Cellular sphingolipid derivatives-ceramides, sphingosine (and other sphingoid bases), and spingosine 1-phosphates are bioactive signaling molecules known to regulate cell growth and apoptosis. Protein Phosphatase 2A (PP2A) is a major cellular phosphatase also shown to function in the regulation of both cell proliferation and apoptosis; in addition, it has been identified as a target of ceramide's pro-apoptotic effects. Ceramide is known to activate PP2A in vitro, an event that is predicted to have anti-growth and pro-apoptosis effects. Thus, PP2A may be an important mediator of the anti-cancer effects of sphingolipid derivatives such as ceramide. PP2A is regulated by a complex set of inputs including binding such as ceramide. PP2A is regulated by a complex set of inputs including binding of lipids (ceramide), covalent modification (e.g. methylation) of the catalytic subunit, and association of a variety of regulatory/targeting subunits. These mechanisms affect the activity, substrate specificity, and cellular localization of PP2A. However, little is known about the upstream signals that feed into these regulatory mechanisms or the roles these regulatory mechanisms play in cell growth control and apoptosis. Although ceramide is known to activate PP2A in vitro, it is not known whether it affects any other PP2A regulatory mechanism in vivo. Also, the effects of other sphingolipid derivatives such as sphingosine and sphingosine 1-phosphate on PP2A in vivo are not known.
The specific aims of this proposal are 1) to characterize the effects of ceramide and other sphingolipid derivatives on PP2A methylation and regulatory subunit association in vivo, 2) to evaluate the role of PP2A in two pathways in colon cancer cells: a) in an anti-growth pathway modulated by sphingolipids: beta-catenin degradation and b) in pro-apoptotic pathway modulated by sphingolipids: the PI 3- kinase/PDK1/Akt/Bad/Bcl-2 pathway, and 3) to screen for promising anti-cancer ceramide derivatives by testing synthetic derivatives made during the progress of this proposal for their ability to activate PP2A in vitro or modulate PP2A made during the progress of this proposal for their ability to activate PP2A in vitro or modulate PP2A regulatory mechanisms or targets in vivo. These studies will help accomplish the broad, long-term objectives of this program which are 1) to investigate the role of PP2A in the anti-cancer (pro-apoptotic) function of bioactive sphingolipid derivatives, 2) to identify PP2A regulatory mechanisms affected by sphingolipid derivatives, and 3) to use current PP2A-based screens, and additional ones developed during the course of these studies, to guide the development of more potent anti-cancer sphingolipid derivatives.
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