The aurora kinase A regulates GSK-3beta in gastric cancer cells.
Aurora kinase A (AURKA) is located at 20q13, a region that is frequently amplified in gastric cancer. In this study, we have investigated the role of AURKA in regulating glycogen synthase kinase (GSK)-3beta and beta-catenin/TCF complex in gastric cancer cells. Our results demonstrate a significant increase in the phosphorylation of GSK-3beta at Ser 9 following the overexpression of AURKA in AGS cells. The immunoprecipitation with antibodies specific for AURKA and GSK-3beta indicated that the two proteins coexist in the same protein complex. The recombinant human AURKA protein phosphorylated the GSK-3beta protein at Ser 9 in a concentration-dependent manner, in vitro. The phosphorylation of beta-catenin (Ser33/37/Thr41) by GSK-3beta is known to target beta-catenin towards degradation. In line with our findings, the increase in phospho-GSK-3beta level was accompanied by a significant decrease in beta-catenin phosphorylation (Ser33/37/Thr41) and accumulation of beta-catenin protein. The knockdown of AURKA reversed the phosphorylation of GSK-3beta and the beta-catenin protein levels. The immunofluorescence analysis demonstrated colocalization of AURKA and GSK-3beta proteins and a significant increase in the nuclear beta-catenin levels in cells overexpressing AURKA. The beta-catenin/TCF transcription activity was measured using the pTopFlash and its mutant pFopFlash luciferase reporter vectors. Indeed, AURKA overexpression led to a significant increase in the pTopFlash reporter activity, whereas kinase dead AURKA mutant (D274A) had no effect. Consistent with these findings, we detected a significant mRNA up-regulation of several direct targets of the beta-catenin/TCF transcription complex (cyclin D1, c-MYC, c-MYC-binding protein, CLDN1, FGF18 and vascular endothelial growth factor), and a two-fold increase in the proliferation rate in AURKA overexpressing cells. We conclude that the AURKA/GSK-3beta interaction is important in regulating beta-catenin, underscoring a novel oncogenic potential for AURKA in gastric tumorigenesis.
Dar
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Department of Surgery, Vanderbilt University Medical Center, Nashville, TN 37232, USA.
Belkhiri
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El-Rifai
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D021581:Active Transport, Cell Nucleus; D064096:Aurora Kinase A; D064127:Aurora Kinases; D045744:Cell Line, Tumor; D002469:Cell Separation; D005434:Flow Cytometry; D015972:Gene Expression Regulation, Neoplastic; D038362:Glycogen Synthase Kinase 3 / Q000378:metabolism*; D000071679:Glycogen Synthase Kinase 3 beta; D006801:Humans; D008856:Microscopy, Fluorescence / Q000379:methods; D008954:Models, Biological; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry; D013274:Stomach Neoplasms / Q000201:enzymology / Q000378:metabolism*; D051176:beta Catenin / Q000378:metabolism
D034741:RNA, Small Interfering; D011994:Recombinant Proteins; D051176:beta Catenin; C576917:AURKA protein, human; D064096:Aurora Kinase A; D064127:Aurora Kinases; C000605506:GSK3B protein, human; D000071679:Glycogen Synthase Kinase 3 beta; D017346:Protein Serine-Threonine Kinases; D038362:Glycogen Synthase Kinase 3