(16)-ceramide. Taken together, these results show that the PP2C family of phosphatases is activated by ceramide, which may have important consequences in mediating the biological effects of ceramide.
authors.affiliation: Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, SC 29425, USA.
authors.forename: David M
authors.initials: DM
authors.lastname: Perry
authors.forename: Kazuyuki
authors.initials: K
authors.lastname: Kitatani
authors.forename: Patrick
authors.initials: P
authors.lastname: Roddy
authors.forename: Mohamad
authors.initials: M
authors.lastname: El-Osta
authors.forename: Yusuf A
authors.initials: YA
authors.lastname: Hannun
mesh_terms: D000818:Animals; D002460:Cell Line; D002518:Ceramides / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D004789:Enzyme Activation / Q000187:drug effects; D007700:Kinetics; D048292:Mitogen-Activated Protein Kinase 13 / Q000378:metabolism; D010749:Phosphoprotein Phosphatases / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D000071636:Protein Phosphatase 2C; D051381:Rats; D015398:Signal Transduction / Q000187:drug effects; D013379:Substrate Specificity
chemical_list: D002518:Ceramides; D048292:Mitogen-Activated Protein Kinase 13; D010749:Phosphoprotein Phosphatases; D000071636:Protein Phosphatase 2C