The structural requirements for ceramide activation of serine-threonine protein phosphatases

Charles E Chalfant,Zdzislaw Szulc,Patrick Roddy,Alicja Bielawska,Yusuf A Hannun

doi:10.1194/jlr.m300347-jlr200

Charles E Chalfant, Zdzislaw Szulc + Show 3 more

Open Access

https://doi.org/10.1194/jlr.m300347-jlr200

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Abstract

The protein phosphatases1 (PP1) and 2A (PP2A) serve as ceramide-activated protein phosphatases (CAPP). In this study, the structural requirements for interaction between ceramide and CAPP were determined. D-erythro-C(6) 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 C(6) ceramide, inhibited PP2Ac (IC(50) = 8.5 microM). Furthermore, phyto C(6) ceramide, D-erythro-dehydro C(6) ceramide, and D-erythro-cis-C(6) ceramide had no effect on PP2Ac activity. Modification of the sphingoid chain also abolished the ability of ceramide 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 C(6) ceramide, L-erythro-urea C(6) ceramide, D-erythro-N-methyl C(6) ceramide, D-erythro-L-O-methyl C(6) ceramide, D-erythro-3-O-methyl C(6) ceramide, and (2S) 3-keto C(6) ceramide. None of these compounds induced significant activation of PP2Ac. Liposome binding studies were also conducted using analogs of D-erythro-C C(6) ceramide, and the results showed that the ability of ceramide analogs to influence CAPP (activation or inhibition) was associated with the ability of the analogs to bind to CAPP. This study demonstrates strict structural requirements for interaction of ceramide with CAPP, and disclose ceramide as a very specific regulator of CAPP. The studies also begin to define features that transform ceramide analogs into inhibitors of CAPP.

Highlights

The protein phosphatases1 (PP1) and 2A (PP2A) serve as ceramide-activated protein phosphatases (CAPP)
To determine the molecular basis of the interaction between PP2Ac and ceramide, we focused on key features in the ceramide molecule required for the activation of the phosphatase
These include the stereochemistry at the C2, C3, C4, and C5 positions, the effect of the functional groups in the sphingoid backbone, the requirement of the secondary amide bond, and the requirement of the primary and secondary hydroxyl groups (Fig. 1)

Summary

Introduction

The protein phosphatases (PP1) and 2A (PP2A) serve as ceramide-activated protein phosphatases (CAPP). 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-Omethyl C6 ceramide, and (2S) 3-keto C6 ceramide None of these compounds induced significant activation of PP2Ac. Liposome binding studies were conducted using analogs of d-erythro-C C6 ceramide, and the results showed that the ability of ceramide analogs to influence CAPP (activation or inhibition) was associated with the ability of the analogs to bind to CAPP.

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