Journal of Cell Science o:10.1242/jcs.113928 doi: 1333–1344 126, Science Cell of Journal 2013 January 2 Accepted al nooa subtypes with endosomal association early its regulates MTMR2 phosphoinositide 3-phosphatase the of phosphorylation Differential Article Research eodmsegr hshioio -hsht P() and [PI(3)P 3-phosphate phosphoinositol PI(3,5)P active lipid messengers the the dephosphorylate second members substrates, family (MTM) as myotubularin proteins use (PTPs) phosphatases phosphotyrosine-containing tyrosine protein of majority the Although Introduction are signaling endosome and maturation endosome words: on Key effects events. results phosphorylation subsequent these differential together, that combinatorial potential Taken and MAPK-mediated of subtypes. binding and through APPL1 analysis endosomal and regulated compartmentalization general Further Rab5 dynamically regulates between S58A. MTMR2 that shuttling MTMR2 endosomal Ser58 that with the of mediates reveal compared status Ser631 phosphorylation activation of the status ERK1/2 led is phosphorylation in variant the it MTMR2 increase that the phosphorylation-deficient demonstrated pronounced to double mutants and phosphorylation-deficient endosomes this phospho-mimetic double sustained Rab5-positive of a at more expression from when a Moreover, MTMR2 shift recapitulated characterized. to was localization phosphorylating mechanism. was shift MTMR2 feedback for S58A/S631A) localization negative a (MTMR2 MTMR2 ERK1/2 responsible in mutant This an kinase resulted through endosomes. regulated APPL1-positive inhibitors the is proximal MAPK MTMR2 is more multiple of ERK1/2 variant targeting with a endosomal treatment that phosphorylation-deficient including the Surprisingly, signaling, that evidence a suggests endosomal provide Using that which in Ser58, increase now and position activation. that an we sequestration shown and ERK1/2 antibody, cytoplasmic depletion have PI(3)P in phosphospecific-Ser58 its we in for resulting Recently, increase endosomes responsible structures. Rab5-positive significant is endosomal to MTMR2 on Ser58 targets enriched on phosphoinositol (S58A) of lipids MTMR2 position D-3 3,5-bisphosphate of the phosphoinositol towards phosphorylation specificity and with phosphatase [PI(3)P] lipid 3-phosphoinositide 3-phosphate a is (MTMR2) 2 Myotubularin-related Summary Canada N9B3P4, Vacratsis Ontario, work O. Windsor, this Panayiotis to Windsor, equally of and ` contributed University authors Xhabija Biochemistry, *These Besa and Chemistry Bonham*, of A. Department Christopher Franklin*, E. Norah eutn rmls fMM2fnto r elestablished, well are function MTMR2 of loss and from muscles, resulting 2002). to al., et (Boerkoel signaling folded atrophy and nerve weakness abnormally muscle inadequate eventual by sheaths, characterized in myelin mutated disorder, is recessive autosomal MTMR2 demyelinating an encoding 4B, disease gene (CMT) al., the Charcot–Marie-Tooth et example, Laporte 2000; three For al., in et 1996). Bolino mutations 2002; al., function et (Boerkoel of disorders is The loss that 2001). phosphorylation fact mtmr al., phosphoinositol the et by regulating Vanhaesebroeck highlighted 2001; of al., importance transduction et Cullen signal 2005; (Balla, proteins module(s) of signaling phosphoinositol-binding distinct appropriate recruiting containing regulation and interacting and by pathways targeting, membrane trafficking, in roles key vesicular reside play mainly and phosphoinositols structures These endocytic on 2001). al., et Walker 2000; uhrfrcrepnec ( correspondence for Author 03 ulse yTeCmayo ilgssLtd Biologists of Company The by Published 2013. ept h atta ahpyilgclconsequences pathophysiological that fact the Despite ee a enascae ihdsic neuromuscular distinct with associated been has genes 2 Boda ta. 00 i ta. 02 alre al., et Taylor 2002; al., et Kim 2000; al., et (Blondeau ] PL,EK TR,Poponstl3popae I3P noyoi,Phosphorylation Endocytosis, PI(3)P, 3-phosphate, Phosphoinositol MTMR2, ERK, APPL1, [email protected] ) nvitro in iaeasy,clua AKihbtr,sRAkokonada and knockdown siRNA inhibitors, MAPK cellular assays, kinase ptal n eprlycnrle.Hwvr h exact the However, be can controlled. substrates lipid temporally its and toward insight activity valuable spatially MTMR2 provides and how the MTMR2 into regulating of mechanism the targeting in critical endosomal notably increase a most represents an reversible phosphorylation pathways, and Therefore, (ERK). vesicles signaling kinase PI(3)P signal-regulated extracellular receptor of Rab5-positive A factor depletion displays with growth efficient conditions. S58A) in localization growth resulting (MTMR2 endosomal variant cell strong deficient normal highly phosphorylation greater be during may was Ser58 MTMR2 Ser58 (Franklin phosphorylated that at evidence substrates providing phosphorylation 50%, of lipid than suggested that stoichiometry analysis its Functional spectrometry the mass to that Specifically, demonstrated endosomal 2011). 2011). access al., MTMR2 thus et al., modification and regulates et localization this PH-GRAM dramatically the (Franklin to phosphorylation prominent of close MTMR2 a is characterization identify of that Ser58 to domain at spectrometry site mass phosphorylation used laboratory TR sntwdl oaie oteedsmlstructures PI(3,5)P endosomal and the PI(3)P to substrates localized its conditions, widely culture containing not cell poorly is remains typical MTMR2 events under trafficking Intriguingly, in understood. participates MTMR2 how ` 2 eety our Recently, . 1333 Journal of Cell Science muoltaayi,caatrzdisphosphospecificity its characterized analysis, designed using and we antibody, end, phospho-specific immunoblot this polyclonal To a generated event. that endosomal and regulatory mechanisms the critical targeted understanding this to the underlie information kinase(s) key responsible of be the and of measure would evidence PI(3)P, pseudo- Furthermore, indirect endosomal the levels. an PI(3)P this both towards provide reflect MTMR2 then also phosphorylation of would monitor state to Ser58 inactive ability MTMR2 The of 2011). levels al., PI(3)P et substrate of endosomal (Franklin physiological sequestration its to from correlates away MTMR2 strongly Ser58 Ser58 of position Phosphorylation at MTMR2 phosphorylates ERK1/2 PI(3)P. Results of pools a different target presenting to different between compartments endosomes shuttle endosomal can APPL1-rich MTMR2 which to through mechanism MTMR2 the of regulates C- that a Ser631 targeting characterized position at have site we phosphorylation Moreover, terminal via loop. MTMR2 these feedback of negative targeting on endosomal a regulate may APPL1 thus and kinase endosomal of factor loss growth off signaling simultaneous turn of the endosomes. to assembly through switch the molecular signaling PI(3)P in a of levels as role increased are pathways, acts functional vesicles Akt and APPL1 MAPK a Since for 2009). complexes play endosomes al., to et PI(3)P/Rab5-positive Zoncu subsequent known 2010; with canonical and al., coincides et EEA1 the (Swan The of centrally into recruitment 2004). move PI(3)P, maturation al., vesicles of et as levels increased (Miaczynska APPL1 of endosomes marker a stage as shedding early a and as effector proximal serves Rab5 APPL1 a for signaling, as endosomal functioning to of addition regulator In (Zoncu 2009). capability al., signaling the et endosomal Interestingly, factor growth 2009). maturation enhanced endosomal and al., in delay Rab5/APPL1- et a displayed to endosomes (Zoncu Rab5/APPL1 Rab5/ endosomes converted wortmannin, early are inhibitor positive endosomes PI(3)K the early when using that EEA1-positive demonstrated depleted been is a has It PI(3)P represent maturation. (APPL1) endosome initiation early motif and signaling of receptor zipper of attenuation leucine during stage a critical and phosphotyrosine domain domain, homology binding pleckstrin a containing endosomes. protein early and Rab5 to Zerial localize with 2004; to association simultaneously al., require PI(3)P are et effectors and These Schnatwinkel (EEA1) 2001). 2003; events McBride, 1 al., fusion effectors endosome et Antigen for endosomal (Merithew important Endosomal critical factors tethering by Early the essential affected and example, dramatically be For Rabenosyn-5 can the levels. that Rab5 reveals PI(3)P of property al., This activities et 2001). Schnatwinkel endosomal McBride, 2003; and al., Zerial et Merithew 2004; PI(3)P 2000; endosomal al., with et whose (Gaullier association effectors requires Rab5 targeting and including endosome membranes (Barr facilitate endosome to of fusion proteins fusion binding endosome collaborates endosome peripheral Rab5 late numerous Furthermore, with to 2009). Spang, early 2010; Lambright, heterotypic as as fusion endosome well early homotypic including events elucidated. maturation be to remains kinase, responsible the identification including of phosphorylation, MTMR2 of mechanism 1334 nti td,w rvd vdneta R12i h Ser58 the is ERK1/2 that evidence provide we study, this In adaptor the in rich endosomes that shown been has it Recently endosome early of coordinator master a is GTPase Rab5 The ora fCl cec 2 (6) 126 Science Cell of Journal nvitro in AK eepromd olwn h iaeasy h proteins the assay, kinase the family Following performed. were MAPKs MAPK distinct and by of phosphorylated ability be the members. examined to antibody, MTMR2 phospho-specific induce Ser58 testing to towards employed our our step we Ser58 first hypothesis, a 2011), phosphorylating As this endosomes. a As al., by from in release MTMR2 loop MTMR2 et regulate family. may feedback ERK1/2 (Franklin negative activated (MAPK) that activation is and hypothesis endosomes kinase ERK1/2 to localizes the enhances protein MTMR2 of members deficient as phosphorylation activated such kinases directed this mitogen proline by that regulated is results like 2011). al., behaves previous et detected functionally (Franklin MTMR2 MTMR2 our also phosphorylated of supporting mutant antibody of phosphomimetic S58E alkaline phospho-specific that with to MTMR2 the recognition. similar treatment phosphate-dependent readily signal Interestingly, emphasizing while the antibody S58A, MTMR2 ablates MTMR2 The completely type 1A,B). phosphatase (Fig. wild Ser58 detects MTMR2 towards sa mlmnigEK/ stersosbekns o Ser58 for kinase responsible the phosphorylation. as the ERK1/2 implementing decreased assay to significantly compared ( Ser58 position levels control at MTMR2 ERK1/2 of S1). levels Fig. in phosphorylation material supplementary reduction 1D; see effectively (Fig. data, This cells the the reduced of conditions in greatly quantitation ERK1/2 EGF for and phosphorylated These active minutes levels of 10 protein levels lysis. the ERK1/2 a total by low cell down followed under knocked before hours grown 6 were stimulation HEK293 for siRNA (siRNA). conditions ERK1/2 RNA serum with interfering transfected have small ERK1/2 cells using of levels reduced expression whose been cells in Ser58 MTMR2 vitro recombinant neither contrast, Ser58 at In p38 MTMR2 panel). nor phosphorylate upper JNK1 our both 1C, effectively using (Fig. that could determined Ser58 analysis ERK2 was immunoblot and It ERK1 1C). by (Fig. determined antibody MTMR2 phospho-Ser58 was of phosphorylation Ser58 and at SDS/PAGE by separated were MK niio 02,t nii h R12ptwy this pathway, ERK1/2 2F,H). the kinase (Fig. inhibit MAPK/ERK MTMR2 the to of with U0126, pattern treated inhibitor were localization (MEK) cells the pattern when 2011). punctate alter However, al., endosomal not et an did (Franklin to the MTMR2 with 2002), Consistent unphosphorylated al., al., by et when Kim et exhibited pattern 2011; al., a Laporte cytoplasmic et of Franklin diffuse 2003; occurrence 2003; al., typical the et by (Berger the phosphorylated evident from be shift would MTMR2 This MTMR2. of activatio type ERK1/2 wild regulator of examine a to attenuation inhibitors as MAPK if various ERK1/2 using cells of HeLa in experiments immunofluorescence potential performed we the localization, into endosomal MTMR2 insight obtain To endosomal to MTMR2 structures targets ERK1/2 of Inhibition eobnn TR a uiidfo atra ellysates cell bacterial from purified was MTMR2 Recombinant MTMR2 that suggesting motif, SP so-called a in resides Ser58 hl h iaeasy eosrt ietphosphorylation direct demonstrate assays kinase the While nvitro in twsipratt xmn h hshrlto ttsof status phosphorylation the examine to important was it , nvitro in , 0 euto) ossetwt our with consistent reduction), 50% iaeasy sn omrilyaalbeactive available commercially using assays kinase . a eeal odtcal hshrlt MTMR2 phosphorylate detectably to able were nvitro in iaeasy,ihbtr oJKadp38 and JNK to inhibitors assays, kinase nvitro in ol le h oaiainof localization the alter would n iaeasy,adusing and assays, kinase nvitro in kinase in Journal of Cell Science eetdb hshrlto eeso R12(i.3; (Fig. increased conditions, downstream also these a levels ERK1/2 Under phosphorylation of (pS6K). Ser58 of those kinase ERK1/2, MTMR2 and S6 levels substrate, S3) activated period ERK1/2 Fig. phosphorylation transiently starvation material supplementary by stimulation serum 30-minute EGF detected a MTMR2 3A). (Fig. conditions, which following Ser58, position control at following decreased phosphorylation Under of phosphorylation levels 3). high Ser58 (Fig. displayed to and on inhibition EGF effects with MEK leads the treated results, for were immunofluorescence MTMR2 examined turns expressing the cells complement and HEK293 in To (Bardwell 2006). substrates target Shah, which downstream by of activated phosphorylation (pERK1/2), as becomes such ERK1/2 factors (EGF), phosphorylation growth factor including growth stimuli epidermal various to response In reduced greatly inhibition is ERK1/2 Ser58 following at MTMR2 of Phosphorylation Ser58 preventing by presumably phosphorylation. pathway targeting ERK1/2 endosomal the MTMR2 endosomal of inhibition to that localization results indicate These MTMR2 the S2). Fig. to of material (supplementary analyzed structures shift punctate we analogous similar we cells, a when HeLa pattern siRNA-treated observed punctate ERK1/2 Moreover, in MTMR2 localization 2D). of endosomal (Fig. localization S58A a to MTMR2 exhibiting MTMR2 structures, re-localized dramatically nvivo in , elevates 2-fold bevdices nSr8popoyainwsstrongly was the with 3). phosphorylation pre-treated (Fig. were inhibitor cells Ser58 MEK stimulated this EGF Importantly, in when 3B). attenuated (Fig. increase samples untreated observed the to compared atr ihasbe fvsce htwr oiiefrboth for Rab5 positive of extent were The panel). that middle vesicles were 4A, punctate (Fig. of cells Rab5 a subset and displayed When MTMR2 a MTMR2 panel). inhibitor, with any MEK pattern upper of the lack 4A, with and (Fig. treated region perinuclear localization the Rab5 in intensity greater pattern for staining with cytoplasmic Untreated, examined characteristic Rab5. the and marker displayed 4; endosome MTMR2 inhibitors (Fig. early MAPK the inhibitors with various colocalization were with MAPK MTMR2 expressing treated cells different we were of HeLa S4). to pattern Fig. Thus, material proline-directed localization response supplementary event. endosomal in may a the phosphorylation member MTMR2 examining family in in this 2008). MAPK interested a regulate al., resides that et also suggesting also a (Dephoure site consensus as Ser631 MTMR2 Ser631 on Interestingly, mapped site have phosphorylation studies phosphoproteomic APPL1 Recent to MTMR2 targets ERK1/2 endosomes and JNK1 of Inhibition oetyrgltn h nooa oaiaino MTMR2. of localization endosomal and the Ser58 position regulating a at potently as MTMR2 phosphorylating ERK1/2 of implicate capable strongly kinase studies, knockdown ERK1/2 the hs eut,tkntgte ihour with together taken results, These TR agt oAP1edsms1335 endosomes APPL1 to targets MTMR2 fvrosknss(o) 200 (top), kinases various of hshrlto fSr8MM2wsdetected was MTMR2 Ser58 of phosphorylation o 0mnts ye n nlzdby analyzed analysis. and immnunoblot lysed EGF minutes, ng/ml 10 5 for with stimulated and were Materials Cells in Methods. described as siRNA cultured ERK1/2 and with MTMR2 or control siRNA with scramble transfected were MTMR2 ( expressing served controls. which loading levels, additional kinase as total for probed were were with levels determined MTMR2 Total antibody. pSer58 using nuae t30 at incubated 5 contained substrate the the in was MTMR2 recombinant ( bacterial untreated. UT, Ser58 pSer58). at (IB: MTMR2 phosphorylated and MTMR2 FLAG) total (IB: for probed and immunoprecipitated FLAG phospho-variants, MTMR2 pSer58 ( with antibody. probed and on membrane spotted nitrocellulose and phosphatase and alkaline with without incubated were peptides phosphoSer58 ERK1/2. by MTMR2 1. Fig. nvitro in nvitro In B E23clswr rnfce with transfected were cells HEK293 ) m frcmiatMM2 0 ng 100 MTMR2, recombinant of g AKkns sa.Ec reaction Each assay. kinase MAPK ˚ o 0mnts The minutes. 30 for C hshrlto frecombinant of phosphorylation a MM2atbd.Reactions antibody. -MTMR2 nvitro in ( A hshSr8adnon- and PhosphoSer58 ) m C iaeasyand assay kinase T n was and ATP M uiidHis Purified ) D E 9 cells 293 HEK ) 6 Journal of Cell Science el xrsigMM2srnl ooaie ihAP1when APPL1 with colocalized HeLa strongly 4B, MTMR2 Fig. expressing for in positive cells shown were As inhibition co-immunofluorescence. MAPK using double APPL1 to response endosomes in MTMR2-positive observed enlarged Thus, the 2009). whether al., of investigated et we reversion (Zoncu enlargement in endosomes vesicle APPL1 accompanying resulted preceding with their endosomes to endosomes. early back early endosomes the PI(3)P/Rab5 PI(3)P-positive at to depletion route PI(3)P en of downstream pits resides clathrin-coated that subpopulation endosome transient the partial early as a S5). phenotype Fig. in similar material resulted a (supplementary alone Rab5, MEK1/2 inhibitor and MEK Additionally, and MTMR2 S1). p38 of colocalization of Table inhibition material 4A, combined supplementary (Fig. 2011). enlarged panel; noticeably al., were bottom and et colocalization Rab5 completely (Franklin of were void vesicles vesicle these pronounced However, a pattern. same displayed punctate MEK1/2 MTMR2 the and JNK1 of of or inhibition Combined adjacent sides directly be opposite to seem on and staining S58A colocalized MTMR2 the with where observed Rab5 that to similar was colocalization 40 at (JNK) SP600125 and minutes MEK 30 the 50 for with at treated (ERK1/2) and U0126 S58A inhibitor FLAG-MTMR2 or FLAG-MTMR2 of with targeting subcellular regions. induces punctate pathway to ERK1/2 MTMR2 the of Inhibition 2. Fig. 1336 neetadaepeetdi xaddves mgswr olce using collected were Images of views. regions expanded indicate in 63 Boxes presented 568. are Fluor and Alexa interest using (red) FLAG-MTMR2 for 6 eety PL otiigvsce eesont eiean define to shown were vesicles containing APPL1 Recently, betvs cl as 15 bars: Scale objectives. ora fCl cec 2 (6) 126 Science Cell of Journal m ( A m. m – o or B050(3)a 20 at (p38) SB203580 hour, 1 for M J eaclswr rninl transfected transiently were cells HeLa ) m o or el eeprobed were Cells hour. 1 for M m M oprsn indicated. comparisons xeiet r ie,wt h ausrpeetn odcag eaieto relative * change inhibitor. fold without representing (s.s) values control the serum-starved with given, western Means are Representative A. experiments (IP). in FLAG shown total are to blots normalized and ImageJ using G o r3 iue t37 at ng/ml minutes 5 30 with or downregulate stimulation 5 to by for U0126 followed EGF inhibitor indicated), for MEK (where (S.S) the activity starved with ERK1/2 serum treated were then Cells minutes hours. 30 42 for FLAG-MTMR2 with rnfcin ees oa R12 ci n LGlstssre sloading as served lysates FLAG ( and controls. equal actin confirm ERK1/2, to Total used levels. was transfections FLAG-IP MTMR2. of Ser58 (IB) of immunoblotting phosphorylation FLAG-MTMR2 for probed then (IP), immunoprecipitated ucaesann atr htwsdvi fAP1co-staining APPL1 a of in devoid inhibitors. resulted was 4B). JNK1 alone that (Fig. pathway and pattern ERK1/2 MEK1/2 staining the the punctate of with inhibition treated However, were cells the ikae n hbln 01.Ntby eosre a observed levels we phosphorylation Notably, 2010; Ser58 al., MTMR2 2011). et in Levin decrease Chibalin, 2003; to significant shown al., and been et also (Hu has Pirkmajer activation starvation serum MAPK types, inhibit cell variety a In Rab5 endosomes to APPL1 localization and MTMR2 promotes starvation Serum Ser58 in decrease a causes pathway ERK1/2 phosphorylation. the MTMR2 of Inhibition 3. Fig. B TR e5 hshrlto a uniidb densitometry by quantified was phosphorylation Ser58 MTMR2 ) ( A 6 ˚ E23clswr rninl transfected transiently were cells HEK293 ) .Clswr ye n FLAG and lysed were Cells C. ..o h eut ftreindependent three of results the of s.d. P , .5 ** 0.05, P # .1frthe for 0.01 Journal of Cell Science eemn h eainhpwt epc oteNtria and N-terminal the to and respect treatments with inhibitor MAPK relationship our the complement determine to effort targeting an endosomal In APPL1 MAPK-mediated in and results Ser58 Ser631 positions that at MTMR2 Phosphorylation-deficient hypothesis MTMR2. of targeting the endosome regulates while phosphorylation supporting conditions similar S7). under further Fig. also performed Ser58 analysis material phospho MTMR2 low MTMR2 immunoblot the supplementary these complement of results 5B; under our (Fig. Collectively, subset endosomes conditions a positive serum APPL1 Furthermore, (supplementary with level S8). colocalizes observation endogenous Fig. This the S7). at material serum Fig. recapitulated material also (quantitation upon supplementary was 5A) data, (Fig. Interestingly, the endosomes of of positive colocalization activation. Rab5 significant with observed MAPK MTMR2 we low conditions starvation to compartments endosomal due to conditions to localization starved MTMR2 targeting serum enhance our would MAPK- its that postulated impedes Since we endosomes, MTMR2 S6). early of Fig. phosphorylation material serum mediated observed (supplementary from also also MTMR2 cells endogenous that We analyzed starved conditions 3A). we (Fig. when serum pattern activation low this ERK1/2 under low grown exhibited were cells when ohMM2adAP1wr nagdol hnMTMR2 when only enlarged were for positive APPL1 were and of which MTMR2 vesicles analysis those both that Quantitative alone revealed APPL1 S1B). area of vesicle those Table to material size 6B, in (Fig. (supplementary comparable and S10) were APPL1 Fig. vesicles material Notably, in these (supplementary occur this for S1). not (S58A/C417S/S631A) enlarged did residue Table enlargement catalytic positive the being material of mutation only supplementary upon vesicles views; those colocalized instead enlarged to the we with comparison colocalized Intriguingly, with strongly 6B; S58A/S631A S9). APPL1, (Fig. MTMR2 Rab5 double that Fig. with colocalize inside observed the not the material did on but just supplementary expressing periphery localized cell be a the to cells of appeared displayed that with HeLa pattern (S58A/S631A) colocalization punctate mutant contrast, significant any phosphorylation-deficient In display vesicles, positive not APPL1. MTMR2 Although of did S9). amount APPL1 Fig. MTMR2 significant S58A with material a supplementary colocalize observed MTMR2 initially we panel; also we top that could thus 6A S58A 2011), (Fig. shown MTMR2 al., et has if (Franklin analyzed or work and Rab5 APPL1 with Ser58 previous with colocalizes colocalize at Our to generated ability mutants Rab5. their we examined site and MTMR2, phosphorylation Ser631 of double sites MTMR2 phosphorylation C-terminal TR agt oAP1edsms1337 endosomes APPL1 to targets MTMR2 betvs cl as 15 63 bars: using Scale collected are were objectives. and Images views. colocalization expanded vesicle in and of presented colocalization lack of indicate regions arrows indicate open APPL1 arrows endogenous white or Solid (red) (A) (B). MTMR2 (green) FLAG Rab5 for endogenous probed and SP600125 were with Cells combined panel). inhibitors: or (bottom following panel) the (middle with alone treated U0126 or were panel) Cells (top microscopy. untreated immunofluorescence hours by 42 analyzed for and FLAG-MTMR2 with transfected transiently targets were pathways endosomes. JNK APPL1-positive and to ERK1/2 MTMR2 of Inhibition 4. Fig. m m. ( A , B eacells HeLa ) 6 Journal of Cell Science htsest emdae ydpopoyaina position at dephosphorylation by inhibitor mediated MTMR2 MTMR2. occurrence MAPK be of of an Ser631 report to double endosomes; first seems early the that the APPL1-positive is to this with localizing together, seen Taken positive treatment. observations MTMR2/APPL1 the of enlargement the observed with contributes vesicles. MTMR2 consistent the of material are activity to phosphatase findings (supplementary the suggestive that intact hypothesis These was S1). activity Table catalytic PI(3)P 1338 motnl,tedul hshrlto uatrecapitulated mutant phosphorylation double the Importantly, ora fCl cec 2 (6) 126 Science Cell of Journal ehshrlto fSr3 sntsfiin omediate punctate characteristic to the display sufficient contrast, did In mutant not phosphorylated. S58A/S631E is the is Ser58 of when mutant Ser631 targeting endosomal S58E/S631A that of suggesting the pattern, punctate 7, dephosphorylation significant Fig. any in lacked MTMR2 shown endosomal for As if sufficient unclear is localization. is Ser631 it position however, at targeting; dephosphorylation Ser58 endosomal at MTMR2 dephosphorylation in that results determined previously have We regulates shuttling Ser631 endosomal of phosphorylation endosomal while general association regulates Ser58 of Phosphorylation 15 were Images 40 views. using expanded collected in of presented Regions are colocalization. interest mark of arrows devoid open vesicles and smaller of vesicles regions enlarged indicate on arrows colocalization Solid and (green). (red) Rab5 FLAG-MTMR2 endogenous for S58A/S631A probed with and transfected phospho-variant APPL1 were and Cells (red) (B) FLAG-MTMR2 (green). for probed phospho-variants were S58A/S631A and or S58A with were transfected Cells by (A) analyzed microscopy. and immunofluorescence hours 42 FLAG-MTMR2 for with phospho-variants transfected transiently were endosomes. cells APPL1 regulates to Ser631 localization and MTMR2 Ser58 of Dephosphorylation 6. Fig. TR ihAP1pstv nooe.Mre images FLAG- Merged of endosomes. colocalization APPL1-positive the with in MTMR2 increase starvation an Serum in (green). resulted FLAG- APPL1 with and probed (red) were MTMR2 Cells (B) Rab5- endosomes. with an positive FLAG-MTMR2 in of colocalization resulted the starvation in Serum increase (green). FLAG-MTMR2 Rab5 for and probed (red) were serum panel) and (lower panel) cells (top starved cells Untreated immunofluorescence by (A) analyzed microscopy. and hours FLAG- 42 with for transfected MTMR2 transiently were cells endosomes. HeLa APPL1-positive and of Rab5 targeting to subcellular MTMR2 induces starvation Serum 5. Fig. betvs rosidct ein fclclzto.Scale 15 colocalization. bars: of regions 40 indicate using Arrows collected objectives. in were represented Images are views. and expanded colocalization of regions display m m. m m. 6 i n 63 and oil 6 betvs cl bars: Scale objectives. ( A 6 , B oil HeLa ) ( A , B ) Journal of Cell Science euaeteeryedsm utp cesbet MTMR2. to to seems accessible subtype Ser631 endosome position early the at endosomal regulate to phosphorylation Ser58 localize that bottom while to notion MTMR2 structures, 7A, the for (Fig. support unphosphorylated be variant further must results prominent active these APPL1 catalytically Thus, observed panel). with the Rab5 we to colocalize and However, S58A/C417S/S631E compared not MTMR2 panel). between did colocalization bottom also to 2001). 7B, mutant ability (Fig. As McBride, the this endosomes. reassessed APPL1-positive 2003; expected, mutant and and and Rab5-positive inactive al., background to Zerial phosphatase colocalize Rab5-positive S58A/S631E et MTMR2 the 2004; a to Merithew in created al., 2000; we localizing Therefore al., et Rab5 for et many Schnatwinkel since PI(3)P (Gaullier MTMR2 endosomal endosomes of require activity to phosphatase effectors the MTMR2 to due targeting for sufficient of structures. dephosphorylation is that at with argue colocalization mutations results As Ser58 of These S11). regions point 7B). any Fig. (Fig. observe single APPL1 material not the did supplementary we 7A; with expected, (Fig. observed (S58A) extent that Ser58 an to to Rab5 with similar colocalization partial and pattern staining epsuae htteprilclclzto ihRb a be may Rab5 with colocalization partial the that postulated We nooe yMM2S8/61 a aentbeen EGFP-2 the not that have fact may the to S58A/S631A due MTMR2 resolved by is early endosomes positive phosphorylation Rab5 at Ser631 depletion PI(3)P endosomes. efficient and for necessary that dephosphorylation of suggest results lack These and panel). Ser58 pattern top 8A, staining (Fig. punctate EGFP- colocalization the any by with displayed mutant as co- the marker contrast, S58A/S631A In MTMR2 of 2011). 2 the al., S58A et loss MTMR2 of (Franklin for marker expression panel) previously significant PI(3)P bottom observed the 8A, had a (Fig. we for what pattern to in similar or staining S58A/ resulted MTMR2 phosphatases punctate 2000). characteristic lipid expression cytoplasmic al., by et to S631E depleted Rab5- (Gillooly is re-distributes to treatment localize wortmannin PI(3)P and largely when endosomes to structures thought early well is a EGFP-2 positive protein of marker, chimeric dispersion protein the This binding structures. analyzing PI(3)P endosomal by from characterized accomplished levels was was mutant PI(3)P This S58A/S631A depleting the of if capable examined we endosomes, early 6 ti osbeta elto fP() nAPPL1-positive on PI(3)P of depletion that possible is It APPL1-postive on MTMR2 of function the define further To YErvae oefc ntelclzto ftePI(3)P the of localization the on effect no revealed FYVE TR agt oAP1edsms1339 endosomes APPL1 to targets MTMR2 niaevsce odo ooaiain mgswr collected were 40 Images using colocalization. of arrows void open vesicles vesicles, indicate colocalized indicate (green). arrows APPL1 Closed endogenous and (red) phospho-variants MTMR2-Rab5 in ( (S58A/ increase colocalization. activity an catalytic in of results Loss C417S/S631E) MTMR2 (green). FLAG Rab5 for and probed (red) and hours 42 for phospho-variants of phosphorylation regulated and Ser631. is Ser58 MTMR2 of of dephosphorylation targeting by endosomal Rab5 7. Fig. 6 ( A i n 63 and oil eaclswr rnfce LGMTMR2 FLAG transfected were cells HeLa ) B el eepoe o FLAG-MTMR2 for probed were Cells ) 6 betvs cl as 15 bars: Scale objectives. 6 YEmre is marker FYVE m 6 m. FYVE. Journal of Cell Science obepopoyainst uatwudfrhrenhance further would vesicles. mutant these from signaling site endosomal phosphorylation to this of MTMR2 expression double targets that signaling Considering postulated Ser631 we 2009). and endosomes, key APPL1-positive Ser58 al., of at et dephosphorylation (Zoncu activation that ERK1/2 and including signaling pathways in resulting receptor endosomes, APPL1 residency extended it within prolongs receptors Interestingly, depletion factor 2011). growth PI(3)P of al., that et demonstrated particularly (Franklin been events has ERK1/2 and signaling to of Ser58 respect enhanced targeting at with in dephosphorylation endosomal results through that subsequently mediated determined is have MTMR2 we Previously activation ERK1/ increases 2 endosomes APPL1 to localization PI(3)P MTMR2 of levels stage. low endosome APPL1- early the proximal deplete to this to localize within order may in MTMR2 showing endosomes with that positive consistent vesicles hypothesis is supplementary pattern working of 8B; colocalization our (Fig. This subset S12). marker Fig. a PI(3)P material the displayed (supplementary S58A/C417S/ as with S13), endosomes colocalization Intriguingly, marker APPL1 Fig. to panel). PI(3)P material localizes lower the which S631A, 8B with S58A/C417S/S631E (Fig. colocalization MTMR2 expected displayed complete endosomes) inactive Rab5-positive nearly to the localizes PI(3)P (which Also, mutant 8B; of material (Fig. S13). (supplementary levels endosomes Fig. that Rab5-positive APPL1 detectable to determined compared functional EGFP-2 contain we the and to do a First accessible endosomes S12). establishing MTMR2 Fig. APPL1 material for between would supplementary sensitivity variants MTMR2 relationship greater that inactive proposed achieve phosphatase we the Thus, endosomes. examining Rab5-positive on enriched 1340 ora fCl cec 2 (6) 126 Science Cell of Journal 6 YEmre,abi tlwrlevels lower at albeit marker, FYVE motn oeo eesbeps-rnltoa oiiain in modifications of post-translational phospho-isomers reversible the of that illustrates subtypes role study endosomal important different this in for endosomal to in localize differences and MTMR2 provides finding rates, to The maturation presumably contributes signaling. endosome This likely destination, that cargo 2006). diversity 2006; al., al., functional et et (Hayakawa surface vesicle phosphoinositol Lakadamyali the and are on protein endosomes composition their early both lipid that of apparent terms become in has heterogeneous it years, recent In APPL1- Discussion of properties signaling APPL1 endosomal endosomes. to positive the MTMR2 Ser631, enhance Altogether, of and Ser58 may targeting 9B). at (Fig. dephosphorylation the by S58A mediated that S58A/S631A endosomes, suggest and expressing results wild-type cells these both for to 60-minute respectively, and compared 15–30 the periods, at difference 80% time there and that 50% a revealed nearly MTMR2 was analysis with densitometry seen activation Accompanying in MTMR2 surprising ERK1/2 S58A. activation of transient mutant the More ERK1/2 to phosphorylation cells sustained compared double S58A. enhanced of the to expressing duration Remarkably, further cells the compared was displayed 9B). ERK1/2 however, (Fig. of S58A/S631A activation wild-type levels with 9A). to (Fig. down transfected with back off wild-type tapered (5-minute consistent eventually cells wild-type which to to period), compared with in time S58A increase compared with observed 40% activation approximate ERK1/2 an as was revealed analysis ERK1/2 Densitometry S58A EGF. of in expressing with increase phosphorylation reproducible stimulated a findings, the were previous our which S58A with MTMR2 Consistent S58A/S631A MTMR2, MTMR2 type wild or expressing cells HEK293 in ots hshptei,w xmndteatvto fERK1/2 of activation the examined we hypothesis, this test To ooaie eils pnarwidct eilsvi of void vesicles indicate arrow open indicate vesicles, arrows colocalized Closed panel). early (lower PI(3)P-rich endosomes to at localizes Phosphorylation (S58A/C417S/S631E) panel). Ser631 (top endosomes colocalization PI(3)P partial with in results MTMR2 of dephosphorylated Loss of (B) (S58A/C417S/S631A) panel). activity (lower catalytic endosomes early PI(3)P at induces depletion S58A/S631E panel). MTMR2 (top of endosomes Expression PI(3)P-rich to (S58A/S631A) localization Ser631 prevents and Ser58 at MTMR2 dephosphorylation (A) microscopy. and immunofluorescence hours by 42 analyzed for phospho-variants (red) EGFP-2 FLAG-MTMR2 with co-transfected were cells betvs cl as 15 bars: Scale objectives. 40 using collected were Images colocalization. i.8 nuil I3Pdpeina al nooe is activity. endosomes catalytic early MTMR2 at on depletion dependent PI(3)P Inducible 8. Fig. m m. 6 YE(re)and (green) FYVE ( A 6 , B i n 63 and oil HeLa ) 6 Journal of Cell Science ** emns essetta hshrlto umnso attenuates or C- augments its phosphorylation that in suspect domains we terminus, interaction in protein–protein MTMR2 C- interested Since multiple the specificity. possesses subtype of also mediates prevents phosphorylation site are which Ser631 potently terminal by so We mechanisms mass the Ser58 of localization. examining of structural details endosomal phosphorylation mechanistic employing MTMR2 how the APPL1-positive and understand currently why to to are studies targeted spectrometry We is if endosomes. endosomes, MTMR2 Rab5-positive PI(3)P-rich unphosphorylated, to targeted phosphorylated, phosphorylation if is destination; MTMR2 the subtype to endosomal seems of early Ser631 of regulate regardless phosphorylation Meanwhile, cytoplasm Ser631. sequestered of status the (S58E) the of mutation is in capacity phosphomimetic Ser58 MTMR2 binding the that endosomal as suggests general MTMR2, the strongly for the data regulator In our master heterogeneity. MTMR2, endosome of early case achieving and regulating in shown are blots western Means and Representative A. levels. ImageJ ERK1/2 using total densitometry to by normalized quantified ( were with expression. levels immunostained protein phosphorylation were equal Lysates confirm control. to Total loading anti-FLAG pERK1/2. EGF as for ng/ml served (IB) 5 immunoblotted levels with were ERK1/2 Lysates treated times. then indicated minutes the 30 for for starved ERK1/2 serum sustained S58A/S631A, elicits Ser631 and Ser58 activation. of Dephosphorylation 9. 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De findings by results study al., APPL1-positive our elegant current on et our Nonetheless, depletion as Kim PI(3)P endosomes. needed, demonstrate 2010; be directly endosome al., will don’t testing in et a further result Berlin However, maturation, to and 2009; endosomal (Akbergenova shown signaling Bykhovskaia, of receptor have continued retardation and others enlargement or which that halting depletion phenotype PI(3)P is MTMR2-mediated to MTMR2/ to possibility due leads One is enlarged effect hypothesis. this observed of this that supports expression the endosomes The upon and APPL1 events. activation S58A/S631A signaling ERK1/2 linked MTMR2 in the increase regulates PI(3)P formation, control part significant temporally these in endosome and which spatially that early to levels means endosomal Rab5/APPL1 is a as proximal precedes potentially explanation a that likely initial represent stage more an PI(3)P endosomes a as a MTMR2/APPL1 in functions regulated However, that events fashion. signaling subtype downstream endosomal for containing endosomes platform distinct MTMR2/APPL1 APPL1 a that sole is are to possibility comparable One endosomes. remaining did change, size vesicle not (S58A/C417S/S631A), activity impaired catalytic was when MTMR2 APPL-positive MTMR2/ of however, of to Importantly, all formation compared alone. endosomes In vesicle when enlarged Rab5-negative. endosomes in are APPL1-positive MTMR2 resulted that this dephosphorylated endosomes cases, doubly APPL1 to this localizes that not but suggesting APPL1 with Rab5, colocalized equivalents stimulated events. or S631A endosomal of diversity by a in attenuated importance levels and the phosphoinositol exemplifying of early levels activity with PI(3)P phosphatase lipid association on MTMR2 RME-8 dependent 2011). was al., endosomes et retrograde (Xhabija and decisions transport sorting endocytosis-8 endosomal receptor-mediated in involved protein, (RME-8), binding identified recently PI(3)P further we novel example, a For effect activity. PI(3)P for this utilize modules which binding Moreover, machinery sorting downstream stages. to transitions particular these through hr r ueosrprsta aedsrbdAP1and APPL1 described have that reports numerous are There upiigrsl rmorsuywsta TR S58A/ MTMR2 that was study our from result surprising A TR agt oAP1edsms1341 endosomes APPL1 to targets MTMR2 Journal of Cell Science h ytei fpDA.-FadpT1 etr otiigwl yeand type wild MTMR2-His containing or vectors pET21a FLAG-MTMR2 and pCDNA3.1-NF mutant of synthesis transfections The and culture cell Plasmids, Methods and Materials pathophysiology the in disease. mediated role CMT ERK1/2 a of sustained plays if MTMR2 Ser58 investigate of to position phosphorylation important at MTMR2, be localized diseases. will phosphorylation cytoplasmic it CMT inactive, that functionally of by cell a fact hallmarks in highlighted results Schwann the main is 2012). the with of and Coupled al., one re-growth et is axonal (Napoli demyelination de- for demyelination critical extensive cell-like triggers al., progenitor is et cues a Napoli into stage environmental 2004; de-differentiation activation cell of al., Schwann et variety 2012). 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P. Wiseman, and S. Costantino, W., J. Comeau, u . al,M,Daosa .H n ae,L. Mayer, and H. W. Dragowska, M., Bally, Y., Hu, Cullen,P.J.,Cozier,G.E.,Banting,G.andMellor,H. Q. Y. Chen, and C. W. Mobley, C., L. McPhail, V., C. Ustach, R., Wu, J., H. Chial, aaaa . enr,D,Mrh,S,Hys . oo . oat,K,Standley, K., Fogarty, M., Soto, S., Hayes, S., Murphy, D., Leonard, A., Hayakawa, eln . ignohm .M,Ds,R . ira .I n ah .D. P. Nash, and I. M. Sierra, S., R. Dise, M., K. Higginbotham, I., Berlin, ii . alj,V,Wshlk,R,Pre,P .adLrjn,B. Larijani, and J. P. Parker, R., Woscholski, V., Calleja, N., Fili, E. Nishida, and K. Kondoh, M., Ebisuya, Ville C., Zhou, N., Dephoure, Cordelie and S. Bolte, M., D. Georgiou, A., M. Salih, E., LeGuern, L., F. Conforti, M., Muglia, A., Bolino, arsnh .C,PrzNdls . akno,D . acl,D . Mudge, S., D. Malcolm, B., D. Parkinson, E., Perez-Nadales, C., M. Harrisingh, egr . cafte,C,Bre,I,Bn .adStr U. Suter, and N. Ban, I., Berger, C., Schaffitzel, P., Berger, G. D. Lambright, and F. Barr, orol .F,Tksia . aca .A,Ony .K,Jhsn . er,K., Berry, J., Johnson, K., R. Olney, A., C. Garcia, H., Takashima, F., C. Boerkoel, oo,Y,Nsia . aaht,T,Hsi . aaai .adSakai, and M. Kawakami, M., Hoshi, T., Yamashita, E., Nishida, Y., Gotoh, lneu . aot,J,Bdn . uet-ug,G,Pyate .adMandel, and B. Payrastre, G., Superti-Furga, S., Bodin, J., Laporte, F., Blondeau, K. Shah, and L. Bardwell, M. Bykhovskaia, and Y. Akbergenova, References at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.113928/-/DC1 online available material Supplementary MOP- Scholarship MOP-89870, Graduate Canadian numbers NSERC C.A.B.). a [grant (to by and (CIHR) P.O.V.]; to Institutes Research 123141 Canadian from Health Grants Operating of by funded was study This Funding iloy .J,Mro,I . ida,M,Gud . rat .J,Gule,J M., J. Gaullier, J., N. Bryant, R., Gould, M., Lindsay, C., I. Morrow, J., D. Gillooly, O. P. Vacratsis, and S. G. Taylor, E., N. Franklin, al,T. Balla, alir .M,Rnig . iloy .J n tnak H. Stenmark, and J. D. Gillooly, E., Ronning, M., J. Gaullier, 20) ebaetreigb PL n PL:dnmcsaflsthat scaffolds dynamic measurements. APPL2: colocalization microscopy and fluorescence APPL1 by phosphoinositides. bind and targeting oligomerize Membrane (2008). hmteaetcefcso 40hmnnnsalcl ugcne el through cells cancer enhances lung cell kinase non-small apoptosis. human kinase of H460 activation signal-regulated on effects kinase/extracellular chemotherapeutic endocytosis. protein for necessary activated endosomes early USA of Sci. class Acad. a Natl. defines Proc. 2 al. et protein C. Mello, containing D., Lambright, K., Bellve, C., n yi .P. S. Gygi, and trafficking. membrane Biol. and Curr. signalling in role domains—their phosphoinositide-binding microscopy. encoding light gene in the analysis in mutations by caused is protein-2. 4B myotubularin-related al. et type A. Charcot-Marie-Tooth Schenone, P., (2000). Mandich, I., Hausmanowa-Petrusewicz, K., Christodoulou, .W n ly,A C. A. dedifferentiation. 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