Regulation of GSK 3B in Xenopus

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Glycogen synthase kinase 3 beta (GSK3b) is an intracellular serinethreonine kinase found at the heart of a number of important signalingpathways. These include embryonic pattern formation, tumor suppression andregulation of neurofibrillary tangles in Alzheimer's disease. A dramaticexample of GSK3 function is in axis formation in the frog Xenopus. Expressionof molecules that act via GSK3b, such as Wnt proteins, or of mutant forms ofGSK3b itself, result in formation of complete ectopic dorsal axes. Thissuggests that reduction in GSK3b activity is sufficient to cause "dorsalness".Preliminary studies indicate that endogenous GSK3b is less abundant on thedorsal side of the embryo than on the ventral side. This contrasts with theeffects of ectopic Wnt expression in which specific activity of GSK3b isreduced but abundance is not. This proposal aims to analyze GSK3b regulationin Xenopus, comparing endogenous mechanisms with classical Wnt regulation, tobetter understand the modes of regulation of this important protein. Endogenous dorsal depletion of GSK3b ccan only be by reduction ofsynthesis or stability or by translocation of protein away from the dorsalside. The recently demonstrated role for a GSK3-binding protein suggests apost-translational mechanism. Therefore, differential stability will be testedby measuring the half-life of epitope-tagged GSK3b in dorsal versus ventrallysates. Translocation of pre-existing GSK3b protein will be monitored byvisualizing localization or dispersion of exogenous, tagged GSK3b versuscontrol proteins. Sites on the GSK3b protein required for endogenous and Wnt-dependentregulation are unknown. They will be mapped, exploiting the advantages of theXenopus system for this purpose. Dorsal-specific and Wnt-dependent proteinmodifications will be coarsely mapped by mass spectrometry of trypticpeptides. Fine mapping and functional correlation will be done bysite-directed mutagenesis and expression in vivo. Mutants will be made atcandidate modification sites and tested in vivo for specific activity with andwithout expression of Wnts. Preliminary biochemical studies reveal proteins associated with GSK3b invivo that do not correspond to known GSK3b-binding proteins. GSK3b-associatedproteins are clearly important and so to identify these novel proteins (and todetermine the association or absence of the known proteins in vivo), directbiochemical analysis of proteins co-immunoprecipitated with GSK3b will becarried out. Gel electrophoresis, Western blotting and microsequencing will beused.