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Papers In Press, published online ahead of print December 1, 2003
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Department of Biochemistry, Medical University of South Carolina, Charleston, SC 29425
Corresponding Author: hannun{at}musc.edu
The search for potential targets for ceramide action led to the identification of the ceramide-activated protein phosphatases (CAPP), protein phosphatase-1 (PP1) and protein phosphatase 2A (PP2A). In this study, the structural requirements for interaction between ceramide and CAPP were determined. D-erythro-C6 ceramide activated the catalytic subunit of PP2A (PP2Ac) approximately 3-fold in a stereospecific manner. In contrast, saturation of the 4-5 double bond, producing D- erythro-dihydro C6 ceramide, inhibited PP2Ac (IC50=8.5 mM). Furthermore, phyto C6 ceramide, D- erythro-dehydro ceramide, and D- erythro-cis-C6 ceramide had no effect on PP2Ac activity, demonstrating a strict requirement for an unmodified 4-5-trans-double bond. The sphingoid chain was required for PP2Ac activation since modifications to this chain producing the truncated, N-hexanoyl-serinol (C6-serinol), or aromatic ceramide analogues, D-erythro-2-(N-hexanoylamino)-1-phenyl-1-propanol (C6-D-MAPP), and L-erythro-2-(N-hexanoylamino)-1-phenyl-1-propanol (C6-L-MAPP),abolished the ability of the ceramide molecule to activate PP2Ac. Further studies demonstrated the requirement for the amide group, the primary hydroxyl group, and the secondary hydroxyl group of the sphingoid backbone for activation of PP2Ac through the synthesis and evaluation of D- erythro-urea C6 ceramide, L- erythro-urea C6 ceramide, D- erythro-N-methyl C6 ceramide, D- erythro-1-O-methyl C6 ceramide, D- erythro-3-O-methyl C6 ceramide, and (2S) 3-keto C6 ceramide. None of these compounds induced significant activation of PP2Ac. These structural requirements for the activation of PP2Ac also applied to the holoenzyme (trimeric PP2A) and to the catalytic subunit of PP1 (PP1c). Liposome binding studies were also conducted using D- erythro-C16 ceramide and D- erythro-C16 ceramide analogs, and the results showed that ability of ceramide and analogs to influence CAPP (activation or inhibition) was associated with the ability of the analogues to interact with CAPP in the liposome binding assay.This study demonstrates strict requirements (structural/stereochemical) of the sphingoid backbone and amide group for interaction of ceramide with CAPP, and disclose ceramide as a very specific regulator of CAPP.
Revised on November 4, 2003
Accepted on November 16, 2003
The structural requirements for ceramide activation of serine-threonine protein phosphatases
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