ah aakl,KrnM ekl,FacsSeaz n uinGomez-Cambronero* Julian and cell Speranza Francis of Src Henkels, activator M. and Karen direct Mahankali, FAK Madhu a PLD, as with A interaction kinase upon Aurora migration for role non-mitotic A ARTICLE RESEARCH ß 516 2014 December 8 Accepted 2014; May 29 attributed. Received properly is work original the Attribution that Commons provided Creative medium the any of distribution in terms use, reproduction the unrestricted and under permits distributed which article (http://creativecommons.org/licenses/by/3.0), Access License Open an is This ([email protected]) correspondence for *Author and USA. Biochemistry 45435, of OH Department Dayton, Medicine, , of Molecular School University State Wright region AURA- a The 20q13.2-q13.3, 2013). chromosome al., on et located Pinyol is in 2000; gene encoding al., assembly et spindle (Yang functional cells mitotic that and through NEDD1, chromatin, of kDa the 46 phosphorylation around 403-amino-acid nucleation a microtubule is promotes STK15), and and AURKA (Vader cells mammalian controls in that division enzymes 2008). cell Lens, cell-cycle-regulated B of of A, aspects group Aurora several a (comprising is kinases family C) aurora kinase the and and Aurora (NIMA) The kinase a (AURKs). gene (CDKs), mitosis kinases in the never cyclin-dependent as the the such (PLKs), kinases, protein kinases different Polo-like many of by dephosphorylation substrates and specific phosphorylation by mediated is Mitosis INTRODUCTION acid Phosphatidic Leukocytes, signaling, Cell Cell polymerization, migration, Tubulin kinase, Aurora WORDS: KEY which lipase, the by E region acid the phosphatidic with AURA, because of to is directly AURA production binds activates PLD the why reason of The cytoplasmic cells. to in colocalize and regions complex and protein–protein AURA loop. a reinforcement form PLD, positive PLD2 a phosphorylates which in engage AURA (PLD2), addition, both D2 so In PTK2), phospholipase activation. by as AURA is by Src causes way regulated, known new is and also that also additional AURA An AURA which movement. (FAK, here cell and of kinase initiation report T288, to leading adhesion We at focal cellular AURA AURA. other activates activates when by and as and activated if phosphorylates known of unclear are progression also still the is functions (AURA, and it formation A Nonetheless, centrosome mitosis. for kinase vital Aurora is AURKA) of activation Timely ABSTRACT hshtdcai r e ptemrgltr fAR that migration. AURA cell of of cellular regulators non-mitotic the upstream in role new its and mediate PLD are and FAK, acid and Src yielding Thus, phosphatidic in migration. cellular direct Src effectual and fully FAK a AURA, this with polymerization synergistically tubulin cooperates Furthermore, enhances and acid phosphatidic pocket. with interaction phosphatidic-acid-binding a 05 ulse yTeCmayo ilgssLd|Junlo elSine(05 2,5656doi:10.1242/jcs.157339 516–526 128, (2015) Science Cell of Journal | Ltd Biologists of Company The by Published 2015. h uoakns sfr AR,as nw as known also (AURA, isoform A kinase Aurora The 171 –E 211 rjce obe to projected smlrt h d1cciB ope)cnb eetdprior detected be can AURA–Ajuba complex) Cdk1–cyclinB1 2011). of al., the complex of et to AURA Jantscher protein–protein (similar 2009; capable of al., heterodimeric et activity are Eckerdt The kinase 2003; which al., and and et (TPX2) (Hirota (CDK1), phosphorylation Xklp2 the 1 for promoting protein kinase targeting cyclin-dependent and Ajuba including Lens, and (Vader cytoplasm is AURA the 2008). Additionally, in the microtubules on 2008). with 2002). mitosis Lens, associated al., and and is G2 et (Vader during spindle (Bar-Shira AURA centrosomes mitotic oncogene to and fide localized bona is malignancies, AURA a be human to in considered amplified frequently ciiyo UAlaigt uui oyeiainadcell the and polymerization affects tubulin positively to intracellular and leading motility. AURA key to of a binds activity acid, phospholipid, phosphatidic signaling Additionally, D2 and phospholipase lipase, Src a (PLD2). motility-related with by along regulated is migration a unexpectedly, that, cell and, FAK found in have implicated have is We perhaps AURA interphase. indeed, during cytoplasm could we the centromeres, in AURA function bipolar the that to modulates reasoned them by still pulling attracting the it detected in chromosomes that participates be of fully indicate AURA could can As functions. still phase cellular and other M AURA AURA cycle the that of outside the fact attention. levels exists The of localization. low much diffuse phases as that immunofluorescence received other clear in has become remain has formation it spindle However, mitotic some with the are processes complex in what AURA or protein. unclear alone or activating enzymatic AURA is factor of it other of its activation this as the of ways to in study, result lead domain intense possible increase The under catalytic other 2005). currently significant al., its but a et of activity, Ferrari is loop AURA 2000; phosphorylation cycle, al., activation T288 et cell conserved the (Walter the the at within of phosphorylation residue by phases regulated specific is function During mitosis. to rae el.Fg Csoswsenbo nlssindicating analyses AURA. increasing overexpressed blot as western well of as shows endogenous, of 1C presence expression Fig. Additionally, of cells. an the migration in treated resulted cell migration. 1B) in of (Fig. increase AURA migration overexpressed AM-8237 reduced of concentrations inhibitor, cell significantly small- enhanced presence AURA-specific the nM) as (30 AURA, the endogenous by mediated towards of fibroblasts was COS-7 EGF of nM migration 3 cell enhanced migration 1A, cell Fig. enhanced In for essential is AURA RESULTS eea ptemrgltr fAR aebe identified, been have AURA of regulators upstream Several h oaiaino UAt etooe ttebgnigof beginning the at centrosomes to AURA of localization The , .-odcmae ihmock- with compared 2.3-fold

Journal of Cell Science EERHARTICLE RESEARCH vrxrse nraigaonso idtp UAhad AURA wild-type of amounts that cells increasing 2B) Furthermore, (Fig. samples. the overexpressed AURA non-silenced of silencing phases to We of growth compared times. result and when a lag later the as at of reaction not terms polymerization in and reduced decrease reaction a was polymerization observed the polymerization were nM in assays 300 tubulin early polymerization to Interestingly, up tubulin with AURA, performed. silenced for were specific that polymerization. siRNA lysates 2000; tubulin cell modulating al., COS-7 AURA by et Using whether migration determined (Ballestrem next another cell organization, we movement regulated Therefore, to 2004). cell al., microtubule to et contributes Yao essential process, also is which AURA physiological the dose- to chemotaxis, important a important being in silencing, of AURA AURA reduced to of process addition significantly result In was a manner. dependent as migration that, cell indicate 2A enhanced 2A Fig. Results Fig. AURA AURA. the in for of specific shown in downregulation (siRNA) RNA confirm shown interfering small silencing analyses As using of cell blot migration. effect western enhanced cell the inset, enhanced determine AURA to on of wanted AURA next overexpression we that migration, tubulin shown modulating by Having migration cell reduces AURA Silencing ftepstv txl n eaie(S)cnrl o tubulin for controls (BSA) negative representative is and respectively. 2E (taxol) polymerization, catalytically Fig. AURA was positive 2D). of (Fig. the process effect of of importance this was to the positive AURA AURA-D274A effect active stresses mutant This which kinase-inactive positive function. overexpressed, the 2C), the when (Fig. cell reversed on confirmed polymerization overexpression further tubulin which increased concomitantly elmgainwsmxmlwt UAadScco- was Src which activity and samples, kinase in AURA increase other substantial with very to the maximal by substantiated compared that was indicate experimentation with overexpression of migration together line present this cell when of results migration Src The that of AURA. shows amount migration further cells the It cell epithelial in AURA. COS-7 enhanced increases of of an observed 3B) overexpression was (Fig. ectopic we activity upon there AURA proteins migration that and two cell shows 3A) (Fig. 3 in these Fig. of increase cell 1. of Fig. the either steps mediate that initial the could hypothesized at at found we are and FAK migration migration, and Src during like adhesions proteins focal motile that fact migration the cell Given AURA-mediated to contributes Src ora fCl cec 21)18 1–2 doi:10.1242/jcs.157339 516–526 128, (2015) Science Cell of Journal etr ltig(...Atni hw sa equal an as control. shown loading is protein Actin by (W.B.). detected blotting as western AURA of endogenous presence and the overexpressed showing controls Positive (C) irtd(ecnaeo oto) * # control). of (percentage migrated mean are the and as triplicate expressed in performed overexpression. were AURA by Transwells, increased using significantly assessed was as post-transfection migration, days cell 2 enhanced of At mock- amount DNA. either indicated plasmid were the AURA cells with COS-7 transfected (B) or inhibitor nM). transfected AURA (20 the a by 8237 as inhibited MLN here was acts this (which and EGF ‘motogen’), with induction Transwell in upon motility settings enhanced migration. had cell fibroblasts COS-7 enhanced (A) induces AURA 1. Fig. P , .5dcessbtensmlsadcontrols. and samples between decreases 0.05 6 ...nme fclsthat cells of number s.e.m. P , .5icessand increases 0.05 517

Journal of Cell Science EERHARTICLE RESEARCH h eeatrato opnnsta nlddradioactive 518 included varying that in components them the of reaction used presence understand [ the in we relevant better interest of kinases, To the kinases other three right). the with the these combinations from of bar dynamics third 3B, (Fig. 2. Fig. 32 P] c T.W hnasydtheir assayed then We ATP. e etpg o legend. for page next See nvitro in iaeatvte by activities kinase eemnn h muto [ of amount the determining ee fpopoyain nteknsscletvl was actual the reaction the collectively and in kinase 3C) kinases each Fig. of phosphorylation overall the ; of (the level filter-binding individual on a incorporated (using was phosphorylation) detected that of radiolabel level of level overall ora fCl cec 21)18 1–2 doi:10.1242/jcs.157339 516–526 128, (2015) Science Cell of Journal 32 P] c T noprtd ohthe Both incorporated. ATP

Journal of Cell Science fclsta irtd(ecnaeo oto)* control) of mean (percentage the migrated # as that expressed cells are Experiments of and siAURA. triplicate amounts in increasing performed with were is treated migration cell cells Enhanced with in (A) transfected decreased (siAURA). were AURA that for lysates (A specific cell siRNA assays. COS-7 increasing and polymerization of SDS-PAGE analyses tubulin blot for for Western siRNA prepared or inset) nM were analysis 300 samples blot to days, western up 3 subsequent with for silenced tubulin. AURA were for modulating that by specific lysates migration cell cell COS-7 reduced Using AURA Silencing 2. Fig. ARTICLE RESEARCH UA A n/rSca hshrlto usrt o h eeatkns fitrs,wihwr hnue o itrbnigasy n subsequent and assays filter-binding for used then were re purified which using interest, activities of mean Kinase kinase (C) the relevant Src. as and the quantified FAK for were AURA, substrate Results recombinant phosphorylation spectrometry. purified of as scintillation states Src Phosphorylation and/or migration. (C,D) FAK cell (B). AURA, AURA-mediated activity to AURA and contribute (A) FAK migration and Src 3. Fig. and membranes we PVDF panels, two to these in visualized transfer shown but As 3C samples and Fig. autoradiography. The from subsequent derived SDS-PAGE 3D). those Fig. are after analysis; 3D Fig. in-gel in an shown (using detected was n r xrse stema eaienme ffursec nt (RFU). units fluorescence of number relative mean triplicate the in as performed expressed were are Experiments and positive controls. (BSA) polymerization negative Tubulin and (E) (taxol) overexpressed. AURA- on was mutant effect (AURA-KD) kinase-inactive positive the D274A The when reversed (D) was on cells. polymerization effect mock tubulin positive to a compared had polymerization (AURA-WT) tubulin samples. AURA result non-silenced a wild-type as to Overexpressed times compared (C) later as at AURA not and silencing reaction of polymerization was AURA the polymerization in for Tubulin early (B) silenced reduced AURA. either (O/E) overexpressed were that that or cells (siAURA) of polymerization Tubulin (B–D) erae ewe ape n otos D hshrlto nglaaye fSc A rAR nteasneo rsneo h te kinase. other the of [ presence containing or buffer absence a the in kinases in other AURA the or of FAK one or Src, alone of buffer analyses with in-gel incubated individually Phosphorylation were (D) proteins controls. Recombinant and samples between decreases fteknsswsosre ySSPG n hnwsenbo nlss E ceai rwn ersnigatvto fusra r rmthe from Src upstream of activation representing drawing Schematic (E) analyses. blot western then and FAK. SDS-PAGE or by AURA observed downstream was kinases the of P , .5dcessbtensmlsadcontrols. and samples between decreases 0.05 P , .5icessand increases 0.05 6 ...number s.e.m. 6 ...pretg ciiycmae otecnrlsml.* sample. control the to compared activity percentage s.e.m. feto vrxrsino elmtlt rtiso UAmdae nacdcell enhanced AURA-mediated on proteins motility cell of overexpression of Effect fAURA). of UA(sAR hshrlto fFKyeddmore yielded FAK of of downstream phosphorylation [ be of A AURA might incorporation FAK AURA. regulating (as kinase whereas and upstream AURA, AURA the FAK and in was shown FAK Src both is both that kinases of suggesting three 3E, these upstream Fig. between to is regulation data of to this Src schematic interpret led We that versa phosphorylation. vice AURA, indicate of and and levels AURA lower FAK of much both phosphorylation phosphorylated FAK strongly whereas Src that found oiieefc nPD ciiy ssoni i.4,AURA 4C, Fig. in shown As a activity. exert AURA PLD2 did on is, that effect investigated true, we positive were Next, scenario activity. inverse AURA the on whether AURA of PLD2 and co-overexpression of by PLD2 produced synthetic inhibitor both gain small-molecule at the a negated a site (FIPI) towards activity Moreover, autophosphorylation 4B). activity its (Fig. mimicked its T288 that measuring substrate this AURA by peptide to of activity specific determined autocatalytic the an on as also using and detected AURA, on as cells residue phosphorylation 4A), epithelial AURA (Fig. on COS-7 T288 effect at in positive concomitant overexpression a PLD2 exerted that found migration cell We AURA-mediated to contributes PLD2 ora fCl cec 21)18 1–2 doi:10.1242/jcs.157339 516–526 128, (2015) Science Cell of Journal 32 P] c T oprdt A phosphorylation FAK to compared ATP 32 P] c T,floigwihphosphorylation which following ATP, P , .5icessand increases 0.05 combinant # P , liquid 519 0.05

Journal of Cell Science [ eutn VFmmrn,w eetdapopoyae PLD2 phosphorylated a detected we membrane, PVDF resulting EERHARTICLE RESEARCH 520 recombinant purified for an substrate in AURA phosphorylation protein control negative the to compared activity sample. lipase used total were in AURA lipase increase overexpressed PLD PLD that the on lysates for effect cell positive when significant activity statistically a exerted FIPI nM 300 of presence or absence the in incubated [ were incorporated PLD2 that plus assay AURA catalytic both AURA or a activit alone for AURA PLD2 used increased overexpression alone, and PLD2 AURA inhibitor) (B) Mock, . small-molecule monoclonal overexpressed (PLD2-specific rabbit that relevant using lysates actin cell or 7 AURA total AURA. (T288), and phospho-AURA PLD PLD2, tagged between activation Reciprocal 4. Fig. hpoiaeadkinase. and phopholipase xeiet eepromdi rpiaeadaeepesda h mean the as expressed are and triplicate in performed were Experiments D.()Idctsteclua ciiyo noeosPDo UAwe ciae ih3n G.At-UAimnpeiiae ranti-PLD or immunoprecipitates Anti-AURA EGF. an nM in 3 AURA with recombinant increas activated for an when and substrate (C) AURA phosphorylation lysates or a cell PLD as COS-7 endogenous used of for activity of was used PLD activity PLD2 were both cellular recombinant immunoprecipitates in the when increase an PLD2 Indicates caused of (E) overexpression phosphorylation (D). AURA of PLD. level on AURA the of in Effects (C,D) controls. and samples roeta esrdpopoiaeatvt ntepeec fP8lpsmsad[ and liposomes PC8 of presence the in activity phospholipase measured that one or 32 sn uiid auoia L2poena full-length a as protein PLD2 baculoviral purified, Using P] c T noPD ysbeun uoaigah fthe of autoradiography subsequent by PLD2 onto ATP nvitro in nvitro in sa,wihwsmnfse sa as manifested was which assay, iaeasymauigicroainof incorporation measuring assay kinase nvitro in sas n htmaue h noprto f[ of incorporation the measured that one assays: A L2oeepeso eutdi UApopoyain VFmmrnswr rbdfrMyc- for probed were membranes PVDF phosphorylation. AURA in resulted overexpression PLD2 (A) . 2-fold 6 ...pretg fcnrl * control. of percentage s.e.m. L2adAR hthsbe ecie eenfrtefrttime first between the loop for activation herein described anti-AURA 4F). continuous been (Fig. a has that of parallel or AURA result in and PLD2 the somewhat run as anti-PLD then activities 4E), and (Fig. both time either that of indicate periods antibodies with several for PLD2 EGF immunoprecipitated nM were wild-type 3 that samples with of cell stimulated similar Further, mass AURA. by molecular phosphorylated expected was which the ( action, AURA at of result present the as band, (phospho-PLD2) , 0 D)(i.4) hsrsl niae htPD was PLD2 that indicated result This 4D). (Fig. kDa) 105 3 ]uao bakcrls.()Idctstercpoa ciainof activation reciprocal the Indicates (F) circles). (black H]butanol 32 P] ora fCl cec 21)18 1–2 doi:10.1242/jcs.157339 516–526 128, (2015) Science Cell of Journal c T oteseii etd usrt o UA(ht squares) (white AURA for substrate peptide specific the to ATP 32 P] c T ntesnhtcAR-28ppiesubstrate. peptide AURA-T288 synthetic the on ATP P , .5icessand increases 0.05 # P , .5dcessbetween decreases 0.05 nvitro in iaeassay. kinase .COS- y. e

Journal of Cell Science EERHARTICLE RESEARCH 08.Teeoe u eut eadn UAcellular also is AURA PLD2, we specifically Additionally, regarding phospholipase, report. a al., earlier that results this et document with (Rannou now line our in cytoplasm are the localization Therefore, and nucleus 2008). the AURA in the of mitosis, than Overexpression centrosomes, rather PLD2. of is cytosol right inhibition as in the nucleus, 5A, upon in the (Fig. of primarily because, nocodazole proximity localized be with is might close treated AURA very This cells two the a the panels). between that in occurred a symbols) suggest proteins of that (* also to localization cell Results cytoplasmic the and in phenotype. shift arrowheads) a (white migratory suggests cells, and membrane the symbols) (* of plasma cytoplasm regions the non-nuclear included at colocalized which PLD2 be and to AURA both found panels), Following were middle symbols). 5A, nuclear PLD2 (# (Fig. symbols), stimulation and Golgi (+ EGF and nucleus AURA the arrowheads) both at (open colocalized membrane panels), be the left to of found in 5A, absence were (Fig. the shown in stimulation 5A, As Fig. any PLD2. in presented endogenous both data 5 immunofluorescence for and Fig. stained proteins. AURA cells of signaling endogenous images cells both immunofluorescence on the of only depicts focused whether co-fluorescence we establishing cell, showed the in that in interested colocalize PLD2 are 1 and we AURA or As with inhibitor). EGF treated mitosis was or nM untreated 3 microscopy were that either immunofluorescence cells COS-7 cell, using the performed and PLD2 other each within of to patterns each localization proximally AURA activate cellular situated the phospholipase be determine To a could other. proteins and both kinase that a indicates that fact The protein–protein a form Pro and heterodimer (C) colocalize respectively. AURA panels, right and B. bottom PLD2 in and co- left the top in the shown in samples antibodies shown of anti-FLAG are set with AURA each probed using and for immunoprecipitated were PLD2 controls was samples both loading PLD2 when for When protein down controls panel). equal pulled P right Positive e and was (top HA-tagged for panel). inputs PLD2 AURA overexpressed 20 left recognizing immunoprecipitated, among FLAG-tagged (bottom antibodies sample was overexpressed AURA anti-HA each AURA phospholipase, with recognizing for inhibition When the probed images (B) upon were to (I.P.). of cytoplasmic that samples specific co- fields fact when close antibodies for representative shown the very two used as to a are were down due suggest Shown post-transfection pulled be also nucleus. was days might the Results 2 This of phenotype. at 10 (whi proximity panels). migratory and membrane bars: the (right PLD2 a plasma Scale in nocodazole of the visualized. than with included that were the rather treated which stimul that to cytosol at cells, cells sample EGF the cell colocalized the the nM in the of be in 3 localized regions in to proteins Following is non-nuclear found shift two panels). at AURA were a colocalized (left the PLD2 suggests be stimulation between to and and of occurred found AURA absence symbols) localization were Both the (* PLD2 in DAPI). and cytoplasm symbols) using to AURA and (# both specific visualized arrowheads) panels), Golgi antibodies nuclei (middle and using blue min arrowheads) fluorescence) and 15 (black yellow PLD2 for membrane (merged for nuclear together labeling symbols), proteins FITC (+ endogenous AURA, nucleus both for or heterodimer. labeling (green) protein–protein (TRITC PLD2 a protein endogenous form (red), and AURA colocalize endogenous AURA for and PLD2 5. Fig. Xenopus el a endcmne ola oislclzto at localization its to lead to documented been has cells m ooaoe( well-characterized (a nocodazole M m .(,)CS7clswr ihrmc-rnfce rtasetdwt lsisepesn LGAR rHA– or FLAG–AURA expressing plasmids with transfected or mock-transfected either were cells COS-7 (B,C) m. A egdimnfursec irsoi mgso O- el stained cells COS-7 of images microscopic immunofluorescence Merged (A) ugssta UAadPD neati h eli manner a in cell signaling. the EGF in by interact mediated PLD2 is 5 that and Fig. samples AURA from of data that set Overall, suggests each co-immunoprecipitations. for the protein controls in the and loading shown left represents protein top 5C equal the Positive and Fig. in inputs respectively. panel). shown panels, are left FLAG-tagged AURA right bottom and bottom 5B, PLD2 overexpressed both also (Fig. for down phospholipase, controls was pulled antibodies interaction the was using down AURA inverse immunoprecipitated to pulled The was specific PLD2 panel). was When right PLD2 tested. top HA-tagged 5B, the (Fig. to overexpressed specific when HA-tagged antibodies shown, kinase, using (either As immunoprecipitated respectively). was protein AURA, AURA FLAG-tagged overexpressed looking or then companion PLD2 endogenous co- and the respectively) by with PLD2, for or determined performed AURA as (either were proteins PLD2 that and immunoprecipitations AURA between chemoattractant interaction of a that migrate. with as to cells stimulated cells the induce cells to in known in locations similar especially to AURA, localized be to found vrxrse UAfo O- el yae on to 6A, Fig. bound in lysates shown cells As COS-7 from acid. AURA phosphatidic protein–protein the of overexpressed and a product reaction, the form by not mediated PLD did was and we interaction this microscopy cell activity, whether AURA know the AURA influenced positively that in that indicate interaction immunofluorescence colocalize 5 Fig. PLD2 in and shown the data immunoprecipitation helps Although and migration dynamics cell tubulin promote changes AURA and involved also activation is AURA acid, in phosphatidic action, PLD of product The i.5 ute ofrsteeitneo protein–protein a of existence the confirms further 5B Fig. ora fCl cec 21)18 1–2 doi:10.1242/jcs.157339 516–526 128, (2015) Science Cell of Journal fmitosis, of each ation 521 LD2 tein ach te

Journal of Cell Science AIt iulz uli(le.Sonaerpeettv ilso mgsaog2 htwr iulzd cl as 10 bars: Scale visualized. were controls. that and 20 (1 samples among acid sensor images phosphatidic acid exogenous of phosphatidic of fields pEGFP-Spo20PABD presence representative the the are in with or Shown transfected days (blue). 2 were nuclei for cells visualize cultured COS-7 and to cells. overexpression DAPI inside PLD2 of visualized presence effectively or be absence can acid phosphatidic that EERHARTICLE RESEARCH 522 polymerization. tubulin AURA-led acid to function AURA-mediated phosphatidic of that on gain indicates a effect provided 6D any Fig. determined polymerization. had we tubulin acid Next, polymerization. phosphatidic biosensor tubulin whether acid on effect phosphatidic positive inside a detected the be using augment also 6C). cells to could (Fig. able and treated 6B) was (Fig. cells similarly exogenous AURA the Additionally, of interaction to incubation activity AURA. added subsequent for acid specific and protein–lipid control, phosphatidic antibody membrane negative an PVDF a with the a using not through detected (but acid phosphatidylcholine) * phosphatidic acid. phosphatidic exogenous nM 300 of presence or absence the post-trans in days 2 Src Transwells with using combination measured was in migration or cell alone Enhanced (E) AURA AURA. overexpressed purified that recombinant cells of absence for or presence the in polymerization expr are increas and of triplicate effect in Positive performed (B) were Experiments control. AURA. negative purified a recombinant as of used activity was catalytic DOPC on polymerization. mean (PA) tubulin AURA. acid affects overexpressing phosphatidic positively cells of and from concentrations AURA lysates activates and and to (DOPA) binds sn-glycero-3-phosphocholine acid Phosphatidic 6. Fig. ssoni i.2 ti la htAR a significant a had AURA that clear is it 2, Fig. in shown As 6 ...pretg fcnrl * control. of percentage s.e.m. P , .5icessbtensmlsadcnrl.()Imnfursec irsoyo oiiecnrl htindicate that controls positive of microscopy Immunofluorescence (C) controls. and samples between increases 0.05 eut,weeAR an ucinlt ntepeec fSrc of presence 6E). the (Fig. migration in acid cell functionality phosphatidic with and gains agreement AURA in where are results, results these Additionally, he ifrn uoaknssaindwl ihteRaf-1 the with well aligned Raf-1 kinases B with Aurora the along in kinase different regions sequence (AURKC) phosphatidic-acid-binding Aurora of three putative C multiple The partner AURA, kinase 7A). 1996), (Fig. Aurora binding with al., and well-known performed (AURKB) et an were (Ghosh is alignments Raf-1 acid As phosphatidic AURA. region AURA, phosphatidic-acid-binding on to putative binds the acid determined phosphatidic next that we indicates 6 Fig. AURA that in Given region phosphatidic-acid-binding putative A ora fCl cec 21)18 1–2 doi:10.1242/jcs.157339 516–526 128, (2015) Science Cell of Journal A rti–ii vra sasuig1,2-dioleoyl- using assays overlay Protein–lipid (A) m m .()Maueeto tubulin of Measurement (D) m. ,1 i) ie n tie with stained and fixed min), 10 M, P , .5icessbetween increases 0.05 se sthe as essed nthe in fection ing

Journal of Cell Science tr oiin1 n position end position-1; start htaesmlrbtenRf1adAR r eitdb h red the by depicted are AURA proposed and Raf-1 the between similar and are regions that Hydrophobic the Raf-1 AURA. of of region sequences of phosphatidic-acid-binding acid amino comparison region the phosphatidic-acid-binding a and phosphatidic-acid-binding the AURA depicts and of 7B,C Raf-1 Fig. of plots regions 7A). hydropathy Fig. the of between right the towards EERHARTICLE RESEARCH yrpoi mn cd uha ecn n aiersde,o h te ad htcudvr elacmoaebt h eaieplrha n the and head wind polar were negative statistics (A the Query region both (C). phosphatidic-acid-binding accommodate AURA well and very (B) hand could Raf-1 one for on that residues, plots hand, lysine hydropathy other and Kyte-Doolittle the histidine respectively. on arginine, acid, re residues, as homologous phosphatidic such valine The of acids Raf-1. and chains amino of leucine side site charged (PA)-binding as positively hydrophobic acid such between phosphatidic fluctuations acids putative hydropathy the amino with acid. showed hydrophobic proteins phosphatidic (C) AURA binds AURA three that and the Raf-1 (B) of on 1 similarity region substantial unique a a shows with Raf-1 homology and shares AURA 7. Fig. 5 0;qeylength query 403; 389 -W 5 423 0;efcielength effective 403; dre leregion blue (darker ) 5 395. cd akdaehdohbcrgosadsvrlhistidine, several and regions phosphatidic hydrophobic to bind are to likely Marked most 6A) acids (Fig. acid. amino regions amino the blue are light corresponding AURA and blue in the the dark the in in Both arrows sequence. underlined green acid similar the and by are graphs depicted that are hydropathy the regions proteins in basic two red the the in between whereas underlined sequence, are and acid graphs amino hydropathy the in arrows ora fCl cec 21)18 1–2 doi:10.1242/jcs.157339 516–526 128, (2015) Science Cell of Journal A lsaWmlil lgmn fArr iae ,BadC and B A, kinases Aurora of alignment multiple ClustalW (A) wsize ow ini Raf- in gion and , 523 5 9;

Journal of Cell Science xesv eunesmlrt ihRf1(A Raf-1 with similarity sequence extensive EERHARTICLE RESEARCH 524 (E AURA on region as well The as head. acid, polar phosphatidic of negative chain accommodate its side potentially hydrophobic could the that both residues lysine and arginine n ai mn cd r eitdi aet n red, amino and basic charged in negatively phosphatidic the magenta colored to of proximity are in head in close phosphate acid in the is depicted phosphatidic acid that of hypothesize are We tails green). acids the and amino respectively, hydrophobic being basic Raf-1, cyan with aligned with and that yellow acids in phosphatidic amino depicted and comparable is the AURA AURA for K171); in facing and acid R179 shown facing and AURA are acid for coordinates phosphatidic supplementary which PDB in contains found which be of S1, can Multiple Table model material the results to acid. access (full the 8A–D phosphatidic binding Fig. generated, potential and were different AURA solutions many acid, consider between phosphatidic to interactions to us AURA allowed docked which theoretically we phosphatidic software, of site binding the for AURA. candidate to prime acid a it makes sn he-iesoa 3)cmue-eeae modeling computer-generated (3D) three-dimensional Using 389 –W 171 423 –E ,which ), 211 has ) cd,sc sK7 Fg AB rR7 Fg 8C,D). (Fig. R179 hydrophobic the or with interact 8A,B) chains could (Fig. acid acid N fatty phosphatidic hydrophobic K171 in the found that as propose we Additionally, such acids, elmgain ssoni h oe eitdi i.8E, Fig. in depicted model the AURA, in activate shown and enhanced As bind an to migration. to leading and cell able polymerization cell tubulin is maximal rapid causing for acid phosphatidic important Phosphatidic product is reaction also acid. report its that and AURA we mechanism D mechanisms, return, phospholipase third migration: two in a these and, yet to Src here addition In of is FAK. presence migration as activates the cell well on as in AURA tubulin of augmented on effect effect The positive polymerization. its tubulin the increase through robust for migration a shown cell induces has expression in study ectopic had AURA Our it that mitosis. that the time to hinted first in unrelated region AURA role perinuclear of and possible staining Golgi a diffuse the in of and to presence defined cytosol The been already extent. has large mitosis during a role AURA of role The DISCUSSION 192 irto smxmlwe hshtdcai n r r present. are Src and acid cell phosphatidic at and when possibly polymerization maximal AURA tubulin is to induces migration acid. binds AURA phosphatidic acid indicated. mediator phosphatidic region lipid that the the shows of also release study the This study. influence of this still means can in by latter data the AURA from PLD2, phosphorylates evident directly is AURA reciprocal AURA Whereas A and positive acid. PLD2 the phosphatidic between of and activation Model PLD2 for (E) AURA, and K171. between R179 facing interactions facing acid acid contains phosphatidic phosphatidic which and and S1, AURA Table AURA material for supplementary coordinates in PDB the found to access be Full can tails. model acid the magenta, phosphatidic being acids; green, acids; cyan amino R179 amino (with basic hydrophobic acids: AURA red, amino yellow, considered); key code: region two Color alignment for (C,D). modes K171 (B,D) and ribbon (A,B) complex and protein–lipid (A,C) AURA–phosphatidic-acid . surface the AURA in of and structure 3D acid the phosphatidic for of Modeling 8. Fig. -I 193 -L ora fCl cec 21)18 1–2 doi:10.1242/jcs.157339 516–526 128, (2015) Science Cell of Journal 194 eino AURA. of region Solutions

Journal of Cell Science cdadScihbtr,frpsil uuetherapeutic future possible for be inhibitors, could That Src tumor. approaches. primary and phosphatidic AURA, the metastatic known acid of from highly combination a of away with ability accomplished move same the very to this diminish cells, cells also dividing arrest could cell to attempt that enhanced drugs cancer and acid. polymerization phosphatidic that PLD-derived Buschhorn shown tubulin through have McKlveen migration we 2004; Additionally, induced al., 2009). al., al., et AURA et et Tong Baba 2005; 2004; (Gritsko al., al., et activity been et to AURA have Li esophageal, inhibit compounds linked 2003; specifically inhibitory prostate, been and to cancers, and designed ovarian, have colon AURA and activity in breast of enzymatic specifically and overexpression its tumorigenesis, its migration mitosis, Besides of 2014). cell in al., upregulation cancer et role (Do as shown well-defined recently such been has other pathways, in adhesion, AURA signaling for role non-mitotic cell a aligned established, well the is division of analysis interesting. mutational be binding. bind by would to acid acid regions determined phosphatidic phosphatidic of as in ability AURA, the involved of be determination Further also might AURA etn.Test fbnigcudb ttehdohbcand hydrophobic the at be could binding of site occurred The AURA acid between setting. As events (Go AURA. phosphatidic binding using indicate, flotillin-1 date and here to presented protein conducted data been raft the have studies lipid no the 2010), by mediated activity catalytic the PLD2. increases of and PLD2 phosphorylates AURA ARTICLE RESEARCH hs sn MMsplmne ih1%ftlcl eu and serum 37 calf at MO) fetal Louis, 10% St log with (Sigma, at gentamicin supplemented culture mg/ml in DMEM 250 maintained were using VA) phase Manassas, (ATCC, cells COS-7 transfection and culture Cell were reagent [ Plus CA), and Mediatech (Carlsbad, from Lipofectamine Invitrogen was Opti-MEM, from (DMEM) VA); medium (Manassas, Eagle’s modified Dulbecco’s Reagents METHODS AND MATERIALS (E AURA on region hydrophilic hshtdcai aeteptnilt neatwt the with in interact found to chains we potential acid and the R195), fatty N have and hydrophobic hydrophobic R180 acid charged the R179, negatively phosphatidic that the (K171, to acids propose proximity phosphatidic amino relative of close head basic in phosphate the was Using that acid found site. we phosphatidic-acid-binding modeling, Raf-1 3D the with similitude cd.Teeoe eseuaeta lhuhti einon region amino this hydrophobic, although as that well speculate (E as we protein, AURA a basic, Therefore, to involves binds acids. acid process phosphatidic when this that suggests model eino a- (A Raf-1 of region ySKW.TeAR niio lsri,MN83,wsfrom pRK5- was 8237, and MLN sensor) TX). Alisertib, (Houston, inhibitor Chemical acid Selleck this AURA in The (phosphatidic used mycS6K-WT. plasmids pEGFP-Spo20PABD The pcDNA3.1-mycPLD2- WT, AL). pCMV6-mycDDK-AURA, follows: (Alabaster, as were Lipids ECL (T288) study was Polar acid TX), AURA phosphatidic Avanti NJ); for (Lewisville, (Piscataway, from Healthcare GE substrate Bio-synthesis from was peptide from reagent synthetic was the (APSSRRTTLCGT) MA); (Waltham, sAR satre oeuefrtedvlpeto anti- of development the for molecule target a is AURA As cell and cycle cell the in AURA of involvement the Although lhuhi skonta h euaino UK is AURKB of regulation the that known is it Although 171 –E 192 211 -I lge ihtephosphatidic-acid-binding the with aligned ) 193 389 -L –W 194 423 eino UA u proposed Our AURA. of region ,ohrbscaioaison acids amino basic other ), 32 P] 171 nvitro in c T a rmPerkin-Elmer from was ATP –E 211 hthsextensive has that ) nalipid–protein a in ˚ n %CO 5% and C ´ e tal., et mez 2 . ltaaye o h rsneo UAado PLD2. and/or AURA of presence western the and for SDS-PAGE analyses by blot analyzed were samples Immunoprecipitated ape,pae nositlainvascnann cnies Iand II with Scintiverse individual protein. (dpm)/mg into washed containing min cut vials was per and disintegrations scintillation air-dried which using were quantified into paper, Filters placed min. filter 5 samples, for Whatman water P81 running onto spotting agdPD,wse gi n hnicbtdi :00dlto of dilution 1:2000 a in incubated then HA- and for again specific washed buffer blocking PLD2, 1:200 in tagged a antibody in incubated anti-HA-TRITC then Myc- of and PBS for dilution with specific times buffer three washed blocking 1:1000 AURA, a tagged in with antibody incubated anti-Myc-FITC were of Cells in (PBS-T). dilution serum post- X-100 calf fetal Triton h 10% 0.1% using 48 and blocked PBS and permeabilized At PBS paraformaldehyde, in coverslips. X-100 4% Triton 0.5% glass using using fixed onto were plated cells and transfection transfected were Cells Immunofluorescence ATP, mM 0.1 mM MgAc, 8 mM of 10 agarose EDTA, presence mM G the 0.2 1 protein in 7.0, to pH. incubated MOPS-NaOH conjugated were Immunoprecipitates antibodies using beads. anti-S6K immunoprecipitation and for h or used 36–48 were for anti-AURA that cells prepared COS-7 were in lysates overexpressed then was S6K or AURA Either assays Kinase sa a efre sotie ytemnfcue Ctseeo,Inc., (Cytoskeleton, manufacturer the by BSA An outlined free as harvesting. fatty-acid performed to was prior 0.5% assay min in 10 acid days for phosphatidic PBS 2 with in for treated pCMV6-mycDDK-AURA were they with or transfected either were Cells assay polymerization Tubulin of solution PVDF ratio 2.0:1.0:0.8 a a onto in spotted dissolved were were MeOH:CHCl AL) increasing lipids (Alabaster, The Lipids Briefly, membrane. Polar 2002). Avanti was from al., 30 (DOPC) binding 1,2-dioleoyl-sn-glycero-3-phosphocholine or or et (DOPA) protein–lipid 10 phosphate (Dowler 3, detecting (0, described and concentrations preparing previously for as method The assay overlay Protein–lipid 7.8, pH HEPES, mM (5 buffer then lysis and Special h 100 with 36–48 for prepared cells were COS-7 lysates in overexpressed was PLD2 Wild-type co-immunoprecipitation a and blotting using Western software. MetaVue obtained and Culture camera were digital Tissue 6 Photomicrographs Spot 50i Instrument filters. Diagnostics Eclipse and fluorescence Upright medium 100 Nikon DAPI mounting Fluor a Plan Vectashield were a using using Coverslips Microscope, viewed dried. slide air then glass and were rinsed a washed onto were mounted Cells PBS. in DAPI o rnfcin prpit lsiswr nuae ih5 h. 36–48 with for incubated incubated were cells Post-transfection, were cells. the plasmids onto appropriate transfection, For o ihrAR T8)(PSRTCT rSK(RSK2) S6K or (APSSRRTTLCGT) (T288) 30 at AURA (KKRNRTLTK) either for at-cdfe S ouin hnicbtdwt h ellst t4 at lysate cell the with incubated then solution, BSA fatty-acid-free H74 n alws ufr(.%NC,01%Ti-C,0.03% 12000 Tris-HCl, at 0.16% sedimented NaCl, Tris-HCl, and (0.6% 1.6% respectively, buffer LiCl, 7.4), wash (2.1% pH buffer NaCl EDTA, wash and LiCl 7.4) with pH washed then were nuae ihaporaescnayatbd n h lt were blots the and antibody and secondary chemiluminescence. washed by was appropriate analyzed membrane The with temperature. room at incubated anti-AURA h with incubated 1–2 and for TBS-T antibody with extensively washed was then ihSeillssbfe.Lstswr rae ih1 10 with and either treated lysed respectively, were antibodies, and Lysates with anti-FLAG harvested buffer. analysis. were lysis performed Special that blot with cells western AURA-transfected were subsequent or untransfected and experiments SDS-PAGE Co-immunoprecipitation to subjected then ioetmn n 5 and Lipofectamine m i[ Ci m oimotoaaae n .%Tio -0)adwere and X-100) Triton 0.1% and orthovanadate, sodium M 32 P] ora fCl cec 21)18 1–2 doi:10.1242/jcs.157339 516–526 128, (2015) Science Cell of Journal c T n 100 and ATP 3 :H 2 .Temmrn a lce vrih iha3% a with overnight blocked was membrane The O. m lsi piMMfr1–0mnadadded and min 15–30 for Opti-MEM in Plus l ˚ o 0mn ecin eesopdby stopped were Reactions min. 10 for C m fasnhtcppiesbtaespecific substrate peptide synthetic a of M 6 13 i betv n IC RT or TRITC FITC, and objective oil /1.30 m )o ihr1,2-dioleoyl-sn-glycero-3- either of g) nvitro in m grs ed t4 at beads agarose l uui polymerization tubulin m niAR or anti-AURA l g o min. 1 for ˚ and C 525 ˚ C, m l

Journal of Cell Science niio rae,ihbtrwsaddt h otmwl tthe at well bottom 37 were assays at the were migration h that Cell to 1 cells figure. For for added relevant plate. the incubated was 24-well in a stated inhibitor of concentration well treated, lower the inhibitor into placed and nacdcl irto assays migration cell Enhanced 10 of total A (ALB). ) mM nM (20 768 buffer and lysis tubulin NaCl, in mM sonicated 20 were Tris-HCl, Cells USA). CO, Denver, ARTICLE RESEARCH 526 http://jcs.biologists.org/lookup/suppl/ at online doi:10.1242/jcs.157339/-/DC1 available material Supplementary material release. Supplementary immediate for number PMC [grant in Association Deposited number Heart J.G.-C.]. [grant American (NIH) to the Health 13GRNT17230097 of and Institutes J.G.-C.]; National to the HL056653-14 by supported was work This Funding and data the analyzed research, the manuscript. this edited the directed and wrote and data planned analyzed J.G.-C. experiments, manuscript. performed F.S. and K.M.H. M.M., contributions Author interests. financial or competing no declare authors The test. (ANOVA) interests Competing variance of analysis factor single of the Probability by mean assessed the as was presented are Data the analysis N773. Statistical using residue visualized site further S1). active grid Table was an material receptor a conformation (supplementary the of in PyMOL docking study, coordinates site present resulting binding the the The or using In space generated protein. search with the was Docking and a of structure. flexible defining region docked have by restricted the to starts refine ligand Vina to the AutoDock used Vina allows was AutoDock receptor which bonds, ligand. as 2010), rotatable as treated Olson, treated was was and AURA partner and (Trott dockings, protein (Ritchie other HEX conformations the the docked whereas docking all the 100 In at accelerate 2000). molecular arrive to Kemp, HEX to the used (SPF) predicting of parameters were fourier control program docking In polar default both The spherical calculations. charge. docking consideration uses a into it electrostatic is juxtapositions, takes HEX and 3D that database. full-length PDB shape program The the from used. docking model obtained was 2010) were macromolecular to al., diC181-PA et order of (Macindoe both In server structures using HEX Vina. the performed AutoDock structure, AURA were and 6.1 studies HEX docking computer-simulated The docking Molecular 5 at resuspended were cells transfection, Post irto ufr(MMpu .%BA.Clswr ihrvehicle- either 37 were at Cells min 20 BSA). for 0.5% inhibitor-treated plus or (DMEM buffer migration elwr ie o yteadto f4 aaomleye The microscopy paraformaldehyde. phase-contrast 4% by fields. of counted separate addition were bottom five the migrated the by of to cells h migrated of 1 cells for number the fixed and were discarded well were inserts migration, ebae G a iue o3n n500 8- from in nM separated 6.5-mm-diameter, 3 were to a diluted that by was inserts EGF wells Transwell membrane. lower of chambers the upper the msinwvlnt f4040n ihabnwdho 0n na in nm 10 the of bandwidth at temperature. a intervals room with the at 1-min and nm Safire2 nm 410–460 TECAN 20 at of of bandwidth min wavelength a with emission 60 nm 340–360 for of wavelength excitation measured 85 was to added polymerization were lysates cell of P , .5wscniee oidct infcn difference. significant a indicate to considered was 0.05 ˚ ne %CO 5% a under C 6 m ftblncnann ufr Tubulin buffer. tubulin-containing of l ...Tedfeec ewe means between difference The s.e.m. ˚ .Attlo 200 of total A C. 2 m m topee fe cell After atmosphere. elmgainbuffer migration cell l 6 -oepolycarbonate m-pore 10 5 m el/li cell in cells/ml a lcdin placed was l m l Go M. R. Bell, and L. Daniel, A., V. Sciorra, C., J. Strum, S., Ghosh, aide . ards . ektaa,V,Dvge,M .adRitchie, and D. M. Devignes, V., Venkatraman, A. L., S. Mavridis, Li, and G., L. Macindoe, J. Salisbury, D., Papa, L., W. Lingle, J., S. Weroha, J., H. J. Sutterluety, Li, and W. Berger, E., C. Mayer, C., Pirker, F., Jantscher, a,L . hn,Z . hn,L . hn .Y,Shte,H n u,Q Y. Q. Sun, and H. Schatten, Y., D. Chen, S., L. Zhang, S., Z. Zhong, J., L. Yao, rtk,T . opl,D,Pcg,J . ag . u,M,Sely .A., S. Shelley, M., Sun, L., Yang, E., J. Paciga, D., Coppola, M., T. Gritsko, M., El-Shemerly, D., Hess, F., Meggio, A., M. Pagano, O., Marin, S., Ferrari, ioa . uiou . aaaa . auoo . hn,D,Nta M., Nitta, D., Zhang, T., Marumoto, T., Sasayama, N., Kunitoku, T., Hirota, ag .C,Hag .H,Ce,N . hu .K n i,C H. C. Lin, and K. C. Chou, J., N. Chen, H., C. Huang, C., S. E. Lees, Yang, and J. Sheung, W., Korver, W., Seghezzi, O., A. Walter, M. S. Lens, and G. Vader, J. A. Olson, and O. Trott, Guo, M., Wang, Z., Liu, M., Fu, Y., Song, L., Dong, J., Kong, Y., Zhong, J. T., Tong, G. Kemp, and W. D. Ritchie, and S. Dimitrov, S., Dutertre, F., Hans, C., Petretti, B., I. M. Troadec, Y., Vernos, Rannou, and J. Scrofani, R., Green, Pinyol, C., M. Hardy, M., S. Chambers, R., R. Klein, H., Buschhorn, McKlveen ced,F,Psra,G,Pity . eeln .L,DPoiRah .A. A. DePaoli-Roach, L., A. Lewellyn, M., Phistry, G., Pascreau, F., R. Eckerdt, D. Alessi, X., and G. Hua, Kular, B., S., H. Dowler, Pathak, J., A. Klein-Szanto, E., L. Bickel, F., Xiao, R., V., W. T. Sellers, Do, M., Goldstein, U., Rozovsky, H., J. A. Pinthus, B. A., Imhof, Bar-Shira, and B. Hinz, B., Wehrle-Haller, C., Ballestrem, J., G. Kirkner, S., Toyoda, S., Kure, N., Irahara, K., Shima, K., Nosho, Y., Baba, References e,V,Sese V., mez, ´ hshtdcai.Popaii cdrgltstetasoaino a- in Raf-1 and of translocation kidney phosphatidylserine the canine cells. for regulates Madin-Darby domains acid 12-O-tetradecanoylphorbol-13-acetate-stimulated Phosphatidic binding acid. distinct phosphatidic possesses kinase rpisprocessors. graphics W. D. rats. ACI female USA in Sci. cancer breast amplification, and centrosome instability, overexpression, chromosomal Aurora-A by mediates entry Estrogen (2004). S-phase with interferes activities. dependent cells D1 primary Cyclin in diminishing Aurora-A of Overexpression 585-598. 20) uoaAi rtclrgltro irtbl sebyadnuclear and assembly embryos. early microtubule and of eggs, fertilized regulator 1392-1399. oocytes, critical mouse in a activity is Aurora-A (2004). irc,J . ioi,S .adCeg .Q. J. Cheng, and V. S. Nicosia, V., J. Fiorica, flotillin-1. protein raft R. lipid Paciucci, and M. T. Thomson, substrates. A. peptide L. synthetic Pinna, and A. Krystyniak, I rti jb,aerqie o ioi omteti ua cells. human in commitment mitotic for required are Ajuba, protein LIM H. Saya, and K. Hatakeyama, cancer. ovarian Res. human Can. in Clin. BTAK/Aurora-A kinase centrosome of overexpression ucinlipiaino ua eietroiekns,hI,i elcycle cell in hAIK, kinase, serine/threonine human progression. of implication Functional and phosphorylation by regulated degradation. is Aurora2/AIK kinase serine/threonine mitotic and optimization, efficient function, cancer. scoring new a with multithreading. docking of accuracy carcinoma. cell squamous esophageal Res. human of al. et development S. Lu, L., correlations. Fourier domain. N-terminal the of and role interphase: nucleation C. microtubule Prigent, chromosomal in A function. Aurora spindle by phosphorylation cancer. prostate and B. lesions R. PIN Nagle, and D. Bearss, S., fArr A. Aurora of L. J. Maller, and adhesion. and migration cell al. STKE cancer et A. ovarian J. epithelial Ecsedy, M., mediates Oncogene cancer Maglaty, A W., prostate S. kinase advanced O’Brien, C., an Howe, in involved are region xenograft. A. Orr-Urtreger, chromosomal and 20q13 Z. Eshhar, Y., retraction Yaron, tail microtubule-dependent and migration. cell formation control-directed lamellipodia cancer. S. dependent colorectal Ogino, in and instability chromosomal S. Neoplasia with C. associated Fuchs, independently A., Goel, 10 .Bo.Chem. Biol. J. 2002 21) eSre:a F-ae rti okn evrpwrdby powered server docking protein FFT-based an HexServer: (2010). 7304-7310. , ici.Bohs Acta Biophys. Biochim. ora fCl cec 21)18 1–2 doi:10.1242/jcs.157339 516–526 128, (2015) Science Cell of Journal 101 acrRes. Cancer 11 33 pl6. , elCycle Cell .Boe.Sci. Biomed. J. during kinases B aurora and A aurora of Localization (2008). 418-425. , Oncogene 539-549. , 18123-18128. , ,M,Santamarı M., ´, .Cmu.Chem. Comput. J. 9 ur Biol. 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