Long chain ceramides activate protein phosphatase-1 and protein phosphatase-2A - Activation is stereospecific and regulated by phosphatidic acid

The search for potential targets for ceramide action led to the identificat ion of ceramide-activated protein phosphatases, which include protein phosp hatase-2A (PP2A) and protein phosphatase-1 (PP1) with roles in regulating a poptosis and cell growth. Thus far, in vitro studies on ceramide-activated protein phosphatases have been restricted to the use of short chain ceramid es, limiting the extent of mechanistic insight. In this study, we show that the long chain D-erythro-C-18-ceramide activated PP2A (AB'C trimer), PP2Ac (catalytic subunit of PP2A), and PP1 gamma c and -alpha c (catalytic subun its of PP1 gamma and -1 alpha isoforms, respectively) 2-6-fold in the prese nce of dodecane, a lipid-solubilizing agent, with 50% maximal activation ac hieved at approximately 10 mu M D-erythro-C-18-ceramide. The diastereoisome rs of D-erythro-C-18-ceramide, D-threo-, and L-threo-C-18-ceramide, as well as the enantiomeric L-erythro-C-18-ceramide, did not activate PP1 or PP2A, but they inhibited PP1 and PP2A activity. The addition of phosphatidic aci d decreased the basal activity of PP1c but also increased the stimulation b y D-erythro-C-18-ceramide from 1.8- to 2.8 fold and decreased the EC50 of D erythro-C-18-ceramide to 4.45 mu M. The addition of 150 mM KCI decreased t he basal activity of PP1 and the dose of D-erythro-C-18-ceramide necessary to activate PP1c (EC50 = 6.25 mu M) and increased the ceramide responsivene ss up to 10-17-fold. These studies disclose stereospecific activation of PP 1 and PP2A by long chain natural ceramides under near physiologic ionic str engths in vitro. The implications of these studies for mechanisms of cerami de action are discussed.