Native and polyubiquitinated forms of dihydroceramide desaturase are differentially linked to human embryonic kidney cell survival

There is controversy concerning the role of dihydroceramide desaturase (Degs1) in regulating cell survival with studies showing that it can both promote and protect against apoptosis. We have therefore, investigated the molecular basis for these opposing roles of Degs1. Treatment of HEK293T cells with the sphingosine kinase inhibitor, SKi (2-(p-hydroxyanilino)-4-(p-chlorophenyl)thiazole)) or fenretinide, but not the Degs1 inhibitor, GT11 (((N-[(1R,2S)-2-hydroxy-1-hydroxymethyl-2-(2-tridecyl-1-cyclopropenyl)ethyl]octan-amide)) induced the polyubiquitination of Degs1 (Mr=40-140 kDa) via a mechanism involving oxidative stress, p38 MAPK and Mdm2 (E3 ligase). The polyubiquitinated forms of Degs1 exhibit ‘gain of function’ and activate pro-survival pathways, p38 MAPK, JNK and X-box protein-1s (XBP-1s). In contrast, another sphingosine kinase inhibitor, ABC294640 (3-(4-chlorophenyl)-adamantane-1-carboxylic acid (pyridin-4-ylmethyl)amide) at concentrations of 25-50 uM failed to induce formation of the polyubiquitinated forms of Degs1. In contrast with SKi, ABC294640 (25 uM) promotes apoptosis of HEK293T cells via a Degs1-dependent mechanism that is associated with increased de novo synthesis of ceramide. These findings are the first to demonstrate that the polyubiquitination of Degs1 appears to change its function from pro-apoptotic to pro-survival. Thus, polyubiquitination of Degs1 might provide an explanation for the reported opposing functions of this enzyme on cell survival/apoptosis