title: Activation of Aurora-A kinase by protein partner binding and phosphorylation are independent and synergistic.
abstract: Protein kinases are activated by phosphorylation and by the binding of activator proteins. The interplay of these two factors is incompletely understood. We applied energetic analysis to this question and characterized the activation process of the serine/threonine kinase Aurora-A by phosphorylation and by its protein partner, targeting protein for Xenopus kinesin-like protein 2 (TPX2). We discovered that these two activators act synergistically and without a predefined order: each can individually increase the activity of Aurora-A, and the effect of both bound together is the exact sum of their individual contributions to catalysis. Unexpectedly, the unphosphorylated enzyme has catalytic activity that is increased 15-fold by the binding of TPX2 alone. The energetic contribution of phosphorylation to catalysis is 2-fold greater than that of TPX2 binding, which is independent of the phosphorylation state of the enzyme. Based on this analysis, we propose a revised, fluid model of Aurora-A activation in which the first step is a reduction in the mobility of the activation loop by either TPX2 binding or phosphorylation. Furthermore, our results suggest that unphosphorylated Aurora-A bound to the mitotic spindle by TPX2 is catalytically active and that the phosphorylation state of Aurora-A is an inaccurate surrogate for its activity. Extending this form of analysis will allow us to compare quantitatively the effects of the whole network of kinase-activating partners. Comparison with other kinases showed that kinetic characterization detects those kinases whose activation loops undergo a rearrangement upon phosphorylation and thus whose unphosphorylated state offers a distinct target for the development of Type II inhibitors.
authors.affiliation: Division of Structural Biology, The Institute of Cancer Research, 237 Fulham Road, London SW3 6JB, United Kingdom.
authors.forename: Charlotte A
authors.initials: CA
authors.lastname: Dodson
authors.forename: Richard
authors.initials: R
authors.lastname: Bayliss
mesh_terms: D064127:Aurora Kinases; D002384:Catalysis; D018797:Cell Cycle Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004789:Enzyme Activation / Q000502:physiology; D006801:Humans; D008869:Microtubule-Associated Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008956:Models, Chemical; D009687:Nuclear Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation / Q000502:physiology; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D018797:Cell Cycle Proteins; D008869:Microtubule-Associated Proteins; D009687:Nuclear Proteins; C122167:TPX2 protein, human; D064127:Aurora Kinases; D017346:Protein Serine-Threonine Kinases
references.citation: Curr Biol. 2003 Apr 15;13(8):691-7
references.pmid: 12699628
references.citation: J Med Chem. 2009 May 14;52(9):2629-51
references.pmid: 19320489
references.citation: Biochemistry. 1983 Jul 19;22(15):3581-6
references.pmid: 6615786
references.citation: J Cell Biol. 2010 Dec 27;191(7):1315-32
references.pmid: 21187329
references.citation: J Biol Chem. 2009 Jun 5;284(23):15880-93
references.pmid: 19359241
references.citation: Cell. 2002 May 3;109(3):275-82
references.pmid: 12015977
references.citation: Biochemistry. 2003 Feb 4;42(4):849-56
references.pmid: 12549901
references.citation: Cell. 1996 Apr 19;85(2):149-58
references.pmid: 8612268
references.citation: Nat Cell Biol. 2011 Jun;13(6):708-14
references.pmid: 21572421
references.citation: Curr Opin Cell Biol. 2003 Apr;15(2):221-31
references.pmid: 12648679
references.citation: Biochemistry. 1995 Feb 28;34(8):2447-54
references.pmid: 7873523
references.citation: ACS Chem Biol. 2010 Jun 18;5(6):563-76
references.pmid: 20426425
references.citation: Biochemistry. 1992 Sep 15;31(36):8516-22
references.pmid: 1390637
references.citation: Biochemistry. 2002 Jul 2;41(26):8528-34
references.pmid: 12081504
references.citation: J Biol Chem. 2008 Sep 26;283(39):26591-601
references.pmid: 18669639
references.citation: Nat Chem Biol. 2006 Jul;2(7):358-64
references.pmid: 16783341
references.citation: Mol Cell. 2003 Oct;12(4):851-62
references.pmid: 14580337
references.citation: Biochemistry. 1998 Dec 22;37(51):17875-81
references.pmid: 9922154
references.citation: Proc Natl Acad Sci U S A. 2002 Nov 26;99(24):15440-5
references.pmid: 12422018
references.citation: J Cell Sci. 2007 Sep 1;120(Pt 17):2987-96
references.pmid: 17715155
references.citation: Biochemistry. 2007 Sep 11;46(36):10287-95
references.pmid: 17705509
references.citation: J Biol Chem. 2001 Jan 5;276(1):275-80
references.pmid: 11029468
references.citation: Cell Cycle. 2007 Nov 15;6(22):2846-54
references.pmid: 18032922
references.citation: Biochem J. 2010 Mar 15;427(1):19-28
references.pmid: 20067443
references.citation: J Biol Chem. 2007 Feb 2;282(5):3173-81
references.pmid: 17095507