Abstract
Although important for cellular stress signaling pathways, the molecular mechanisms of acid sphingomyelinase (ASMase) activation remain poorly understood. Previous studies showed that treatment of MCF-7 mammary carcinoma cells with the potent protein kinase C (PKC) agonist, phorbol 12-myristate 13-acetate (PMA), induces a transient drop in sphingomyelin concomitant with an increase in cellular ceramide levels (Becker, K. P., Kitatani, K., Idkowiak-Baldys, J., Bielawski, J., and Hannun, Y. A. (2005) J. Biol. Chem. 280, 2606-2612). Here we show that PMA selectively activates ASMase and that ASMase accounts for the majority of PMA-induced ceramide. Pharmacologic inhibition and RNA interference experiments indicated that the novel PKC, PKCdelta, is required for ASMase activation. Immunoprecipitation experiments revealed the formation of a novel PKCdelta-ASMase complex after PMA stimulation, and PKCdelta was able to phosphorylate ASMase in vitro and in cells. Using site-directed mutagenesis, we identify serine 508 as the key residue phosphorylated in response to PMA. Phosphorylation of Ser(508) proved to be an indispensable step for ASMase activation and membrane translocation in response to PMA. The relevance of the proposed mechanism of ASMase regulation is further validated in a model of UV radiation. UV radiation also induced phosphorylation of ASMase at serine 508. Moreover, when transiently overexpressed, ASMase(S508A) blocked the ceramide formation after PMA treatment, suggesting a dominant negative function for this mutant. Taken together, these results establish a novel direct biochemical mechanism for ASMase activation in which PKCdelta serves as a key upstream kinase.
Highlights
Contrast to the multistep de novo pathway of ceramide biosynthesis, breakdown of SM is catalyzed by a single class of enzymes, the sphingomyelinases (SMases)
In Vitro Kinase Assays—In vitro phosphorylation reactions were performed as follows: 6 g of purified acid sphingomyelinase (ASMase) were incubated with 25 ng of recombinant protein kinase C (PKC)␦ (Calbiochem) at 30 °C in a kinase buffer (20 mM HEPES, pH 7.4, 10 mM MgCl2, 0.1 mM EGTA, 0.1 mM ATP), 0.5 Ci of [␥-32P]ATP, and where indicated, the buffer was supplemented with lipid-detergent mixed micelles consisting of 100 g/ml phosphatidylserine and 10 g/ml diacylglycerol sonicated in 0.3% Triton X-100 solution
phorbol 12-myristate 13-acetate (PMA) Induces Activation and Membrane Translocation of ASMase—Previously, we reported that PMA treatment induces an elevation in ceramide levels that persisted after blocking the de novo pathway with myriocin but was inhibited by fumonisin B1, suggesting operation of the salvage pathway [1]
Summary
Materials—Cell culture material, including RPMI medium, fetal bovine serum (FBS), phosphate-free medium, and dialyzed serum, were from Invitrogen. The program selects regions of a protein that are candidates for raising antibodies, based on antigenicity, flexibility, and -turn Based on this analysis, the following peptide sequence was chosen and synthesized: CVGELQAAEDRGDKV (15 mer, residues 361–374). In Vitro Kinase Assays—In vitro phosphorylation reactions were performed as follows: 6 g of purified ASMase were incubated with 25 ng of recombinant PKC␦ (Calbiochem) at 30 °C in a kinase buffer (20 mM HEPES, pH 7.4, 10 mM MgCl2, 0.1 mM EGTA, 0.1 mM ATP), 0.5 Ci of [␥-32P]ATP, and where indicated, the buffer was supplemented with lipid-detergent mixed micelles consisting of 100 g/ml phosphatidylserine and 10 g/ml diacylglycerol sonicated in 0.3% Triton X-100 solution. The samples were washed with 1.5% FBS solution three times This was followed by incubation with secondary antibodies for 1 h at room temperature. A p value of 0.05 or less is considered as statistically significant and marked in the figures with an asterisk
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
