title: Identification of phosphorylated residues that affect the activity of the mitotic kinase Aurora-A.
abstract: The activity of the kinase Aurora-A (Aur-A) peaks during mitosis and depends on phosphorylation by one or more unknown kinases. Mitotic phosphorylation sites were mapped by mass spec sequencing of recombinant Aur-A protein that had been activated by incubation in extracts of metaphase-arrested Xenopus eggs. Three sites were identified: serine 53 (Ser-53), threonine 295 (Thr-295), and serine 349 (Ser-349), which are equivalent to Ser-51, Thr-288, and Ser-342, respectively, in human Aur-A. To ask how phosphorylation of these residues might affect kinase activity, each was mutated to either alanine or aspartic acid, and the recombinant proteins were then tested for their ability to be activated by M phase extract. Mutation of Thr-295, which resides in the activation loop of the kinase, to either alanine or aspartic acid abolished activity. The S349A mutant had slightly reduced activity, indicating that phosphorylation is not required for activity. The S349D mutation completely blocked activation, suggesting that Ser-349 is important for either the structure or regulation of Aur-A. Finally, like human Aur-A, overexpression of Xenopus Aur-A transformed NIH 3T3 cells and led to tumors in nude mice. These results provide further evidence that Xenopus Aur-A is a functional ortholog of human Aur-A and, along with the recently described crystal structure of human Aur-A, should help in future studies of the mechanisms that regulate Aur-A activity during mitotic progression.
authors.affiliation: Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA.
authors.forename: Laurie E
authors.initials: LE
authors.lastname: Littlepage
authors.forename: Hua
authors.initials: H
authors.lastname: Wu
authors.forename: Thorkell
authors.initials: T
authors.lastname: Andresson
authors.forename: Julia K
authors.initials: JK
authors.lastname: Deanehan
authors.forename: Laufey T
authors.initials: LT
authors.lastname: Amundadottir
authors.forename: Joan V
authors.initials: JV
authors.lastname: Ruderman
mesh_terms: D016475:3T3 Cells; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D064173:Anaphase-Promoting Complex-Cyclosome; D000818:Animals; D064096:Aurora Kinase A; D064127:Aurora Kinases; D018797:Cell Cycle Proteins / Q000737:chemistry* / Q000502:physiology; D002471:Cell Transformation, Neoplastic / Q000235:genetics; D004527:Egg Proteins / Q000737:chemistry / Q000378:metabolism; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D008025:Ligases / Q000502:physiology; D051379:Mice; D008819:Mice, Nude; D008938:Mitosis; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009368:Neoplasm Transplantation; D009374:Neoplasms, Experimental / Q000201:enzymology / Q000209:etiology / Q000235:genetics; D009865:Oocytes / Q000201:enzymology; D010749:Phosphoprotein Phosphatases / Q000378:metabolism; D010766:Phosphorylation; D010768:Phosphoserine / Q000737:chemistry; D010769:Phosphothreonine / Q000737:chemistry; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases / Q000737:chemistry* / Q000502:physiology*; D011499:Protein Processing, Post-Translational; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000502:physiology; D011518:Proto-Oncogene Proteins / Q000737:chemistry* / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000502:physiology; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D013329:Structure-Activity Relationship; D025801:Ubiquitin / Q000378:metabolism; D043743:Ubiquitin-Protein Ligase Complexes; D029867:Xenopus Proteins; D014982:Xenopus laevis / Q000378:metabolism
chemical_list: D018797:Cell Cycle Proteins; D004527:Egg Proteins; D004791:Enzyme Inhibitors; D047428:Protein Kinase Inhibitors; D011518:Proto-Oncogene Proteins; D011993:Recombinant Fusion Proteins; D025801:Ubiquitin; D029867:Xenopus Proteins; D010769:Phosphothreonine; D010768:Phosphoserine; D043743:Ubiquitin-Protein Ligase Complexes; D064173:Anaphase-Promoting Complex-Cyclosome; D011494:Protein Kinases; C112134:AURKA protein, Xenopus; C576918:Aurka protein, mouse; D064096:Aurora Kinase A; D064127:Aurora Kinases; D017346:Protein Serine-Threonine Kinases; D010749:Phosphoprotein Phosphatases; D008025:Ligases