Sphingosine kinase (SphK) is a central enzyme regulating the levels of sphingosine-1-phosphate (S1P), a bioactive sphingolipid metabolite and a ligand for a family of 5 S1P receptors. We have previously cloned and characterized 2 distinct isoforms, SphK1 and SphK2. SphK1 is activated by a numerous stimuli to promote cell proliferation, cell survival, migration and tumor formation. SphK1 and intracellularly generated S1P can signal """"""""inside-out"""""""" to regulate cytoskeletal rearrangements and cell movement, yet stimulates cell growth and suppresses apoptosis independently of S1P receptors. Remarkably, although highly similar in sequence to SphK1 and possessing the same 5 conserved domains, we recently found that SphK2 rather than promoting growth and survival, suppressed growth and also enhanced apoptosis. The goal of this proposal is to determine how these 2 very closely related and similar isoenzymes that can produce the same product can have such different functions. We will examine the hypothesis that the opposite actions of SphK1 and SphK2 result from their specific regulation and/or translocation to distinct cellular compartments to produce distinct pools of S1P or other phosphorylated sphingoid bases that have unique functions, and thus affect cellular processes that are particularly important for the transformed phenotype. Biochemical and genetic approaches will be utilized to determine whether endogenous SphK1 and SphK2 have redundant, overlapping, complementary, or antagonistic functions in growth, survival and motility of cancer cells. We will also elucidate their unrecognized roles in regulating ceramide biosynthesis and the sphingolipidome and determine whether SphK2 is a dual function BH3-only protein that links the Bcl-2 family, calcium homeostasis, and regulation of apoptosis. Collectively, these studies will add a new dimension to the physiological roles of SphK1 and SphK2 whose activities are central and obligatory in controlling levels of S1P, a potent lipid mediator that regulates many cellular processes important for cancer.
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