ARD1-mediated Aurora kinase A acetylation promotes cancer cell proliferation and migration

학위논문 (박사)-- 서울대학교 대학원 약학대학 약학과, 2017. 8. Kyu-Won Kim.Aurora kinase A (AuA) is a prerequisite for centrosome maturation, separation, and mitotic spindle assembly, thus, it is essential for cell cycle regulation. Overexpression of AuA is implicated in poor prognosis of many types of cancer. However, the regulatory mechanisms underlying the functions of AuA are still not fully understood. Here, we report that AuA colocalizes with arrest defective protein 1 (ARD1) acetyltransferase during cell division and cell migration. Additionally, AuA is acetylated by ARD1 at lysine residues at positions 75 and 125. The double mutations at K75/K125 abolished the kinase activity of AuA. Moreover, the double mutant AuA exhibited diminished ability to promote cell proliferation and cell migration. Mechanistic studies revealed that AuA acetylation at K75/K125 promoted cell proliferation via activation of cyclin E/CDK2 and cyclin B1. In addition, AuA acetylation stimulated cell migration by activating the p38/AKT/MMP-2 pathway. These findings indicate that ARD1-mediated acetylation of AuA enhances cell proliferation and migration, and probably contributes to cancer development.INTRODUCTION 1 I. Protein acetylation 1 II. ARD1 5 III. Aurora kinases 9 1. Aurora kinase family 9 2. Structure of Aurora kinases 10 3. Subcellular localizations and functions of Aurora kinases 12 4. Activation and degradation of Aurora kinase A 15 5. Mitoticfunction of Aurora kinase A 18 6. Non-mitotic function of Aurora kinase A 22 PURPOSE OF THIS STUDY 25 MATERIALS AND METHODS 27 1. Antibodies 27 2. Plasmid construction .28 3. Cell culture 29 4. Cell synchronization 29 5. Immunoflourescence 30 6. Immunoblotting and immunoprecipitation 31 7. In vitro acetylation assay 32 8. ATP-binding assay 33 9. ATPase activity assay 33 10. Protein sequence alignment 34 11. Cell proliferation assay 34 12. Clonogenic assay 35 13. Cell cycle analysis 35 14. Wound healing assay 36 15. Transwell invasion assay 36 16. Statistical analysis .37 RESULTS 38 1. ARD1 has centrosome-like localization during cell cycle progression 38 2. ARD1 colocalizes with Aurora kinase A during cell cycle progression 41 3. ARD1 colocalizes with Aurora kinase A during cell migration 43 4. Aurora kinase A is acetylated by ARD1 45 5 Acetylation of Aurora kinase A is dependent on ARD1 catalytic activity 49 6 Aurora kinase A is acetylated at at lysine residues at position at 75 and 125 52 7 Double mutation at lysine 75 and 125 abolished acetylation of AuA 56 8 Acetylation of Aurora kinase A increases the phosphorylation of Aurora kinase A 60 9 Acetylation of Aurora kinase A increases the kinase activity of Aurora kinase A 62 10 Acetylation of Aurora kinase A promotes cell proliferation 68 11 Acetylation of Aurora kinase A increases G2/M cells population 73 12 Acetylation of Aurora kinase A promotes cell proliferation by increasing expression level of cell cycle check point proteins 76 13 Acetylation of Aurora kinase A increases during cell migration 79 14 Acetylation of Aurora kinase A promotes cell migration 81 15 Acetylation of Aurora kinase A promotes cell migration via p38/AKT/MMP2 pathway 84 DISCUSSION 87 REFERENCES 94 ABSTRACT IN KOREAN (국문초록) 114Docto