I 80 Australia. 3800, VIC [PtdIns(4,5) 4,5-bisphosphate phosphatidylinositol PtdIns(3,4) laV Plotnikova V. Olga stability signaling phosphoinositide primary linking to AURKA, with interacts INPP5E ARTICLE RESEARCH ß 364 2014 November 4 Accepted 2014; August 7 Received 1 [PtdIns(3,4,5) (3,4,5)-trisphosphate cilia. primary phosphatidylinositol to function INPP5E linking (mental thereby MORM 2009), and that al., (Jacoby et micropenis) 2009) ( and shown dystrophy al., retinal obesity, et E have truncal retardation, (Bielas polyphosphate-5- studies Syndrome Joubert Recent inositol cause others. in include and mutations that Bardet- Syndrome disorders (PKD), the disease developmental Biedel kidney and polycystic inherited (JBTS), Syndrome of cycle Joubert group division a c cell human 2011), in Nachur of the pathogenesis resorb the and from and underlies (Seeley grow arising which cues environment structures, to dynamic response are cilia Primary INTRODUCTION cilium Primary INPP5E, AURKA, WORDS: KEY ( E 5-phosphatase polyphosphate inositol in Mutations ABSTRACT Ato o orsodne([email protected]) correspondence for Australia. *Author 3800, VIC Clayton, University, Monash Biology, Developmental nvriyo oa oaCt,I 52242. IA City, Iowa Iowa, of University salse ii fe nuto fclaydsseby(aoye al., pre- et (Jacoby of disassembly ciliary stability of induction decreased after cilia exhibit established and have cilia also They shortened 2009). abnormally al., et (Jacoby defects polydactyly closure tube neural kidneys, and polycystic including phenotypes ciliopathic epne ovrossiui(odi ta. 02.INPP5E 2012). and al., cell cilium, et primary mediate (Conduit the that stimuli to various localizes messengers to second responses creating respectively, h ucino NPEi ii sa es atymdae yits by mediated partly least at is cilia AURKA. in with INPP5E interactions that of suggest function and signaling the link phosphoinositide direct and first AURKA AKT- the between establish an findings through These partly mechanism. dependent AURKA, of of promotes stability and downregulation turn in the transcriptional INPP5E which activity, for phosphorylates 5-phosphatase its important increases AURKA is thereby Furthermore, interaction cilia. this that primary disassembly, show ciliary and a we mitosis AURKA, and regulates and INPP5E that between kinase interaction centrosomal understood. well an not syndromes; describe is we biology MORM ciliary Here, and in INPP5E Joubert of role as the however, known the cause hitn .Mitchell A. Christina eateto iceityadMlclrBooy oahUiest,Clayton, University, Monash Biology, Molecular and Biochemistry of Department 05 ulse yTeCmayo ilgssLd|Junlo elSine(05 2,3432doi:10.1242/jcs.161323 364–372 128, (2015) Science Cell of Journal | Ltd Biologists of Company The by Published 2015. NPEi etkonfrisrl nhydrolyzing in role its for known best is INPP5E P 2 n hshtdlnstl4popae[PtdIns(4) 4-phosphate phosphatidylinositol and 2 eateto ptamlg n iulSciences, Visual and Ophthalmology of Department 1 enjnSeo Seongjin , 1 n a .Smyth M. Ian and 3 eateto ntm and Anatomy of Department ,21) iir dysfunction Ciliary 2010). y, loahe Hlernte al., et (Hildebrandt iliopathies Inpp5e 2 en .Cottle L. Denny , 2 / 2 1,3, iedevelop mice P * 2 ]toform INPP5E P INPP5E 3 ]and P 1 ], ) ) aa Conduit Sarah , hshrlto fteplcsi inydsaepoenPD also PKD2 Ca intracellular disease influences central kidney a polycystic plays the of it AURKA-mediated Furthermore, phosphorylation stability. way, cilia this of regulator In a as 2007). role al., et (Pugacheva factor growth stimulation to response in of resorption ciliary body induces basal and disassembly. cilium the the cilia to localizes of AURKA regulation cells, mammalian the in Indeed, in AURKA an for suggest observations role these important and , cilia-associated of function rhlg AK euae h eopino h lgla( structure (a related flagella the distantly of resorption a the regulates CALK, However, ortholog, body. spindle the of stability and assembly and maturation centrosome with associated cilia. primary understood. poorly decreased of very to stability leads is INPP5E stability the of cilia loss of which by regulation mechanism the to Indeed these linked of been none However, have 2012). al., proteins et to (Humbert centrosomal targeting cilium INPP5E the mediate and that CEP164) ciliary and PDE6D several (ARL13B, cells. with quiescent growth-factor-stimulated interacts in INPP5E length cilia and stabilizes structure also INPP5E that suggest observations These 2009). epnet lee oi odtoso usfrmtn Pne al., et in (Pan cilium) mating mammalian for cues the or 2004). to conditions ionic analogous altered respects to some response in is that tblt,w is se hte NPEadAURKA and INPP5E cilia whether regulating asked in first overlap functional we apparent stability, their INPP5E Given with interacts directly AURKA RESULTS ihdseuaino ohpoen n h elsgaigpathways signaling cell control. associated the they and diseases proteins the both understanding of better dysregulation and with to direct crucial normal be first will that under the observations signaling; establish phosphoinositide and stability AURKA studies between link ciliary These conditions. regulating is pathological interaction this for that demonstrate important we and AURKA, and INPP5E characterised. be (Yeh to remain the involved, cilium pathways between signaling inter-relationships the cell the of importantly, cilium and, base for basis disassembly mechanistic the the findings, these Despite at 2013). al., et present Tctex-1 phosphorylated resorption, organism ciliary ciliated highly regulate the found proteins in to of family thought (NRK) kinase NIMA-related are the including that molecules of number a other are there AURKA, from Aside 2011). al., et (Plotnikova eetr ywyo o-aoia G non-canonical its of of way activation of (IGF-1)-mediated by result a 1 receptor, as factor cells mammalian growth in regulated insulin-like be to shown been also has progression G1-S of accelerated surface and the resorption Cilia on organism. cilia the of sets specific of resorption the induce NRKs uoakns sa motn euao fmtss hr tis it where mitosis, of regulator important an is A kinase Aurora nti ae,w eotadrc ucinlitrcinbetween interaction functional direct a report we paper, this In Chlamydomonas 1 adaHakim Sandra , a rvnivlal npeitn the predicting in invaluable proven has 2+ eestruhaclabsdmechanism cilia-based a through levels Tetrahymena 1 enfrM Dyson M. Jennifer , bc Woae l,2008). al., et (Wloga inln n T94- and signaling Chlamydomonas 1 ,

Journal of Cell Science oaiaino UK n iir xnm oaiaino NPE vrapn ihARAsga tbsledo iim(rohas.4 (arrowheads). cilium of end basal at signal AURKA with overlapping INPP5E, of localization ciliary and AURKA of localization oani nylo.Teetn owihec osrc oaie oteclu n id oARAi niae ntergt ,wa idn;+,mdu b medium 10 ++, binding; bar: weak Scale +, (blue). right. nucleus the the an on indicates indicated blue staining is light (DAPI) AURKA in phenylindole to is binds motif and CaaX cilium the red, to in localizes domain construct (IPPc) each binding; catalytic which strong phosphatase to +++, polyphosphate extent inositol The yellow. blue, in in is indicated domain is (PRD) domain proline-rich The htedgnu UK n vrxrse NPEco- INPP5E overexpressed and AURKA established endogenous also We HA-tagged S1A). that Fig. and material (supplementary AURKA INPP5E co-overexpressed AURKA between the also were interactions confirmed to Reciprocal 1A). not (Fig. alone but domain catalytic domain, regulatory HA-tagged N-terminal non- overexpressed its catalytic and AURKA in RFP-tagged that full-length both to showed bound INPP5E experiments cells Co-immunoprecipitation kidney transfected interact. themselves ARTICLE RESEARCH r niae nka h rp niae h ai fpeiiae ottlIP5,qatfe rmaayi fbosfo he needn experim independent three from blots of analysis from quantified Mole INPP5E, I indicated. total whole as to precipitated from blotting of western AURKA ratio endogenous by immunoprecipitate mean the followed indicates the to mutants, graph used deletion The s INPP5E was kDa. is antibody and in material Anti-AURKA INPP5E indicated Starting full-length (D) are panel). FLAG-tagged IgG. alone, left of (anti-GST, vector chain blotting expressing heavy western the lysates the by from cell indicates indicated probed immunoprecipitated asterisk as were was The immunoprecipitates blotted AURKA (Input). and and right (C) His–AURKA, HA–INPP5E the immunoprecipitation. recombinant expressing for and cells control only GST IMCD3 negative or from as INPP5E immunoprecipitated used was was AURKA (IgG) were Endogenous Immunoprecipitates antibody. G (B) anti-RFP Immunoglobulin with indicated. (IP) antibodies immunoprecipitated and the cells with HEK293 in blotted HA–INPP5E with coexpressed were domains INPP5E. 1–131) with acids interacts directly AURKA 1. Fig. that established studies preliminary These 1C). (Fig. GST– recombinant INPP5E purified with co-immunoprecipitated AURKArobustly recombinant purified as direct, interaction was the proteins that these demonstrated between then We 1B). duct (Fig. collecting cells medullary (IMCD3) inner murine from immunoprecipitated 6 ... * s.e.m.; P 2 , bec fbnig F muoloecn eeto fARA(re)adIP5 rd nIC3clsdmntaigbslbody basal demonstrating cells IMCD3 in (red) INPP5E and (green) AURKA of detection Immunofluorescent (F) binding. of absence , .0 oprdt ullnt NPE(Student’s INPP5E full-length to compared 0.001 A F-ue ullnt F)ARAadctltc(a;aioais1243 n o-aayi nnct amino cat; (non non-catalytic and 132–403) acids amino (cat; catalytic and AURKA (FL) full-length RFP-fused (A) t ts) E ceai ersnaino NPEvrat sdfrARAbnigstudies. binding AURKA for used variants INPP5E of representation Schematic (E) -test). m .Ist hwamgiiaino h aa oyadcilium. and body basal the of magnification a show Insets m. oani ED sal omdaeisbnigt AURKA to the between SH3 binding points contact its sites additional the CaaX creating mediate and by of SH3 binding to the enhance presence Whether able 2005). Golemis, the is and (Pugacheva that NEDD9 consistent is showing in This domain studies and 1D,E). sites previous (Fig. the binding binding SH3 with to the enhanced AURKA of domain adjacent presence with CaaX the an interaction although with region, for IPPc motif required the mapped domain CaaX We 2012). minimal a al., phosphatase et and (Humbert polyphosphate sequence ciliary-targeting (IPPc) inositol the domain sites, catalytic direct binding through SH3 two likely most domain. regulatory AURKA, AURKA the with with binding interacts INPP5E NPEcnit fapoierc oanta encompasses that domain proline-rich a of consists INPP5E ora fCl cec 21)18 6–7 doi:10.1242/jcs.161323 364–372 128, (2015) Science Cell of Journal nvitro in itr otiigGST– containing mixture 9 ,6-diamidino-2- ns aashow Data ents. ua masses cular h SH3 the d onon hown western inding; MCD3 . 365

Journal of Cell Science ftepoenbtalaetogtt irp h phosphatase the disrupt to thought are PtdIns(3,4,5) towards all INPP5E Importantly, domain but of 5-phosphatase activity. the activity protein of the parts and different of in binding are changes AURKA these conformational influence INPP5E in specific mutations a could missense whether a (JBTS) tested Syndrome we Joubert from mutation, of derived range D480N not was the with binding and AURKA associated in INPP5E change activity change of bind this altered to version that able from confirm less kinase-dead To was 2E,F). 2002) a (Fig. al., AURKA and et to (Cheetham bind abrogated, (K162D) to also D480N-INPP5E of INPP5E, T288was capacity catalytic the and AURKA on as dependent increase AURKA function, be to 5- not both appeared its interaction did For protein–protein 2007), on 2D–F). activate (Fig. al., dependent phosphorylation to et be INPP5E, (Horan to INPP5E of variant D480N inactive appeared catalytically for a context as activity, phosphatase capacity this signaling but in the INPP5E-mediated INPP5E, the AURKA by downstream Interestingly, directly of from events. induced that phosphorylation not results indicate is results instead in AURKA These 2E,F). of increases (Fig. activation HH3 as concomitant substrate 2D), (Fig. AURKA cells as in AURKA auto-phosphorylation well and AURKA by INPP5E of in coexpression altered increases upon marked demonstrably observe T288did not proteins Although recombinant auto- between 2000). was interactions al., through A phosphorylation et cells. is We (Walter in AURKA activation T288 case AURKA. AURKA the of of was phosphorylation for activation this mechanism whether the determine common affect to not wished next does INPP5E 2C; (Fig. with activity 5-phosphatase its S2B–D). a Fig. with material in caused supplementary AURKA cells decrease HEK293 of INPP5E-transfected dose-dependent inhibition in required as C1368 enhancement inhibitor activity, This catalytic S2A). AURKA Fig. material supplementary hte h iir oaiaino NPEwsdpneton dependent was INPP5E examine of To localization compartment. ciliary subcellular the other showing any whether of consistently in colocalization material negligible proteins cilia, supplementary observed the of we 1F; Importantly, (Fig. end S1B). AURKA Fig. basal with the overlap spatial to extent, lesser determined. be to proper remains to contributing conformation by protein or colocalization enhancing by proteins, ARTICLE RESEARCH 366 the PtdIns(3,4,5) not towards INPP5E was enhanced activity reverse AURKA recombinant phosphatase the with However, incubation reaction 2A). as the (Fig. case, in phosphorylate (HH3) recombinant substrate, INPP5E control H3 a to of histone on activity presence AURKA able affect the not did and was 2A), (Fig. AURKA INPP5E activated Recombinant we al., AURKA, for canonical an et substrate a other (Nikonova performed is INPP5E regulation to whether examine mitotic and To 2013). as disassembly such ciliary functions, of AURKA regulation the relevant substrates to protein was of range wide a phosphorylates AURKA also activity the proteins affect both AURKA INPP5E of and AURKA of between 3A). Interactions (Fig. Fig. localization INPP5E of material absence body the (supplementary in did unchanged this Basal localization but 2011), INPP5E inhibitor, al., S1C,D). et AURKA alter Plotnikova specific 2006; not al., a et employed (Zhang C1368 we activity, AURKA hs bevtosidct ht nioain ietinteraction direct isolation, in that, indicate observations These NPElclzdpicplyt h iir xnm n,t a to and, axoneme ciliary the to principally localized INPP5E nvitro in iaeasyuigrcmiatproteins. recombinant using assay kinase P 3 nvitro in P Bea ta. 2009). al., et (Bielas 3 nvitro in Fg A,we 2A), (Fig. Fg 2B; (Fig. rmrl euae K ciiytruhhdoyi of hydrolysis through activity INPP5E AKT 2009). PtdIns(3,4,5) regulates al., et of primarily 5- (Bielas levels lacking elevated (phospho)-AKT in mutants results phosphorylated JBTS cells HEK293 of in expression activity phosphatase or INPP5E of depletion cell in overexpressing al., reported been et lines has (Liu phosphorylation AKT signaling Decreased phosphoinositide 2008). of studies mediator Previous that downstream 3E). (Fig. shown cells wild-type have of that with compared aeilFg 3) nta,w on that found we experiments Instead, (supplementary block-chase INPP5E in S3B). by expression cycloheximide affected Fig. not but was material this 2000), that indicated al., (Walter AURKA of et decreased degradation proteasome-mediated mutant) through is inactive catalytically 3D). (Fig. 3C; a interfering levels (Fig. protein not small AURKA protein (but by AURKA of overexpression INPP5E of cells Conversely, S3A). amount Fig. IMCD3 material supplementary the in increased levels, of INPP5E protein (si)RNA reflection AURKA of As a regulates protein. cell. INPP5E depletion the actually the within that protein was evidence of AURKA this of further levels activation that total the specific apparent in increases of phosphorylation was AURKA consequence it of Instead, a levels not absolute the was in increase the oaie oteclaybslbd nmmaincls hr it where AURKA cells, mammalian 2009). in al., body basal et ciliary the (Jacoby to previously localises described models hshrlto fARAaelne oicesdcilia 2012), in increased al., Increases et to Plotnikova 2007). linked 2007; al., al., are whereas et et (Pugacheva AURKA disassembly (Pugacheva of proteins NEDD9 phosphorylation with other interactions through and disassembly ciliary regulates Jcb ta. 09.I eu-tre rmr irbat from fibroblasts primary serum-starved In Inpp5e 2009). al., et (Jacoby nueclaydsseby(i.3) nxetdy nlssof analysis in Unexpectedly, to levels 3A). serum AURKA (Fig. total with disassembly treatment ciliary cells. upon induce wild-type exacerbated of was that differential This with compared AURKA phosphorylated eaielvl fIP5 eaoie.T netgt this 4-phosphatase investigate PtdIns(3,4) the of To with levels reduces AURKA metabolites. the which coexpressed by INPP4B, INPP5E way we some of in possibility, mediated levels be might relative interaction and activity o hneteitrcinbtenARAadINPP5E presence and the required AURKA activation only between PtdIns(3,4) that of interaction indicating did but 2I), the AURKA (Fig. of activation change the blocked not we so, doing In 2002). hsooia etn,w bandmrn mroi fibroblasts embryonic a from murine in (MEFs) obtained interactions we INPP5E–AURKA setting, of physiological relevance the INPP5E establish by To regulated are levels AURKA PtdIns(3,4,5) K Kseeae l,20) ossetwt hs previous these with Consistent 2002). al., et (Kisseleva AKT aaiyo NPEt ehshrlt rbn oPtdIns(3,4,5) by to bind AURKA or dephosphorylate the of to upon relied INPP5E activation proteins of the capacity the of interaction changes, physical both the these with and that of INPP5E interact suggested nature disparate to findings the our or Given 2H). 2G) (Fig. (Fig. AURKA phosphorylation to of AURKA unable were versions induce mutants missense mutant JBTS that and demonstrated INPP5E wild-type V5-tagged of Overexpression n ehns ywihARApoenlvl r regulated are levels protein AURKA which by mechanism One ie hs bevtos ehpteie httecagsin changes the that hypothesized we observations, these Given 2 / 2 ora fCl cec 21)18 6–7 doi:10.1242/jcs.161323 364–372 128, (2015) Science Cell of Journal Inpp5e ie eosre nices ntelvlof level the in increase an observed we mice, P P Inpp5e P 3 3 Inpp5e hc sesnilfrteefcieatvto of activation effective the for essential is which , 2 yrlssmdae yIP5 Kseeae al., et (Kisseleva INPP5E by mediated hydrolysis . 2 / 2 Aurka 2 2 Eshv iir iasml defects disassembly ciliary have MEFs / 2 / 2 Inpp5e Inpp5e mro,wihpeooytoemouse those phenocopy which embryos, irbat a infcnl increased significantly was fibroblasts xrsini euae yAT a AKT, by regulated is expression 2 / Kseeae l,20) and 2002), al., et (Kisseleva 2 Es(i.3)idctdthat indicated 3B) (Fig. MEFs P 2 h rdc of product the , Aurka mRNA P 3 .

Journal of Cell Science nlsso rcpttsadlstsfo E23clsc-rnfce ihRPARAadteidctdpopaae.Mlclrmse r indicat are masses Molecular . indicated the Weste (I) and immunoprecipitation. RFP–AURKA for with control negative co-transfected a cells as used HEK293 was from kDa. V5-LacZ lysates Plasmid in and mutants. JBTS precipitates INPP5E of indicated the analysis or INPP5E (WT) wild-type either * H ottlH3(pe ae)ado muorcpttdIP5 oipti ellsts aawr uniidfo he lt n hwtemean the show and blots three phosphorylated from of quantified ratio were the Data indicate lysates. cell Graphs in (F) kinase input phosphorylation. The to AURKA HA–INPP5E. INPP5E of with immunoprecipitated specificity of immunoprecipitation for and AURKA control panel) assess negative (upper to a HH3 used as an total was in used to used fraction was HH3 was second K162D sample A the mutant of HH3. AURKA fraction towards dead a activity and (IP) AURKA immunoprecipitated and was AURKA auto-phosphorylation plasmids. indicated the with transfected h niae rtis ellstswr eaae n rbdwt h nioisidctd lt rmtreidpneteprmnswr quanti mean were experiments the independent show three Data exp from plasmids panel). Blots with (lower indicated. co-transfected antibodies calculated were the cells was with HEK293 AURKA probed (D) of and S2D. separated Fig. phosphorylation were material relative lysates supplementary Cell in shown proteins. are indicated data the Non-normalized cells. the in phosphatases * EERHARTICLE RESEARCH pnARAihbto ihteidctdcnetain fC38i E23clsoeepesn AIP5 repyvco.Dt hwtemean the show Data vector. empty or HA–INPP5E overexpressing cells HEK293 in C1368 of concentrations indicated the with inhibition AURKA upon proteins. both of activity the affect INPP5E and AURKA between Interactions 2. Fig. h oe ae hw nu rtiswsenbotda niae.()Rcmiatatv UK ciae h yrlsso PtdIns(3,4,5) of hydrolysis the activates AURKA active Recombinant (B) indicated. as blotted western proteins input shows panel lower The orsodn opopoyae S–NPE( GST–INPP5E phosphorylated to corresponding ( n P P 5 , , o ahsml) aaso h mean the show Data sample). each for 3 .5(Student’s 0.05 .1(Student’s 0.01 t t ts) GH etr ltaayi flsts()adimnpeiiae H rmHK9 el otasetdwt F–UK and RFP–AURKA with co-transfected cells HEK293 from (H) immunoprecipitates and (G) lysates of analysis blot Western (G,H) -test). ts) eut eenraie otoeo h etrepesn el ordc akrudcue ytepeec fother of presence the by caused background reduce to cells vector-expressing the of those to normalized were Results -test). 6 ... * s.e.m.; , 1 D) uopopoyae p)UK ( (ph)AURKA auto-phosphorylated kDa), 110 P 5 .1 (Student’s 0.014 t ts) C uniiaino NPE5popaaeatvt gis PtdIns(3,4,5) against activity 5-phosphatase INPP5E of Quantification (C) -test). 6 ... * s.e.m.; (A) nvitro In ora fCl cec 21)18 6–7 doi:10.1242/jcs.161323 364–372 128, (2015) Science Cell of Journal , P 0ka n hshrltdhsoeH (phHH3, H3 histone phosphorylated and kDa) 50 5 iaeasy h pe ae indicates panel upper The assay. kinase .0 (Student’s 0.002 t ts) E E23clswr co- were cells HEK293 (E) -test). nvitro in iaeasyt eetAURKA detect to assay kinase c P -[ 3 32 yINPP5E by ]T signal P]ATP idadthe and fied , 6kDa). 16 6 6 ressing s.e.m.; s.e.m.; nblot rn ed 367 P 3

Journal of Cell Science K n UK in AURKA and AKT n NPEwr uniidfo h eut fteeprmn rsne nF aaaeepesda h mean the as expressed 10 are with Data F. h phosphorylat in relative 12 presented the for and experiment treated levels the AURKA then of (G) transfected results kDa. in siRNA the indicated were from are quantified masses Molecular were antibodies. INPP5E indicated and the with performed was blotting Western mean EERHARTICLE RESEARCH 368 INPP5E. by regulated are acetylated levels AURKA 3. Fig. he lt;Dt hwtemean the show Data blots; three * lte sidctd I h ai fARAo hshrltdATt odn oto ci ne ifrn odtosfloigAKAihbto,a inhibition, AUKRA following conditions different under actin mean control the loading show to AKT Data phosphorylated blots. or three AURKA of of analysis ratio from The (I) indicated. as blotted cabe oto iN Sr.Gah niaetertoo UK otblnlaigcnrl uniidfo he lt.Dt hwtemean the show Data blots. three from quantified control, loading tubulin to AURKA of ratio the mean indicate the Graphs show (Scr). Data siRNA blots. control three scrambled of analysis from control, ** fpopoyae p)UK rARAi h aa oyfo he rpiaeasy esrn 0clsprasy aaso h mean the show Data assay. per cells 50 measuring assays triplicate three from body basal the in AURKA or 10 (ph)AURKA bar: phosphorylated Scale of cilia. primary indicate Arrowheads indicated. Inpp5e P P 5 5 .05 D MD el eetasetdadwsenbotda niae.Gah niaetertoo UK otblnlaigcnrl quantified control, loading tubulin to AURKA of ratio the indicate Graphs indicated. as blotted western and transfected were cells IMCD3 (D) 0.0045. .1 frpopoyae UK)ad* and AURKA) phosphorylated (for 0.013 6 2 ... * s.e.m.; / 2 mroi irbatclue yra-ieR-C,addt r rsne stefl hnerltv otewl-yecnrl aaso the show Data control. wild-type the to relative change fold the as presented are data and RT-PCR, real-time by cultures fibroblast embryonic a tbln(ii,rd n AI(N,bu)i rmr irbat from fibroblasts primary in blue) (DNA, DAPI and red) (cilia, -tubulin P 5 .1.()Pamd xrsigH–NPEwr rnfce noHK9 el,adlstswr olce tteidctdtime-points. indicated the at collected were lysates and cells, HEK293 into transfected were HA–INPP5E expressing Plasmids (F) 0.017. Inpp5e +/+ and 6 ... * s.e.m.; Inpp5e P 2 / # 2 P .02(oprdwt cN) E RAepeso eesof levels expression mRNA (E) pcDNA). with (compared 0.0032 mroi irbat.Gah niaetertoo ihrARA hshrltdATo K oatnloading actin to AKT or AKT phosphorylated AURKA, either of ratio the indicate Graphs fibroblasts. embryonic 5 6 .02 ** 0.0012, A muoloecnedtcino UK gen rT8-hshrltdARA(hUK,green), (phAURKA, AURKA T288-phosphorylated or (green) AURKA of detection Immunofluorescence (A) ... * s.e.m.; m K niio K26o MO yae eecletda h niae iepit n western and time-points indicated the at collected were Lysates DMSO. or MK2206 inhibitor AKT M 6 m .Ist hwmgiiain ftecnrsm n iim h rpsso h eaieintensity relative the show graphs The cilium. and centrosome the of magnifications show Insets m. ... * s.e.m.; P P 5 , .1 frARA Student’s AURKA; (for 0.014 0.01. P , .5 C MD el eetasetdwith transfected were cells IMCD3 (C) 0.05. Inpp5e +/+ W)and (WT) t ora fCl cec 21)18 6–7 doi:10.1242/jcs.161323 364–372 128, (2015) Science Cell of Journal ts) B etr ltaayi fSer473-phosphorylated of analysis blot Western (B) -test). Inpp5e 2 / Aurka 2 6 ( ...(he xeiet) H MD cells IMCD3 (H) experiments). (three s.e.m. 2 INPP5E / 2 eedtrie from determined were mro iho ihu eu as serum without or with embryos ) seii iN sIP5)or (siINPP5E) siRNA -specific 6 ... * s.e.m.; Inpp5e 6 s.e.m.; quantified s o fAKT of ion +/+ P 5 0.003, and from

Journal of Cell Science eitd tlati at yATactivity. are AKT regulates by that part, INPP5E mechanisms AKT in that transcriptional least at another by confirmed mediated, levels results using protein material These AURKA results (supplementary S3E–I). 2009) Fig. al., similar Fig. et material (Bertotti obtained AKTiX supplementary inhibitor, We 2010). 3H,I; (Fig. al., S3C,D). manner et the si dependent abolished upon (Pal MK2206 levels AURKA with in MK2206 increase phosphorylation AKT inhibitor of Inhibition AKT well-validated ehns,w xmndARAlvl nIC3clstreated cells IMCD3 si in with levels INPP5E AURKA examined how we mechanism, evaluate further To governs 3F,G). AKT (Fig. decreased by phosphorylation a accompanied in was AURKA and of manner amount time-dependent the AURKA decreased overexpression. INPP5E in INPP5E of following changes Overexpression test time-points dynamic To different the at signaling. examined levels AKT we by that hypothesis, mediated suggesting this were 3B), AURKA (Fig. in phospho-AKT increases and AKT steady-state idns efudthat found we findings, ARTICLE RESEARCH iha nraei ii iasml Pgceae l,2007; al., et (Pugacheva disassembly associated cilia are in phospho-AURKA increase of an levels with cellular in Increases AURKA–INPP5E cilia the primary of in role interaction the of dissection Functional INPP5E Aurka rsrmldcnrlsRAwt rwtotthe without or with siRNA control scrambled or xrsintruhaphospho-AKT-dependent a through expression Inpp5e 2 / 2 INPP5E Eshdicesdlvl of levels increased had MEFs rnfcin natime- a in transfection, ltioae l,21)adsmlrdfcsaeosre in observed are defects similar and 2012) Inpp5e al., et Plotnikova ea yt) nadto oohrcloah features, other to in (and addition cells In kidney cultured cysts). in renal formation lumen and polarity stability. cell cilia controlling an in proteins demonstrating two (supplementary INPP5E, the lacking restore for line cells to role opposing able in cell is experiments stability AURKA cilia IMCD3 of in normal inhibition MLN8237 Thus, the also S4B). Fig. inhibitor, in and material INPP5E AURKA S4A), obtained Fig. depleting another We material 4A). using (Fig. (supplementary INPP5E results of disassembly ciliary loss similar the with and rescued associated 2009) inhibition defects al., AURKA et (Bielas that controls showed wild-type of that with compared K Ponkv ta. 01 n nterglto fprimary of regulation the in and 2009). 2011) al., in al., AURKA frequently et for et role is (Plotnikova (Jacoby described that PKD previously defects the phenotype account cilium into a Taking primary cysts, with renal associated develop also mice ii tblt Pgceae l,20;Ponkv ta. 02,we 2012), al., et Plotnikova 2007; al., et (Pugacheva stability cilia rfldcladssebyuo h diino eu nthe in number vehicle ciliated reduced or serum the (C1368) of confirmed of inhibitor experiment This AURKA addition (DMSO). an control of the absence upon or presence disassembly cilia profiled a hrdfntoa ai oteepeoye,w serum we and phenotypes, wild-type these to from basis fibroblasts functional starved shared a was rmr ii r lokont lyacnrlrl nmediating in role central a play to known also are cilia Primary 2 / 2 ora fCl cec 21)18 6–7 doi:10.1242/jcs.161323 364–372 128, (2015) Science Cell of Journal el Bea ta. 09.T eemn hte there whether determine To 2009). al., et (Bielas cells Inpp5e 2 / 2 Esa – fe eu application serum after h 2–4 at MEFs el ih2 with cells IMCD3 (siINPP5E) siRNA-treated of images Immunofluorescence (B) INPP5E. and AURKA the between of interaction role Functional 4. Fig. el aaso h mean * the show Data per well. numbers spheroid of Quantitative analysis (C) (blue). staining DAPI, DNA membrane); cell (red, E- cadherin E-cadh, cilia); (green, tubulin acetylated AcTub, RNA; scrambled control Scr, culture. in Matrigel grown 3D control, DMSO or inhibitor (Student’s significant not marker body basal and centrosome marker ciliary acetylated the of panels show merged images All control. DMSO of presence 2 the in disassemble to induced were conditions) under low-serum h (72 fibroblasts embryonic and hwtemean Data the assay. show per cells 100 counting undertaken, Three were right. assays the triplicate on graph the in indicated are after treatments indicated and the before times at cells ciliated 10 bars: Scale cilia. primary indicate (blue). Arrowheads DAPI and (red) tubulin P m , UK niio C38 or (C1368) inhibitor AURKA M .1(Student’s 0.01 Inpp5e A ii in Cilia (A) a 2 m tbln(re) the (green), -tubulin Inpp5e / 2 16 AURKA C1368 M m 6 ( 2 .Tepretg of percentage The m. ... * s.e.m.; / 2 t 2 rmr mouse primary ) Inpp5e -test). / 2 P t Inpp5e -test). 6 ieand mice , +/+ s.e.m.; .1 ns, 0.01; (WT) 369 2 c / 2 -

Journal of Cell Science edfradlo htdwrgltsATatvt n feed- reduces a and that activity loop AKT back downregulates that loop feed-forward ciiyadtehdoyi fPtdIns(3,4,5) phosphatase of its hydrolysis increasing the and thereby activity and to this INPP5E, binds and AURKA which Activated phosphorylates determined. by INPP5E be through mechanism to protein remains The latter occurs between the 5A). doing, of (Fig. interaction activation so auto-phosphorylation the in in functional resulting and, AURKA, species the phosphoinositide mediates different of levels ihirglro o-xsetlmn,rnol oriented E-cadherin randomly of expression lumens, polarized of non-existent lack a and or cilia cells of shortened colonies clusters irregular as cell developed three- instead resulting with a and The spheroids in form them matrix. to failed grew Matrigel and (3D) cells dimensional IMCD3 we from features, cellular INPP5E these upon a depleted impacted in whether complex examine AURKA–INPP5E function To the formation. 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Journal of Cell Science h aucit etakteMns ir mgn ltomfrterassistance, their about for Platform discussions Imaging helpful Micro for Monash Wicking the Carol thank We and manuscript. Golemis the Erica to grateful are We Acknowledgements Software) (GraphPad software. InStat (Microsoft) the Excel using or made were significance statistical Student’s two-tailed a using made were comparisons Statistical analysis Statistical ARTICLE RESEARCH 372 T. Sasaki, E., J. Rasko, G., C. Bailey, M., A. Kong, K., Watanabe, A., and K. Horan, Jr N., P. N. Lara, Katsanis, and T. R., Benzing, F., P. Hildebrandt, Purnell, C., P. Mack, J., Heighway, O., Gautschi, L., A. C. Swenson, Mitchell, and B., M. S. J. Dyson, Renwick, E., S. T., Conduit, J. Coll, M., R. Knegtel, M., G. Cheetham, Sztriha, L., Al-Gazali, V., M. Kisseleva, F., Brancati, L., J. Silhavy, E., L., S. Medico, Bielas, D., Torti, F., Galimi, S., Gastaldi, F., M. Burbridge, A., Bertotti, References at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.161323/-/DC1 online available material Supplementary material Supplementary University Monash a and an Fellowship; Fellowship. from Council Future support Research Council acknowledges Medical Research I.M.S. and Australian APP1046174]. Health number National [grant a grant by project supported was work This Funding analysis. data to and contributed writing I.M.S. manuscript on reagents; design, commented provided research C.A.M. and paper reagents; design the provided research to J.M.D. manuscript, contributed and the D.L.C. S.H. on design; S.C., commented research writing; S.S. to paper; contributed the and wrote manuscript data and performed figures experiments, prepared conducted analysis, design, research to contributed O.V.P. contributions Author interests. financial or competing no declare authors The interests Competing for respectively. Australia) inhibitors, of Melbourne, AURKA (University Institute, and James AKT (Bio21 David Ng providing and Dominic assays and phosphatase Australia) with Sydney, help for Ooms Lisa xeietlvle r eotda h mean at significant the considered as were reported values mean are values Experimental ifrnilrcuteto hs -hshtsst h hgctccup. phagocytic the by to phagocytosis 5-phosphatases mediated these 110 3 of recruitment receptor 72-kDa differential the complement not but regulates SHIP1, 5-phosphatase: 5-phosphatase, polyphosphate inositol 72-kDa the by A. C. Mitchell, and 364 Res. R. D. Gandara, ciliopathies. and cilia of regulation 586 the in players new phosphatases; kinase. A. serine/threonine D. oncogenic Austen, an and 2, A. J. Lippke, P., al. Weber, et link ciliopathies. O. E, the R. polyphosphate-5-phosphatase to Rosti, signaling inositol A., inositol Abdel-Aleem, encoding phosphatidyl S., INPP5E, M. in oncogene Zaki, Mutations A., sustain R. to Bayoumi, required L., are pathways Met-activated al. et of P. addiction. B. subset Lockhart, a G., Rolland-Valognes, Only S., Corso, S., Giordano, 4480-4491. , 1533-1543. , 2846-2857. , 14 1639-1648. , c.Signal. Sci. 20) uoaknssa niacrdu targets. drug anticancer as kinases Aurora (2008). 20) euaino camRsiuae phagocytosis FcgammaR-stimulated of Regulation (2007). 2 ra80. , .Bo.Chem. Biol. J. 21) Ciliopathies. (2011). 20) rsa tutr faurora- of structure Crystal (2002). 21) nstlplpopae5- polyphosphate Inositol (2012). P , a.Genet. Nat. .5 l acltosof calculations All 0.05. 6 ...Dfeecsin Differences s.e.m. 277 42419-42422. , 41 .Eg.J Med. J. Engl. N. 1032-1036. , ln Cancer Clin. ESLett. FEBS (2009). (2009). t Blood -test. a,S . ekm,K,Y,H n iln .A. R. Figlin, and and H. Jr Yu, K., L., Reckamp, R. K., S. 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C. and phosphorylation by regulated degradation. is Aurora2/AIK kinase serine/threonine mitotic 5- 3,4,5-trisphosphate phosphatidylinositol SHIP2 activity. on phosphatase lipids anionic of influence cilium. primary the of disassembly Cell Biol. Mol. nstlplpopae5popaaeI niisAtpoenkns B kinase death. Akt/protein cell apoptotic inhibits to IV leads and 5-phosphatase al. phosphorylation et polyphosphate P. PI3K Waring, inositol IA A., class B. through Peters, tumorigenesis promote A., p85alpha activation. H. in Dbouk, mutations Z., Somatic Kan, W., Wang, and instability mouse. ciliary and defects, human signaling in cilium ciliopathies primary Compe cause V., mutations M. INPP5E Kisseleva, E., Pernot, targeting. ciliary 19696. INPP5E S. for Seo, network and C. V. ciiyifune acu inln nkde cells. kidney in signaling calcium influences activity Chlamydomonas. in disassembly flagellar 1366. 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