| One of the most intriguing enzymes of sphingolipid biology is acid sphingomyelinase (ASMase). Similar to a phospholipase C reaction, ASMase catalyzes the cleavage of the phosphocholine head group of sphingomyelin to generate ceramide. Cumulative efforts of various laboratories over the past 40 years have placed ASMase and its product ceramide at the forefront of lipid research. Activation of the ASMase/ceramide pathway is a shared response to an ever growing list of receptor and non receptor mediated forms of cellular stress including: death ligands (TNFalpha, TRAIL, Fas ligand), cytokines (IL-1, IFNgamma), radiation, pathogenic infections, cytotoxic agents and others. Given the strategic role of ASMase in lipid metabolism and cellular stress response, the objective of the current work is to investigate the molecular mechanisms underlying ASMase activation. In pursuit of this objective, an initial step was to generate the appropriate tools necessary to investigate the biology of ASMase. This entailed generation and optimization of a specific ASMase antibody, subcloning of full length ASMase cDNA into various fluorescent and non fluorescent tagged plasmids and development of RNA interference approach for genetic targeting of ASMase. Our findings support a model for ASMase activation in which protein kinase C delta (PKCdelta) serves as a key upstream kinase. In particular, PKCdelta mediates phosphorylation of ASMase at 5508 within the catalytic domain of the enzyme. This phosphorylation event is shown to be necessary not only for agonist-dependent activation of ASMase but also for its relocation to the plasma membrane. Functionally, the PKCdelta/ASMase pathway of ceramide generation is shown to be pivotal for response of mammary carcinoma cells to the chemotherapeutic agent cisplatin as well as UV radiation-induced mitochondrial injury. In sum, these studies identify a novel mechanism for ASMase regulation and elaborate on the translational role of the ASMase/ceramide pathway in cancer biology and therapy. |
