Involvement of the binding residues in GSKIP with GSK3beta - comparison to AxinGID and FRATtide
| Autor: | Meng-Yu Hsu, 徐夢妤 |
|---|---|
| Rok vydání: | 2008 |
| Druh dokumentu: | 學位論文 ; thesis |
| Popis: | 96 Glycogen synthase kinase 3β (GSK3β) is a multifaceted serine/threonine kinase and has been implicated in many biological processes, such as insulin-glycogen and Wnt signaling pathways. The specificity and regulation of GSK3β are thought to be involved in the docking interactions at core kinase domain because of the particular amino acid residues. Recent X-ray diffraction studies illuminated the relative binding residues on AxinGID and FRATtide for GSK3β docking and appeared that GSK3β Val267Gly (V267G) and Tyr288Phe (Y288F) could distinct the direct interaction between AxinGID and FRATtide. The previous study revealed a new GSK3β binding protein – GSKIP, binding to the overlapping site as AxinGID and FRATtide on GSK3β, however, the binding mode of ?nGSKIP to GSK3β?nhas not yet been determined. In this study, based on the sequence alignment of AxinGID and FRATtide, four mutants were generated to determine the binding sites of GSKIP to GSK3β according to the corresponding sites. We showed that the binding of GSKIP to GSK3β was completely abolished at F122P, L126P, L130P and the corresponding sites on AxinGID and FRATtide. On the other hand, mutants of putative salt-bridge site at R129A of GSKIP showed no effect. These results indicated that all three binding proteins shared similar binding sites on GSK3β. Meanwhile, in a parallel study, we found that the binding to GSKIP and AxinGID in GSK3β?nV267G mutant, but not Y288F, was affected. Electrostatic potential map apparently supported our contention as it showed that the binding region of GSKIP could be likely replaced with AxinGID, not FRATtide, to dock the same sites on GSK3β. In addition, pull-down assay showed that GSKIP binds to both GSK3β?nV267G (but less) and Y288F mutants, indicating that these three proteins still possess some distinct binding modes. To search for different binding modes, more GSK3β binding proteins were performed. The data showed the other two possibilities, including CABYR had no effect by both V267G and Y288F, whereas Astrin affected by either V267G or Y288F. Moreover, using the truncated and site-directed mutagenesis assay, we discovered that C-terminal helix region of GSK3β?n(354-383 residues) would act as an important region for the differential recognition of GSK3?猀]to FRATtide from GSKIP and AxinGID. Our data further showed that C-terminal helix region single-point mutation of GSK3β?nL359P, F362A,?nE366K and?nL367P were able to eliminate the binding with FRATtide, but not AxinGID or GSKIP. These observations were also supported by other examined GSK3β?nbinding proteins including, CABYR, hNinein, dynamin-like protein and Astrin which GSK3β?nbindings ability were abolished, whereas GSK3β?nL377P only affected the interaction with Dynamin-like protein and Astrin. Taken together, we unravel that in addition to scaffold binding region, the C-terminus helix region of GSK3β may also participate in the recognition and specificity of GSK3β with regard to its particular binding proteins. |
| Databáze: | Networked Digital Library of Theses & Dissertations |
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