NSF Award
9817393
Glycogen synthase kinase 3 beta (GSK3b) is an intracellular serine<br/>threonine kinase found at the heart of a number of important signaling<br/>pathways. These include embryonic pattern formation, tumor suppression and<br/>regulation of neurofibrillary tangles in Alzheimer's disease. A dramatic<br/>example of GSK3 function is in axis formation in the frog Xenopus. Expression<br/>of molecules that act via GSK3b, such as Wnt proteins, or of mutant forms of<br/>GSK3b itself, result in formation of complete ectopic dorsal axes. This<br/>suggests that reduction in GSK3b activity is sufficient to cause "dorsalness".<br/>Preliminary studies indicate that endogenous GSK3b is less abundant on the<br/>dorsal side of the embryo than on the ventral side. This contrasts with the<br/>effects of ectopic Wnt expression in which specific activity of GSK3b is<br/>reduced but abundance is not. This proposal aims to analyze GSK3b regulation<br/>in Xenopus, comparing endogenous mechanisms with classical Wnt regulation, to<br/>better understand the modes of regulation of this important protein.<br/><br/> Endogenous dorsal depletion of GSK3b ccan only be by reduction of<br/>synthesis or stability or by translocation of protein away from the dorsal<br/>side. The recently demonstrated role for a GSK3-binding protein suggests a<br/>post-translational mechanism. Therefore, differential stability will be tested<br/>by measuring the half-life of epitope-tagged GSK3b in dorsal versus ventral<br/>lysates. Translocation of pre-existing GSK3b protein will be monitored by<br/>visualizing localization or dispersion of exogenous, tagged GSK3b versus<br/>control proteins.<br/><br/> Sites on the GSK3b protein required for endogenous and Wnt-dependent<br/>regulation are unknown. They will be mapped, exploiting the advantages of the<br/>Xenopus system for this purpose. Dorsal-specific and Wnt-dependent protein<br/>modifications will be coarsely mapped by mass spectrometry of tryptic<br/>peptides. Fine mapping and functional correlation will be done by<br/>site-directed mutagenesis and expression in vivo. Mutants will be made at<br/>candidate modification sites and tested in vivo for specific activity with and<br/>without expression of Wnts.<br/><br/> Preliminary biochemical studies reveal proteins associated with GSK3b in<br/>vivo that do not correspond to known GSK3b-binding proteins. GSK3b-associated<br/>proteins are clearly important and so to identify these novel proteins (and to<br/>determine the association or absence of the known proteins in vivo), direct<br/>biochemical analysis of proteins co-immunoprecipitated with GSK3b will be<br/>carried out. Gel electrophoresis, Western blotting and microsequencing will be<br/>used.
