Mi Kaihong, Dolan Philip J, Johnson Gail V W
Department of Psychiatry, University of Alabama at Birmingham, Birmingham, Alabama 35294-0017, USA.
J Biol Chem. 2006 Feb 24;281(8):4787-94. doi: 10.1074/jbc.M508657200. Epub 2005 Dec 19.
Glycogen synthase kinase 3 (GSK3) is a widely expressed Ser/Thr protein kinase that phosphorylates numerous substrates. This large number of substrates requires precise and specific regulation of GSK3 activity, which is achieved by a combination of phosphorylation, localization, and interactions with GSK3-binding proteins. Members of the Wnt canonical pathway have been shown to influence GSK3 activity. Through a yeast two-hybrid screen, we identified the Wnt canonical pathway co-receptor protein low density lipoprotein receptor-related protein 6 (LRP6) as a GSK3-binding protein. The interaction between the C terminus of LRP6 and GSK3 was also confirmed by in vitro GST pull-down assays and in situ coimmunoprecipitation assays. In vitro assays using immunoprecipitated proteins demonstrated that the C terminus of LRP6 significantly attenuated the activity of GSK3beta. In situ, LRP6 significantly decreased GSK3beta-mediated phosphorylation of tau at both primed and unprimed sites. Finally, it was also demonstrated that GSK3beta phosphorylates the PPP(S/T)P motifs in the C terminus of LRP6. This is the first identification of a direct interaction between LRP6 and GSK3, which results in an attenuation of GSK3 activity.
糖原合酶激酶3（GSK3）是一种广泛表达的丝氨酸/苏氨酸蛋白激酶，可磷酸化众多底物。大量的底物需要对GSK3活性进行精确且特异的调控，这可通过磷酸化、定位以及与GSK3结合蛋白的相互作用来实现。Wnt经典途径的成员已被证明会影响GSK3活性。通过酵母双杂交筛选，我们鉴定出Wnt经典途径的共受体蛋白低密度脂蛋白受体相关蛋白6（LRP6）为一种GSK3结合蛋白。LRP6的C末端与GSK3之间的相互作用也通过体外GST下拉实验和原位共免疫沉淀实验得到了证实。使用免疫沉淀蛋白进行的体外实验表明，LRP6的C末端显著减弱了GSK3β的活性。在原位实验中，LRP6显著降低了GSK3β介导的tau蛋白在引发位点和未引发位点的磷酸化。最后，还证明了GSK3β可磷酸化LRP6 C末端的PPP(S/T)P基序。这是首次鉴定出LRP6与GSK3之间的直接相互作用，这种相互作用导致了GSK3活性的减弱。
