fBooia cecs nvriyo ascuet oel oel A01854, MA Lowell, Lowell, Massachusetts of USA. University Sciences, Biological of agokLee Sangmook in phosphatases and kinases dynamics for neurofilament roles convergent and Divergent ARTICLE RESEARCH ß 4064 2014 July 1 Accepted 2014; March 17 Received 1 with Pant along 1992; Shea, 1995), and light, Veeranna, (Nixon and mass) medium and molecular heavy, their to to (corresponding according NF-L NF- and termed NF-M subunits, H, three of consist neurofilaments Mammalian INTRODUCTION Cytoskeleton Axonal stability, Phosphatase, Kinase, Neurofilament, WORDS: KEY 187 distal-most Li PD98059, the roscovitine, with lacking treated NF-H were NF-H, Cultures and acids. GFP-tagged amino sidearms expressing NF-H constructs cellsisolated with NB2a/d1 transfected differentiated Herein, were interaction. and neurofilaments extensive they to because display addition confounded in been substrates multiple have phosphorylate kinases these 3 of kinase mitogen- contributions synthase and (CK1 glycogen (CDK5), 2 and and 5 1 CK2) (NF-H), kinase casein kinase subunit (MAPKs), kinases protein neurofilament activated protein heavy cyclin-dependent the including the of the into domains phosphorylate kinases C-terminal incorporation Multiple cytoskeleton. neurofilament axonal stationary to leading self-association cation-dependent mediates phosphorylation neurofilament C-terminal ABSTRACT Atosfrcrepnec [email protected];[email protected]; [email protected]) ([email protected]; correspondence for *Authors USA. 20892, h o-rln-ietdC1adGSK3 of and by CK1 neurofilaments phosphorylation non-proline-directed CDK5 of phosphorylation the and of that pathway impact MAPK the GSK3 indicate counterbalance proline-directed the and findings by CK1 neurofilaments These not neurofilament– but proteolysis. restricted pathway CDK5, MAPK and The and neurites. axonal transport to associations axonal neurofilament promoted Sequential which respectively. GSK3 downregulate to found ERK1/ was 2) to (PP1), MKK1 (i.e. pathway MAPK The 1 associations. neurofilament GSK3 and phosphatase CDK5 by phosphorylation protein active GSK3 MAP2K1), are as and known which (also MKK1 calyculin, CDK5, against and tetrabromobenzotriazole D4476, omto ftesainr yokltn hc provides which cytoskeleton, the stationary C- mediate the NF-M and divalent-cation-dependent of that NF-H 2012). formation fosters interactions al., et neurofilament–neurofilament phosphorylation Yuan 2006; terminal al., et (Yuan etrfrClua erbooyadNuoeeeainRsac,Department Research, Neurodegeneration and Neurobiology Cellular for Center 04 ulse yTeCmayo ilgssLd|Junlo elSine(04 2,46–07doi:10.1242/jcs.153346 4064–4077 127, (2014) Science Cell of Journal | Ltd Biologists of Company The by Published 2014. 2 yokltlPoenRglto eto,NH ID,Bted,MD Bethesda, NINDS, NIH, Section, Regulation Protein Cytoskeletal 1, ,Hrs .Pant C. Harish *, b n K ciae P,bt of both PP1, activated CK1 and , b a eitdteneurofilament– the mediated itrei n peripherin and -internexin b b b . niie neurofilament inhibited , 2, (GSK3 n hmsB Shea B. Thomas and * b .Terespective The ). b K,CK2 CK1, , + , 1, n P nnuoiaetdynamics. neurofilament in PP1 and GSK3 CK1, for divergent roles convergent elucidated combination and approach a of reductionist and analyses allow This fractions. immunological to kinases cellular and bulk analyses in of immunofluorescence grown of be manipulation can readily and selective are phosphatases, cells These allowing neurites. axonal transfected, within phase stationary establish and a subunits neurofilament all phosphorylate and express 1999; (Goldberg, kinases 2005). al., neurofilament et the Adams regulate Finally, al., 2001). negatively et al., positively and Hergovich et also can 2002; (Liu neurofilaments dephosphorylate al., activity CK1- that phosphatases et CDK5 2007). Wang regulates al., 2002; and 2006) Harwood, et 2002; (Zheng al., 2012) GSK3 et several al., primes et phosphorylation mediated (Noh activity pathway pathway MAPK MAPK GSK3 display activates downregulates are and MKK1 ERK1/2) 2013), (i.e. pathway dynamics al., to MAPK the et example, neurofilament Pan For crosstalk. 2012; 2009; of considerable al., al., independently et et Shukla Galletti activated 2012; 2001; al., al., et et Pucilowska (Maccioni medi homeostasis they neuronal because and Jung 2011). confounded 1997; al., been et al., Veeranna et 2003; Strack al., 1995; PP2B) et al., and Gong et PP2A 1999; Saito Shea, (PP1, 1993; 2B al., and et 2A dynamics (Shea 1, neurofilament Holmgren phosphatase regulate 2008; protein that al., include Phosphatases et Barry 2012). Perrot 2004; 2007; al., al., Shea, et et and and Chan DeFuria Li 2003; 2007; Giasson al., Herdegen, 1999; Kesavapany et and 1997; al., 2003; Waetzig al., 2003; et et Miller, Ackerley al., 2000; Bajaj et al., 1992; and 1998; et al., Ackerley al., 1999; Bajaj et al., et et Link 1996; Veeranna 1991; 1997; al., al., Mushynski, et et (Guan (JNK) Guidato kinase terminal Jun xrse yadicroae notectseeo of cytoskeleton the into incorporated and by are NF-H expressed phosphorylated extensively containing Neurofilaments RESULTS iae MPs,csi iae1ad2(K n K) glycogen CK2), protein and 3 (CK1 kinase mitogen-activated 2 synthase and (CDK5), 1 kinase casein 5 (MAPKs), kinases kinase protein dependent Sihag (ALS) 1998; sclerosis Mushynski, and resembling lateral neurofilament 2007). Julien al., amyotrophic axons 1995; et of Veeranna, during proximal and occur accumulation (Pant and that aberrant perikarya those disruption within fosters phosphatases, and spheroids through which kinases Shea axons of 2001b; of within interaction al., complex segregated et a phosphorylated normally Yabe Extensively 2013). are 1998; Lee, neurofilaments and (Nixon, Shea 2011; axon Lee, and the to support * osron hs ifclis euiie Bad el,which cells, NB2a/d1 utilized we difficulties, these surmount To con respective the of Elucidation iae euaignuoiaetdnmc nld cyclin- include dynamics neurofilament regulating Kinases a n 3 and b (GSK3 a b n GSK3 and hra D5ihbt the inhibits CDK5 whereas , rbto fteeknsshas kinases these of tribution b t utpeitga oe in roles integral multiple ate Dn ta. 05.GSK3 2005). al., et (Ding h AKptwy CDK5 pathway, MAPK the , b ,p8MP n c- and MAPK p38 ), b usrts(Hagen substrates b

Journal of Cell Science yohs hypophosphorylated. hypophos, total and phospho- aha aepeiul endmntae ihnteecells these within demonstrated been previously we have phospho-epitopes, pathway MAPK, CDK5, neurofilament kinases GSK3 neurofilament known studied generation the identify overexpressed for commonly to responsible efforts predominantly of In were 1A,B). kinase(s) (Fig. which cells NB2a/d1 differentiated ARTICLE RESEARCH ihnuoiaet eadeso hshrlto tt,a odn oto.Nt vrxrsino K1icesdSI3,SI3 n RT97 and SMI-34 SMI-31, increased MKK1 of overexpression Note control. accomp loading The control. a loading as a GSK3 state, as whereas phosphorylation immunoreactivity, DM1A of antibody regardless anti-tubulin GSK3 neurofilaments with GSK, MKK1; probed with zym (MAPk, sample gel kinases same representative indicated the A the of controls. overexpressing aliquot mean untreated an the versus of presents phosphorylation replica graph casein nitrocellulose decreased, a D4476 with inhibitor along CK1 presented the whereas increased, mean overexpression the indicate present are graphs (M) accompanying NF-M and (H) GSK3 NF-H of of levels migration increased antibodies; significantly above the neurofilame situated with GSK3 centrally probed expressing the homogenates construct Note a of indicated. with as replicas Transfection epitopes Nitrocelluose (C) (SMI-32) responsible. (B) nonphospho- kinases (arrows). and RMO-24) the neurites SMI-34, and along SMI-31, cells (RT97, NB2a/d1 phospho- against in directed phospho-epitopes Neurofilament 1. Fig. ta. 96] n erae eeso h GSK3 the of levels decreased and 1996)], al., et b n K.Icesdatvt fCK n h MAPK the and CDK5 of activity Increased CK1. and b ctni lbldpctnnadctnn respectively). catenin, and p-catenin (labeled -catentin 6 ...o aenpopoyainvru nrae otosfrtresc es E ircluoerpia fhomogenates of replicas Nitrocellulose (E) gels. such three for controls untreated versus phosphorylation casein of s.e.m. b nrae M-4 l ape rbdwt h aeatbd nec ae r rmtesm e.po,phosphorylated; phos, gel. same the from are panel each in antibody same the with probed samples All RMO-24. increased b hshrltda e9[hc nciae GSK3 inactivates [which Ser9 at phosphorylated 6 ...frtreimnbosfrttladphospho-GSK3 and total for immunoblots three for s.e.m. b b substrate GK infcnl ( significantly (GSK) b ctni pe9GK hc spoelzdfloigGSK3 following proteolyzed is which GSK, (pSer9 -catentin b ,poe ihteaoenuoiaetatbde n oylnlatbd R9 htreacts that (R39) antibody polyclonal a and antibodies neurofilament above the with probed ), P , .5 nrae vrl GSK3 overall increased 0.05) b tbln(rbdwt nioyD1)wsicue salaigcnrl The control. loading a as included was DM1A) antibody with (probed -tubulin hs iae eesbetdt muoltaaye.Gvnthat Given analyses. immunoblot to subjected were kinases overexpressingthese cells of Homogenates 1C,D). (Fig. treatment D4476 olwn rnfcin n hi niiinfloigLi following inhibition GSK3 their of al., et and activity Shea transfection, increased 2004; following confirmed al., CDK5) et We of (Chan regulator 2004). MKK1 positive active (a constitutively and p25 with transfection following b ora fCl cec 21)17 0447 doi:10.1242/jcs.153346 4064–4077 127, (2014) Science Cell of Journal LmreadDsoir,20) n rvne erdto of degradation prevented and 2008)] Desrosiers, and (Lamarre A ersnaiedfeetae el rbdwt antibodies with probed cells differentiated Representative (A) b n w o hsh-adtotal and phospho- for two and b ees[hc nrae e GSK3 net increases [which levels b mdae hshrlto) Li phosphorylation). -mediated b ctni.()CK1 (D) -catentin. b ciiy(Wagner activity b n CK1 and gahis ograph tbundle nt + anying d. 4065 and +

Journal of Cell Science M-4eioewspeoiatygnrtdb GSK3 by generated predominantly were epitopes was RT97 the epitope contrast, and By pathway. RMO-24 SMI-34 MAPK the SMI-31, by generated analysespredominantly the These 1E). that (Fig. detectable indicated barely total non- were activation, in levels cells any the transfected kinase that such of restricted, selective were gels detection onto following loaded facilitate aliquots epitopes to nonphospho- in order and changes in phospho- 1B), of (Fig. levels neurofilaments high contain cells NB2a/d1 ARTICLE RESEARCH 4066 Li and untreated for was isoform immunoreactivity 260-kDa Phospho-NF-H the to 2011). also hypophosphorylated fused at al., was NF-H concentrated GFP–NF-H to et phosphorylated GFP; (extensively (Lee inhibitor corresponding kDa 35-kDa GFP) 260 (CK2) to at 2), fused 2 detected (Fig. NF-H kinase kDa) kDa (160 195 casein the migratedat above GFP–NF-H of the bulk plus The with tetrabromobenzotriazole (tBBT). responsible GFP–NF-H inhibitors the expressing determine cells kinase To treated 1992). Shea, we kDa and 160 kinase(s), (Nixon from kDa gels 200 SDS on to migration NF-H alters on Phosphorylation migration NF-H of gels retardation SDS major the kinase mediates CK1 of influence the monitor interference neurofilaments. endogenous the closely of without dynamics more neurofilament endogenous on to manipulation constructs phosphorylated us GFP-tagged neurofilaments of allowed expression expressed differentially addition, newly In neurofilaments. of of distribution independently and levels GFP- the with of monitoring cells the facilitated which constructs, transfected neurofilament tagged we phosphorylation, neurofilament 1E). (Fig. itiuin niaigta hssiti hsh-eciiywsnot NF-H. total was of phospho-reactivity migration altered in GFP–NF-H to shift alter due this 260-kDa not that did the indicating D4476 reduced distribution, 2). and (Fig. reactivity reactivity phospho-NF-H increased phospho-NF-H D4476 195-kDa homogenates. the roscovitine-treated and PD98059- oivsiaefrhrteptnilrlso hs iae on kinases these of roles potential the further investigate To + -, b ptpscnb eeae nteasneo h irtr shift. migratory the of absence the phospho- in RT97 generated and be SMI-34 SMI-31, can the epitopes (3) and shift, migratory ta. 01.Teest-pcfcmttosalwdmntrn of monitoring allowed (Lee mutations site-specific mimic states consensus These to 2011). nonphosphorylated al., sidearm) CDK5 et or NF-H termed normal phosphorylated residues, of the permanently Ala (i.e. residues or sidearms wild-type Asp with GFP–NF-H site-directed Ser along by sidearms, GFP–NF-Hala replaced and the including GFP–NF-Hasp been NF-H which had sidearms, on sequences kinases in NF-H GFP- above mutants expressing C-terminal the constructs with of tagged cells transfected impact we the migration, further phospho- spans the kDa examine example, 260 for at and phospho-isoforms, band; migrating broad (Lewis resultant isoform relatively acrylamide a its as of and appear amount phospho-dependent also GFP–NF-H, low multiple 1988). a into with Nixon, resolved gels be NF-H on can phosphorylated isoforms gels extensively for SDS observed on band broad gels The SDS on migration neurofilament GSK3 and CDK5 ugssta h irtr hf fti sfr rm10to 190 from isoform This this of affected. shift 3D). not (Fig. migratory the were isoform that isoforms 200-kDa suggests RT97-reactive immunoreactive and RMO-24 isoform SMI-31- 210-kDa the a at a revealed migrating substitutions Ala isoform and with unique construct isoform at sidearm The 205-kDa migrating 3C). (Fig. the isoforms with distinct subtle associated two to into , gels sensitive SDS 3B). more Fig. acrylamide (e.g. were addition, constructs full-length In constructs were migration. than shifts only’ isoform migratory on ‘tail CDK5 these of impact potential 0 D,()popoyaineet eitdb h MAPK the by to GSK3 NF-H mediated and of events shift CDK5 migratory phosphorylation pathway, the (2) inducing kDa, for 200 crucial are CK1 by 0 n 0 D.RO2 muoeciiywsexclusively was immunoreactivity RMO-24 kDa. 205 and 200 M-1adR9 nwl-yesdam eovdo low- on resolved sidearms wild-type in RT97 and SMI-31 hsfnigsget ht()popoyaineet mediated events phosphorylation (1) that suggests finding This ora fCl cec 21)17 0447 doi:10.1242/jcs.153346 4064–4077 127, (2014) Science Cell of Journal b eit eaieymnrcagsin changes minor relatively mediate homogenates. untreated versus immunoreactivity mean the represent Values conditions. * other all versus immunoreactivity isoform, phospho- 195-kDa 195-kDa the increased did of but treatment levels D4476 total included. alter are not B conditions and all A not in NF-H; 195-kDa of analyses densitometric presents graph accompanying D4476 The only migration. Note GFP–NF-H indicated. inhibited are kDa) (260 195 GFP and to fused NF-H hypophosphorylated and extensively The of cells. migration control untreated with of along homogenates incubation inhibitors, of h indicated 2 the final with the for treated GFP– and expressing NF-H construct a h with 24 previously transfected homogenates of replicas NF- gels. of SDS on retardation migration the H mediates CK1 2. Fig. P , .1 NV ihps-o analyses. post-hoc with ANOVA 0.01, b , 9 D,culdwt h osof loss the with coupled kDa, 190 r o ufcett nuethis induce to sufficient not are , 0ka(..Fg A.To 3A). Fig. (e.g. kDa 10 AB Nitrocellulose (A,B) 6 s.e.m.

Journal of Cell Science 1-D sfr nadto ota fGSK3 of that to mutated addition were in the that sidearms isoform of generation indicates in 210-kDa finding mutant role a this plays sites, Asp phosphorylation CDK5-mediated consensus CDK5 that at contrast, sidearms exclusively By Given mutant Asp kDa. the 210 3E). in depleted to (Fig. not isoform was isoform 205-kDa 210-kDa the the of migration GSK3 the that phosphorylation indicate (R39) findings of antibody These regardless state. following polyclonal neurofilaments a observed all with against also directed replicas was additional reactivity of pattern isoform probing This in 3E). the reduction (Fig. and increased of relatively depleted, was severely isoform more 205-kDa was isoform RMO-24-reactive EERHARTICLE RESEARCH 61% GSK3 2004). al., et Shea 2011; al., et (Lee endogenous cells NB2a/d1 of in presence activity CDK5 wild-type the and with difference consistent GFP–NF-Hasp No sidearms, of GFP–NF-H 3D). migration (Fig. between activity detected CDK5 was upon dependent substitution migration not its Ala that was suggesting affected, the not was that isoform 205-kDa given the CDK5, of by Migration phosphorylation. CDK5-mediated blocked mediated specifically is kDa 200 Li with treatment without or with (G) construct NF-H no or isoforms. (C–F) NF-H sidearms generate terminal kinases Multiple 3. Fig. ooeae rmnntasetdclswt rwtottetetwt ra h tcigglwsrtie o rnfrt ircluoea clear as nitrocellulose SMI-34 to with transfer reactive for is material retained aggregated was This gel electrophoresis. of stacking to replicas the prior Nitrocellulose urea urea; with from (G) M homogenates isoforms isoform. 8 of clear 205-kDa with treatment however, the following treatment increased detected without only and or are isoform with isoforms 210-kDa cells RMO-24-reactive non-transfected the from depleted homogenates treatment PD98059 (F) sidearm. ramn lodpee h M-4ratv 1-D isoform, 210-kDa RMO-24-reactive the depleted also treatment el xrsigteGPN-aasdamlc h 0-D sfr u ipa 9-D sfr hti eciet M-1adR9 u o RMO-24. not but RT97 and Li SMI-31 without to or reactive with is sidearms that GFP–NF-Hasp isoform or antibod 190-kDa sidearms indicated a GFP–NF-H the display wild-type with but expressing probed isoform homogenates sidearms 200-kDa of GFP–NF-Hala the replicas or lack Nitrocellulose GFP–NF-Hasp sidearm (E) GFP–NF-H, GFP–NF-Hala wild-type the expressing expressing homogenates Cells of replicas Nitrocellulose (D) niiae,sdamol osrcsmgaefse nSSgl.()SI3 n T7ratv sfrsi idtp F–FHsdam eovdon resolved sidearms GFP–NF-H wild-type in As isoforms indicated. RT97-reactive (160 as RT9 and NF-H sidearm, with SMI-31 of at GFP–NF-H probed (C) range migrating respective replicas gels. migratory isoforms their Nitrocelluose SDS the into or (B) on to gels GFP–NF-Hala, isoforms. faster corresponds SDS or slowest-migrating migrate which GFP–NF-Hasp the constructs kDa, GFP–NF-H, within sidearm-only 195–260 (wt) concentrated anticipated, of wild-type are range full-length Phospho-epitopes a GFP. expressing over 35-kDa homogenates migrates the GFP–NF-H plus (A) kDa) neurofilaments). 210 total against (directed R39 M-4o 3.Li R39. or RMO-24 h aoiyo M-4imnratvt a eie from derived was immunoreactivity RMO-24 of majority The 6 b (mean 6 eas M-4imnratvt a eue by reduced was immunoreactivity RMO-24 because , 6 + ... olwn Li following s.e.m.) eltdte20kaioomadicesdte25kaioomi idtp F–FHsdam u i o fetteGFP–NF-Hasp the affect not did but sidearms GFP–NF-H wild-type in isoform 205-kDa the increased and isoform 270-kDa the depleted , 1 o20ka(ros r eetbei h bec fue (arrowheads). urea of absence the in detectable are (arrows) kDa 200 to 210 , 0 n 0 D.RO2 muoeciiywsascae ihioom irtn t25ad20kDa. 210 and 205 at migrating isoforms with associated was immunoreactivity RMO-24 kDa. 205 and 200 + b ramn;te210-kDa the treatment; lydarl nshifting in role a played ircluoerpia fti D eso ooeae xrsigGPN- AB,GPtge C- GFP-tagged (A,B), GFP–NF-H expressing homogenates of gels SDS thin of replicas Nitrocellulose b PD98059 . eryalRO2 muoeciiywti h axonal the within immunoreactivity 1). (Fig. the analyses RMO-24 immunofluorescence of in detection this bundle neurofilament with all within consistent observed also is was nearly This reactivity 3G). anticipated (Fig. SMI-34 aggregate and the SMI-31 Notably, with some NF-H. phosphorylated to extensively for associated position urea migratory immunoreactivity M and stack 8 increased the within immunoreactivity with 1990), RMO-24 eliminated Hartley, preparations which and of (Marston cytoskeletal complexes neurofilament majority of dissociate treatment the confirmed was by possibility that This 3F). with suggesting (Fig. associated complexes SDS-resistant are stack, isoforms NF-H RMO-24-reactive the was endogenous immunoreactivity within by RMO-24 detect nitrocellulose, observed onto to transfer difficult retained for was is gel stacking cells the when non-transfected However, analyses. immunoblot in NF-H RMO-24 of the immunoreactivity with involves antibodies, isoforms neurofilament NF-H dependent of GSK3 range and CDK5 full pathway, demonstrate MAPK the findings these of generation 2), (Fig. that CK1 by mediated apparently form. 210-kDa the of contributes generation also that pathway to suggesting MAPK isoform, the 205-kDa by the mediated phosphorylation in increase an with coupled + D85 rue,adpoe ihat-F,SI3,SI3,R9 or RT97 SMI-34, SMI-31, anti-GFP, with probed and urea, or PD98059 , lhuhtemjrsiti irto fN- nSSgl was gels SDS on NF-H of migration in shift major the Although ora fCl cec 21)17 0447 doi:10.1242/jcs.153346 4064–4077 127, (2014) Science Cell of Journal b ncnrs oohrphospho- other to contrast In . + ramn rbdwith probed treatment n SMI-31; and NF-H 7of 4067 thin ies. –

Journal of Cell Science EERHARTICLE RESEARCH n ihu uiidknss fe hc eqatfe the quantified or three 4068 we with associated which were after that neurofilaments kinases, of purified percentage from with without incubated recovered and Rhodamine and with neurofilaments conjugated were Individual cords spinal 4E). (Fig. neurofilament– analyses decreased pathway activity MAPK 4C,D). CK1 and associations, (Fig. associations contrast, neurofilament CDK5 neurofilament–neurofilament by extent, GSK3 lesser whereas, increased that a found activity to on and inhibition and, or distribution, activity overexpression GFP–NF-H kinase of intracellular impact crucial the the compared mediate we associations, kinase(s) neurofilament–neurofilament cross- promote which that investigate events cation-dependent To 2001a). by al., Yabe 2009; et mediated al., et (Kushkuley are phospho-neurofilaments associations of sucrose, bridging bundling Neurofilament–neurofilament al., to et over leading (Kushkuley 4A,B). (Fig. associated surrounding sedimentation physically are 2009) from they by separated that indicating neurofilaments be can individual 1A). neurofilaments (Fig. closely neurofilaments individual Bundled of surrounding more the bundle turnover than and situated slowly transport undergo centrally that a neurofilaments opposed contain neurites bundling neurofilament Axonal to contribute kinases Multiple eas xmndteipc fkns ciiyi cell-free in activity kinase of impact the examined also We b pfursec mgso erflmnsfo hs preparations. these from representative neurofilaments and of phospho-epitopes, images (M) epifluorescence SMI-31, NF-M with and probed (H) preparation NF-H this depicting of replica nitrocellulose a present GSK3 bevdwt D5aoe(* alone CDK5 with observed (** GSK3 + CDK5 associations(* neurofilament–neurofilament ERK1/2). increased of CDK5 addition with exogenous Incubation or (MAPk, with kinases h indicated 2 the for without incubated neurofilaments cord spinal Rhodamine-conjugated essutetdnuoiaetadnuoiaet nuae ne l other all under * incubated conditions neurofilaments and neurofilament untreated versus erflmns( neurofilaments untreated versus associations neurofilament–neurofilament GSK3 increased with incubation experiments; independent two niiulyo ncmiain ossetwt ro studies an GSK3 prior and induced CDK5 contrast, CDK5 with By associations. 2009), neurofilament–neurofilament Consistent nor al., combination. et ERK1/2 (Kushkuley in neither or analyses, individually multiple cell-free within with transfection attempt In GSK3 require to constructs. would practical it kinase not neurofilaments because was isolated cells, treat this intact to kinases; able multiple also cell- with were these In we 2009). analyses, al., free et (Kushkuley neurofilaments other more niiulyo ncmiain oevr vrxrsinof overexpression compounds Moreover, these combination. with in with treatment or following reduction treatment individually observed contrast, By not 5A). combined (Fig. was GFP–NF-H levels total NF-H RT97-reactive reduced However, and significantly PD98059 and 5A). roscovitine (Fig. isolation rnfce ihkns osrcs ausrpeettemean the not represent values those constructs; versus kinase with constructs transfected transfected kinase cells neurofilament in indicated neurofilaments the bundled:individual with of ratio the in change constructs GSK3 with MKK1, co-transfected previously p25, or expressing h (–) 24 alone transfected constructs cells GFP–NF-H from with (B) fractions homogenates bundle-enriched of anti-GFP, and with (H) bundle probed the replicas, into Nitrocellulose incorporated (C) is (arrows). and Note cells GFP–NF-H. throughout expressing distributes cell GFP–NF-H A a (B) of markers. image mass epifluorescent molecular representative MW, R39. with non-bundled detected individual, neurofilaments) the in at enriched remaining (Super, or interface neurofilaments) buffer–sucrose sucrose bundled M in 0.8 enriched a (Pellet, through cushion sedimented supernatant that Triton-soluble neurofilaments the and in (Super), remaining or in recovered (Pellet) (M) cytoskeletons NF-M and total (H) bundling. NF-H endogenous neurofilament of replicas promote Nitrocellulose (A) activities Kinase 4. Fig. rtcinaantpoelss oacmls hs etreated PD98059, roscovitine, we inhibitors this, therefore Li pharmacological accomplish the We with To 1995). cells proteolysis. Veeranna, to against kinases and neurofilament the protection of Pant contribution relative 2001; the proteolysis compared al., neurofilament et inhibits (Grant phosphorylation C-terminal prevent pathway MAPK proteolysis the neurofilament and CDK5 by Phosphorylation erflmn associations. neurofilament GSK3 with of consistent with specifically impact interfered is the pathway MAPK This the GSK3 alone. that possibility CDK5 the and with incubation CDK5 followingfollowing with observed incubation incubation associations Notably, GSK3 neurofilament–neurofilament bundling. CDK5, neurofilament with on kinases these , P 0 fnuoiaet,idctn yegsi matof impact synergistic a indicating neurofilaments, of 90% P + , , .5vru nrae erflmns Student’s neurofilaments; untreated versus 0.05 n 47,nn fwihatrdGPN- eesin levels GFP–NF-H altered which of none D4476, and .5vru D5aoe Student’s alone; CDK5 versus 0.05 b b b n K1peetdteGSK3 the prevented MKK1 and ora fCl cec 21)17 0447 doi:10.1242/jcs.153346 4064–4077 127, (2014) Science Cell of Journal nue erflmn–erflmn soitosfor associations neurofilament–neurofilament induced P rmtdnuoiaetnuoiaetassociations neurofilament–neurofilament promoted , b .3 NV ihps-o nlss E uniiainof Quantification (E) analyses. post-hoc with ANOVA 0.03; P ute nrae erflmn–erflmn associations neurofilament–neurofilament increased further , .5 NV) K erae erflmn associations neurofilament decreased CK1 ANOVA). 0.05, b b n o hto D5 nneurofilament– on CDK5, of that not and , n R12atnae h xetof extent the attenuated ERK1/2 and P , b .5vru nrae erflmns.Insets neurofilaments). untreated versus 0.05 GK n K.()Tepercentage The (D) CK1. and (GSK) b mdae nraebyn that beyond increase -mediated t ts) nuainwt CDK5, with Incubation -test). b otelvlobserved level the to b t 2 rMKK1 or p25 , ts) nuainwith incubation -test); , 0 nraein increase 40% 6 h ag from range the

Journal of Cell Science K,poetnuoiaet rmpoelss ossetwith Consistent proteolysis. from GSK3 events neurofilaments by not phosphorylation protect but pathway, that CK1, MAPK PD98059 the indicate and and CDK5 findings by mediated roscovitine These with 5B). (Fig. treatment combined following GSK3 ARTICLE RESEARCH b rC1cudntpeettedpeino GFP–NF-H of depletion the prevent not could CK1 or b or hrfr ananaciia ee fnuoiaet,wihwould which bundling. might neurofilaments, neurofilament pathway promote of indirectly MAPK level critical the a and maintain CDK5 therefore 5C). (Fig. homogenates within neurofilamentsin phospho-neurofilaments were than proteolysis 2012), to resistant al., more were bundles et (Rao studies recent ora fCl cec 21)17 0447 doi:10.1242/jcs.153346 4064–4077 127, (2014) Science Cell of Journal graphs, Ros+PD+GSK3 versus olwn ramn ihRsP iho without GSK3 or of with overexpression Ros+PD with bundles treatment versus following homogenates in depletion increased or GSK3 presence of the absence in homogenates in with immunoreactivity treatment B, phospho-neurofilament in reduced As Ros+PD condition. each under The immunoreactivity present B. total the in present as graphs treated accompanying cells from fractions derived bundle-enriched (B) RT97 and or (H) SMI-34 homogenates with of probed replicas Nitrocellulose (C) ( treatment this for GSK3 compensate of overexpression that and rsn ne ahcniin oerdcini levels in reduction Note condition. each under immunoreactivity present the presents graph accompanying of overexpression without or GSK3 with cells Ros+PD from with (H) treated non- homogenates or of anti-GFP SMI32 with phospho-specific probed analyses). replicas post-hoc Nitrocellulose (B) with ANOVA (* conditions; PD other + all Ros for with immunoreactivity treated RT97 cells and specific GFP–NF-H the in Note reduction condition. each for experiments olwn ramn ihRs+P (* PD + Ros with treatment following )peetudrec odto;vle represent values condition; mean each + under (H present GFP–NF-H B) total the The present (Untreat). graphs controls accompanying or untreated (PD) with PD98509 along (Ros), both, roscovitine with treated from (B) cells fractions bundle-enriched and (H) homogenates neurofilaments protect proteolysis. MAPK against and CDK5 5. Fig. 6 b ...imnratvt rmtoo three or two from immunoreactivity s.e.m. GK rC1aogwt nrae otos The controls. untreated with along CK1 or (GSK) P , .1 ANOVA). 0.01; b rC1(* CK1 or A ircluoerpia of replicas Nitrocellulose (A) b b rC1 ANOVA). CK1; or rC1(seik between (asterisks CK1 or P P , P , .1 NV) Note ANOVA). 0.01; , .3 n .1versus 0.01 and 0.03, b .9frRos+PD for 0.19 rC1ddnot did CK1 or P , .2 ANOVA), 0.02; 4069

Journal of Cell Science ih rcueiaporaepraylnuoiaetbundling. neurofilament perkaryal inappropriate preclude might erflmn cuuain,adaecnitn ihour with 4). (Fig. analyses consistent cell-free in associations are neurofilament and GSK3 that demonstration accumulations, GSK3 neurofilament and CDK5 of ehns ean nla.Ntby h AKpathway responsible MAPK the the but GSK3 that 2004) demonstrated Notably, GSK3 al., unclear. et inactivates (Chan remains axons mechanism along and neurofilaments of transport into for required is activity pathway MAPK bundling neurofilament GSK3 inhibiting neurofilament by anterograde transport mediates pathway MAPK The ARTICLE RESEARCH 4070 of extensive and within levels NF-H bulk the RT97-reactive to examined and (GFP–NF-H) al., therefore the total We attributed of et 2001a). distribution contain al., (Kushkuley was et NF-H Yabe bundles neurofilaments 2009; that in and phosphorylated shift gels extensively 2), the (Fig. SDS for phosphorylation responsible on was counterintuitive, migration activity seemed inhibits apparently CK1 finding CK1 because This that indicate bundling. 4) neurofilament (Fig. results above The activity phosphatase perikarya maintaining within by phosphorylation neurofilament inhibits CK1 bundles. neurofilament Li perikaryal with with treatment cells However, of GSK3 and percentage p25 the Both GSK3 harvest. of to prior (inhibitors roscovitine GSK3 overexpressed or or we p25 not with possibility, along this or GFP–NF-H investigate whether To accumulation. questioned we GSK3 GSK3 accumulation, for role crucial neurofilament a Given of 2004). demonstration al., above phospho- et the (Shea of perikarya accumulation within bundles the neurofilament induces CDK5 of Overexpression GSK3 GSK3 inhibiting by neurofilament transport promotes These pathway axonal 6C). MAPK (Fig. the that bundles indicate neurofilament findings perikaryal observed in we cells increase PD98059, when an with However, treated image). were representative GSKala for overexpressing 6C and 2004) inhibits Fig. al., transport also et (which (see (Chan neurofilament accumulation PD98059 neurofilament inhibits perikaryal fosters activity) with axonal pathway Treatment along MAPK and 6B). (Fig. into transport constitutively neurites therefore neurofilament is inhibited and active) phosphorylation by inactivated be GSK3 inhibiting through be pathway might MAPK GSK3 transport the which neurofilament by mediates mechanism we the 2011), Lee, that and hypothesized (Shea pool transporting the bundling GSK3 from undergo withdrawn that are neurofilaments attenuated that Given activity 4). (Fig. bundling pathway MAPK whereas niie eiaylnuoiaetacmlto nue by induced roscovitine accumulation with neurofilament GSK3 treatment perikaryal and inhibited accumulation, neurofilament hspsiiiy efrtcnimdta h AKpathway MAPK the that confirmed first GSK3 phosphorylated we possibility, this with GSK3 coupled of PD98059 overexpression with treatment by transport neurofilament fti eeide h ae ehpteie htihbto of inhibition that hypothesized we case, the indeed were this If b b b b b mdae erflmn–erflmn interactions. neurofilament–neurofilament -mediated oetae D5idcdnuoiaetbundling neurofilament CDK5-induced potentiates nwihSr a uae oAa(Saa hc cannot which (GSKala, Ala to mutated was Ser9 which in atcptdi D5idcdprkra neurofilament perikaryal CDK5-induced in participated Fg D.Teefnig niaeta combination a that indicate findings These 6D). (Fig. b b Dn ta. 05.Hri,w have we Herein, 2005). al., et (Ding b b ciiyi eurdt nueperikaryal induce to required is activity b b oetae D5idcdneurofilament– CDK5-induced potentiated tSr Fg A.Oeepeso of Overexpression 6A). (Fig. Ser9 at atcptsi erflmn bundling, neurofilament in participates ih aea diieefc.T test To effect. additive an have might + b niie 2-nue perikaryal p25-induced inhibited -mediated b b b b n rae el ihLi with cells treated and , n D5 respectively) CDK5, and ciiy n ndigso, doing in and activity, vrxrsinincreased overexpression b nperikaryal in b -mediated + 47 ttsial nrae oa T7immunoreactivity RT97 RT97 total total increased reduced ( statistically statistically D4476 CK1 that ( revealed immunoreactivity of analyses, densitometric cells Visual overexpression by 7A). (a from (Fig. D4476 confirmed CK1) with inspection, derived against treatment active fractions and inhibitor CK1 pharmacological bundle of overexpression and following homogenates total D es hc orsod oteatcptdmigratory amino anticipated 187 terminal the the lacking to (i.e. NF-H corresponds 115-kDa at of which position migrating gels, species SDS GFP-reactive been had the prominent 2000)] (NF-H sidearm al., C-terminal et which (Chen the bundling deleted of in for essential region be GFP–NF-H [the to full-length reported acids of amino of 187 incorporation fraction terminal with bundle-enriched the GFP–NF-H GSK3 into of role incorporation the further the address essential To is to C-terminal NF-H bundling indirectly the for of portion or distal The directly bundles. axonal contributes into neurofilaments kinases of each incorporation that these confirm findings and These of 8A,B). bundles (Fig. bundles with neurofilament NF-H GSK3 displaying and CK1 neurites CDK5, the increased of both this kinases these of percentage and of support each establishment In of overexpression bundles. the notion, neurofilament to axonal directly would of kinases or maintenance an these indirectly of foster we either each of neurites, contribute would activity axonal increased functions that within considered diverse phospho-neurofilaments these in and increase of 6,7) neurofilament Figs impact CK1; of collective GSK3 and (CDK5, (MAPK bundling restriction neurofilament of neurites promotion of axonal 5), promotion into Fig. proteolysis transport perikarya, of CDK5; within MAPK phosphorylation inhibition and the including of GSK3 (MAPK activity dynamics, and the CK1 neurofilament that CDK5, highlight pathway, findings above The bunding GSK3 neurofilament and axonal CK1 to CDK5, pathway, MAPK The nrae T7imnratvt ihnprkraadaxonal and and perikarya 7D), (Fig. within immunoreactivity SMI-32 RT97 the not increased but antibody RT97 with high- reactive nonphospho-neurofilament agent SDS-resistant was of pharmacological that formation material a the molecular-mass fostered Calyculin, PP1, against 7C). active (Fig. neurites axonal dynamics neurofilament significantly treatment regulated ( D4476 activity. CK1 PP1 of not modulation through treatment or whether D4476 probed following reactivity 7B). RT97 (Fig. perikaryal axonal in NF-H and increase an RT97-reactive demonstrated significance, and analyses towards Immunofluorescence total (trend of bundles amount within the (trend bundles increased within D4476 NF-H RT97-reactive significance, and towards total of amount K ih etitnuoiaetpopoyainthrough phosphorylation neurofilament restrict PP1. of activity activation that might PP1 and perikarya, CK1 that within indicate restricting accumulation including phospho-neurofilament findings phosphorylation, C-terminal These NF-H regulates 7E). (Fig. neurites P P , , ie htC1atvtsP1(er n illa 93,we 1993), Killilea, and (Henry PP1 activates CK1 that Given .5 eue P u o PAatvt nprkraand perikarya in activity PP2A not but PP1 reduced 0.05) .1 NV) vrxrsino K erae the decreased CK1 of Overexpression ANOVA). 0.01, ora fCl cec 21)17 0447 doi:10.1242/jcs.153346 4064–4077 127, (2014) Science Cell of Journal D 8) el xrsigH expressing Cells 187). P , P .1 NV) hra ramn with treatment whereas ANOVA), 0.01, , b .6 NV) hra ramn with treatment whereas ANOVA), 0.06, ahicesdteascaino GFP– of association the increased each b b b nbnln,w compared we bundling, in xr iegn oe on roles divergent exert is67.Gvnta the that Given 6,7). Figs ; P b , ahcontribute each D .8 ANOVA). 0.08, 8 generated 187 , 5 D on kDa 150

Journal of Cell Science upre omdaenuoiaetnuoiaetbundling, sidearm profiles NF-H neurofilament–neurofilament the of mediate filamentous portion the to with of the purported deletion within Despite colocalization distribution 8D). its (Fig. and by shown cytoskeleton as network neurofilament EERHARTICLE RESEARCH eosrtsisaiiyt neg phospho-mediated undergo full-length and Shea 1995; of Veeranna, 2008). and to retardation (Pant Chan, gels SDS foster sidearm. on migration ability that NF-H the alterations of H its conformation portion phospho-reactive of proximal of demonstrates migration retention the of confirming within Retardation SMI31), epitopes and C-terminal these SMI34 antibodies neurofilament (RT97, with phospho-dependent epitopes and immunoreactivity 8C), against (Fig. prominent kDa directed 175 displayed and 155 GFP-reactive these between slower-migrating observed Additional were GFP. species to fused acids) GFP–NF-H D 8 oasmldwt h endogenous the with co-assembled 187 D 8 isoforms 187 ohtss.RsoiiepeetdteGKaamdae nrae whereas increase, Li GSK3ala-mediated the prevented Roscovitine tests). both ifrne ewe en,adFshrlatsgiiatdfeec (LSD) difference significant least Fischer and means, between differences neurofilament perikaryal increased ( CDK5 bundles or GSKala of Overexpression (D) mean P el vrxrsigCK ifrdfo hti nrae el (* cells untreated in in that and from GSKala differed overexpressing CDK5 cells overexpressing cells in bundling perikaryal that revealed each h FHCtria alpasacuilrl nneurofilament in role crucial a plays tail of bundling. residues C-terminal acid amino NF-H 187 distal the the GSK3 within that sites of suggest phosphorylation axonal GFP–NF-H findings of with These association associated the bundles. alter was not did that but GFP–NF-H bundles, of amount relative GFP–NF-H otiigprkra erflmn bundles. neurofilament perikaryal containing nrae h ecnaeo el otiigprkra erflmn bundles neurofilament perikaryal (* containing cells of percentage treatment the GSKala increased + PD98059 GSKala. expressing cells PD98049-treated in (arrows) bundles neurofilament perikaryal immunoreactivity and treatment, GFP PD98059 following neuritic decreased and treatment. perikaryal PD98059 increased and co-expression Note label) (GSK3B GSKala without GFP–NF-H or expressing with cells of images epifluorescence Representative (C) itiuinwti ude esstesronigaxoplasm. surrounding GSK3 the of versus overexpression bundles However, within distribution GFP–NF-H and GFP–NF- and GFP–NF-H al., GFP–NF-H of et bundling Sasaki on 2000; 1996; H 1997), al., al., al., et et et Shaw Hollander (Chen 2002; 1992; acids Bennett, amino and 187 Hollander distal-most the within kinases these exist for sites consensus NF-H of majority 2009; the because CK1, al., same et the (Kushkuley 2011). al., previously into et among Lee shown co-assembled crosslinking as C-terminal NF-H neurofilaments by (full-length) mediated endogenous a was in that 5B,C), (Fig. manner analyses immunofluorescence and fractionation eandsaitclyietclt hto nrae el AOAwt above mean with the (ANOVA represent cells Values untreated analyses). post-hoc of that to identical statistically remained ramn infcnl (* GSK3 of significantly neuritis. percentage treatment the axonal presents with graph GFP right-most The total segments. versus axonal ten GSKala the expressing mean of cells the represent of Values segments cells. ** in untreated cells; GFP untreated in versus decrease GSKala the expressing cells of segments GSK3 6. Fig. hs vnswti xn,wtotwihtedvlpn xnwill in axon developing the result which promoting without yet axons, ALS, within otherwise as events such these conditions would of characteristic phospho-neurofilaments, neurofilament– are of which which spheroids perikaryal perikarya, of the those accumulation phase. eliminating within or preventing stationary of foster task events the the with generate faced are events Neurons that associations phosphorylation neurofilament Key DISCUSSION B h etms rp rsnsqatfcto fGPN- ln axonal along GFP–NF-H of quantification ( phospho-GSK3 presents neurites increased graph MKK1 left-most of The overexpression (B) Note phospho-GSK3 levels. presents actin graph RMO24. and accompanying with The probed control. loading and as treatment used GFP–NF-H PD98059 expressing without homogenates or GSK3 and with against (p-GSK), directed Ser9 of antibody at overexpression an phosphorylated without with or probed with (MEK1), cells MKK1 pathway. from MAPK homogenates the of by replicas inhibited are and CDK5 require , P + D enx oprdteifuneo vrxrsino GSK3 of overexpression of influence the compared next We , .6frP909+GKl essutetd.Vle ersn the represent Values untreated). versus GSKala + PD98059 for 0.06 rvne h D5mdae nrae ausfrteeconditions these for values increase; CDK5-mediated the prevented 8 Fg E.I h bec fkns vrxrsin both overexpression, kinase of absence the In 8E). (Fig. 187 .5 ANOVA). 0.05, 6 ...pretg fcls( cells of percentage s.e.m. ora fCl cec 21)17 0447 doi:10.1242/jcs.153346 4064–4077 127, (2014) Science Cell of Journal P n , 5 .0;AOA.Ps o nlsswt ukyKae etfor test Turkey–Kramer a with analyses hoc Post ANOVA). 0.001; 9frec odto) * condition). each for 19 D b mdae erflmn–erflmn associations neurofilament–neurofilament -mediated 8 a on ihnbnlsbt ncellular in both bundles within found was 187 P , .1frP909+GKl essP909alone; PD98059 versus GSKala + PD98059 for 0.01 P , .5 eue vrl F–FHlevels. GFP–NF-H overall reduced 0.05) D n 8 ipae nietclrelative identical an displayed 187 § P b 0clsprcondition). per cells 20 , vrxrsinpu PD98059 plus overexpression .5frteices nGPin GFP in increase the for 0.05 b 6 rC1ec nrae the increased each CK1 or ...pretg F neach in GFP percentage s.e.m. 6 ...pretg fcells of percentage s.e.m. b n CK1-mediated and - A Nitrocellulose (A) b D atnwas -actin P b 8 within 187 P , , .0,for 0.001, .5for 0.05 b 4071 and b b .

Journal of Cell Science eue hsh-erflmnswti erts(arrowheads). and neurites perikarya within within phospho-neurofilaments treatment phospho-neurofilaments reduced calyculin of Note accumulation RT97. the or with induced with probed (GFP-H) treatment, GFP–NF-H calyculin H, expressing without nonphos Cells NF-H; (E) phosphorylated NF-H. the H, unphosphorylated penetrate Phos to (Stack). unable gel SDS-resistant were stacking of that probed formation phospho-neurofilaments treatment induced of calyculin Calyculin aggregates without SMI-32. or and with RT97 cells with of analyses Immunoblot (D) ramn.Arw eoeteprkro.()D46rdcdP1activity PP1 reduced (* D4476 soma (C) in D4476 perikaryon. preferentially without the and denote with Arrows immunoreactivity analyses). treatment. RT97 post-hoc of with image (ANOVA Representative cells (B) untreated versus NF-H * reactive and decrease the for EERHARTICLE RESEARCH 4072 in seen readily is which phase, stationary the into neurofilaments of incorporation to leading interactions neurofilament–neurofilament the of phosphatases. for among and direct interactions maintenance kinases by by also part but roles and/or in neurofilaments, mediated of phosphorylation were establishment functions divergent These the phase. axonal stationary encompass elucidate and that phosphatases transport The herein and 2013). kinases Lee, and neurofilament presented Shea 2011; and Lee, findings Pant and 1998; Shea Mushynski, 1995; and Veeranna, (Julien stabilization undergo not mean the represent H Values the indicated. within as amount fractions, the B the separately, and present (and, graphs immunoreactivity CK1 accompanying cellular without The total or treatment. with D4476 GFP–NF-H or expressing overexpression cells from of (B) activation fractions by phosphorylation neurofilament PP1. inhibits CK1 7. Fig. edmntae eenta GSK3 that herein demonstrated We A ircluoerpia fttlhmgnts()adbundle and (H) homogenates total of replicas Nitrocellulose (A) P P , , .5frteices nGPN- rRT97- or GFP–NF-H in increase the for 0.05 .1 Student’s 0.01; t et u o PAactivity. PP2A not but test) b ciiyi seta for essential is activity 6 ..** s.d. P , 0.05 ciiywti eiay hnwti xnlnuie.This neurites. axonal by within kinase achieved to than phosphatase is perikarya of ratio axons within higher and to activity relatively phosphorylation a bundling extensive maintaining of neurofilament suggests segregation finding resultant normal This neurites; fostered the perikarya. PP1 that axonal and within of immunoreactivity bundles inhibition into neurofilament phospho-neurofilament direct and or of activity accumulation perikarya CK1 of of inhibition out neurofilaments within perikarya. bundles neurofilament of accumulation and transport axonal GSK3 of potentiated activity inhibition pathway MAPK whereas association, neurofilament–neurofilament of transport GSK3 GSK3 by for transport increased axonal essential cells neurofilament GSK3 NB2a/d1 promotes MAPK inhibiting of 2004), is al., neurites the et axonal which that (Chan along and of herein into neurofilaments GSK3 occurred demonstrated activity also However, but We 2009). pathway, neurites perikarya. axonal al., to et within restricted not Yuan was 2001a; bundling al., tightly of Yabe 2009; et appearance al., the et (Kushkuley by neurofilaments ‘bundled’ neurons associated cultured and cells NB2a/d1 etitdt h xnlsat(ugadSe,19;Sa normally 1999; within Shea, is 2000). NF-H and al., RT97 (Jung et 200-kDa Yabe shaft is whereas 2000; axonal al., of reasoning et hillocks, the of to observation axonal restricted line isoforms and the This on perikarya by CK1. detected by supported be phosphorylated routinely already kDa 200 would this to immunoreactivity, immunoreactivity RT97 160 activity; alters from MAPK manipulation of MAPK NF-H independently whereas of and constitutively shift occur the would Given for phosphorylation activity. responsible CK1-mediated MAPK active, events constitutively and is CK1 CDK5 2000). that proline-directed al., et CK1 with manipulated Yabe simultaneously along 2008; not most al., have studies et the prior Veeranna However, with 1999; at Shea, associated migrating and normally isoforms, pathway (Jung NF-H isoform, is MAPK phosphorylated 2008), the 160-kDa highly al., by RT97 et the generated because (Veeranna is unexpected, including which was This immunoreactivity, isoforms, inhibition. entire CK1 NF-H the following Herein, with of kDa. immunoreactivity 200 to RT97 range kDa prominent 160 observed from gels we SDS on shift migratory neurofilamentH of axons. cycles within ongoing dephosphorylation are and there phosphorylation that al., et suggests Shea and (herein 1993), PP2A and with along PP1 phospho-neurofilaments coupled of increased inhibition finding, following are axons This there 2009). 2007; al., that finding et al., the Yuan et 2001a; Trivedi al., 1986; et Logvinenko, Yabe and Nixon GSK3 1988; and Nixon, Lewis of 2009; al., et (Kushkuley inhibition cytoskeleton and stationary the enter leave neurofilaments respective transporting However, along their PP1. of neurofilaments activation by of neurites transport continued axonal mediated pathway CK1 MAPK of the and out not or transport whether neurofilament determine not of did We promotion perikarya. in CK1 and pathway 1999). Shea, and (Jung axons PP2A proximal and perikarya ganglion cell retinal rapid within the immunoreactivity phospho-neurofilament with consistent is possibility K ciae P,wihwsesnilfrtasoainof translocation for essential was which PP1, activated CK1 nadto orglto fP1atvt,C1mdae nNF- an mediated CK1 activity, PP1 of regulation to addition In MAPK the for roles essential demonstrate findings above The b ora fCl cec 21)17 0447 doi:10.1242/jcs.153346 4064–4077 127, (2014) Science Cell of Journal Steln ta. 93]adpeetdGSK3 prevented and 1993)] al., et (Sutherland b b ciiy nraigMP aha activity pathway MAPK increasing activity: hshrlto tSr wihinactivates [which Ser9 at phosphorylation nsitu in enovo de olwn niiinof inhibition following b idcdihbto of inhibition -induced cuuainof accumulation § b b 0 kDa 200 -induced -induced ´ b nchez and

Journal of Cell Science EERHARTICLE RESEARCH erna 95.Orfnig aepoie ute nih into insight further provided have findings Our GSK3 1995). Veeranna, and MAPK of isoform. combination this generated a activity that slowest-migrating this 1988). indicating depleted Nixon, pathway isoform, and MAPK (Lewis the pathway of optic Inhibition in isoform to 210-kDa corresponding the gels. isoform, neurofilament SDS slowest-migrating on the of migration induced NF-H of sites those GSK3 migration in to phosphorylation alterations similar CDK5-specific minor isoforms Site-directed of 1988). NF-H Nixon, and mutagenesis (Lewis of pathway optic array in observed an revealed content hshrlto niisnuoiaetpoelss(atand (Pant proteolysis neurofilament inhibits Phosphorylation acrylamide low relatively a containing gels SDS of use The b ciiyidcdRO2 eciiyadteappearance the and reactivity RMO-24 induced activity b npoelssadbnln sta h -emnl17aioacid one amino 187 2000), C-terminal kinases the al., these that is of et bundling impact and (Chen differential proteolysis on the bundling GSK3 from residues, for and arising acid CK1 speculation essential the amino is of 187 majority sequences, terminal the contains the which against that that protection indicate Given provides findings domains proteolysis. These KSP pathway. the MAPK of inhibition phosphorylation the following and proteolysis NF-H CDK5 prevent of to failed CK1 and not but GSK3 pathway GSK3 of following MAPK inhibition the and/or upon proteolysis and CDK5 NF-H CK1 of inhibition increased by simultaneous observed not We CDK5 and protection. by phosphorylation MAPK, that and demonstrating by regulation this ora fCl cec 21)17 0447 doi:10.1242/jcs.153346 4064–4077 127, (2014) Science Cell of Journal pce,crepnigt h irtr oiino h NF- the of position migratory the to corresponding species, NF- expressing to H cells fused from NF-H Fractions of GFP. position 30–35-kDa migratory the to corresponding at GFP-reactive migrating GFP- prominent species expressing displayed cells NF-H from full-length the Fractions tagged along panel. indicated each are of protein right fusion each of positions vrxrsinicesdti ai o FHbtntfrNF- for not but NF-H H for ratio this GSK3 increased H overexpression. overexpression and kinase NF-H of for absence identical the and was Materials ratio the This in Methods. surrounding described the as versus calculated bundle axoplasm within the intensity depicts GFP graph of accompanying ratio The indicated. as CK1 full-length H GFP-tagged or expressing of NF-H cells region of central the neurites of axonal images Representative filamentous (E) depicting profiles. neurites axonal higher of Inserts: magnification bundle. neurofilament the situated with association centrally their including axons, along distribution and GFP–NF-H NF-H overexpressing cells kDa). Representative (125–140 (D) NF-M and NF-H kDa) endogenous (160–205 to correspond species immunoreactive additional Minor neurofilaments. with endogenous the co-assembly and indicating cytoskeleton fraction, the bundle-enriched into incorporated and antibodies kDa. 175 NF-H and Note 155 between observed GFP- were slower-migrating species additional reactive GFP; to fused fragment H rmclsepesn FHo NF-H or fractions NF-H (B) expressing bundle cells and from (H) nitrocellulose homogenates presents initial row of lower replicas The SMI-31. and GFP– 34 expressing cells GFP-NF-H from and fractions NF-H (I) insoluble Triton-soluble and of (S) upper replicas The nitrocellulose (C) presents experiments. row duplicate from mean cells the multiple represent Values MKK1. except NV) h ai fGPi ude essaols was axoplasm versus ( bundles increased in GFP of ratio The ANOVA). nrae olwn vrxrsino l iae ( kinases all was of bundles overexpression with following cells increased of (MEK1, percentage overexpression The kinase MKK1). without expressing or cells with for GFP–NF-H bundles axoplasm within surrounding GFP the of versus intensity and densitometric bundles relative neurofilament the GFP-labeled displaying cells of GFP–NF-H GSK3 expressing without cells or of with neurite axonal and hillock to contribute bundling. kinases neurofilament neurofilament Multiple 8. Fig. D D 8 ipae rmnn 5-D GFP-reactive 150-kDa prominent a displayed 187 8.* 187. D 8,adimnsandwt T7 oethe Note RT97. with immunostained and 187, P D , D P 8,wt + n ihu – vrxrsinof overexpression (–) without and (+) with 187, b 8 eandimnratvt ihall with immunoreactivity retained 187 .5(Student’s 0.05 , rC1 n vrxrsino GSK3 of overexpression and CK1, or .5 olwn vrxrsino l kinases all of overexpression following 0.05) , b D 8 ( 187 vrxrsin B h percentage The (B) overexpression. 9–5 D nSSgels, SDS on kDa 195–250 A ersnaieiae fthe of images Representative (A) D t 8)poe ihR9,SMI- RT97, with probed 187) test). D 8.Migratory 187. b eitdthis mediated , 6 ...from s.e.m. b b consensus rCK1 or D P 8 in 187 , 4073 0.05; b

Journal of Cell Science S-ihrgo ol lo rtoyi fbundled of localized axonal required for proteolysis any means including a cytoskeleton, allow the provide of the might could remodeling of which dephosphorylation case, region neurofilaments, the protease potential indeed to were KSP-rich exposed this it, of If proximal portion more activity. a the or leaving the 2009), region, al., KSP-rich mediate neurofilaments et might (Kushkuley incorporates bundles regions, into that KSP-rich crosslinking the cation-dependent than rather residues, ARTICLE RESEARCH xld h osblt htmnplto fkns and/or kinase of manipulation 4074 provides that cannot we possibility 1995), clearly example, phosphatase Veeranna, the For and and/or (Pant exclude phosphorylation pathways. proteolysis cellular kinase to derived resistance neurofilament other of was on hererin, although effects impact seen indirect as the by neurofilaments, of on at manipulation that some possibility the exclude least cannot we such, As neurofilaments. more or two impractical. require plus is which would GFP–NF-H constructs), kinases (i.e. kinase multiple transfection of triple consequence successful of construct. the neurofilament examine GFP-tagged consequences To respective the have the versus of the more construct times GFP– We kinase two examine with utilizing by co-transfected kinases. GFP–NF-M to cells or NF-H in multiple unable kinases manipulated of routinely were overexpression we simultaneous Veeranna, limitation that further neurofilament- and A of 2009). is (Pant al., because et conditions cells (Kushkuley proteases cell-free associated within under proteolysis and 1995) rapid dephosphorylation in neurofilament kinases.results the individual experimental of to manipulation Unfortunately, contributed kinase following the events of observed phosphorylation exclude changes impact prior completely the that cannot possibility of we examination Accordingly, manipulation. to prior neurofilaments CDK5. and pathway neurofilament MAPK proline-directed the GSK3 the of and to by kinases CK1 neurofilament regulated kinases contribute on functionally non-proline-directed are impact the could the of that prior dynamics kinases represent indicate and collectively these CDK5 findings CDK5, by which and GSK3 least by pathway neurofilament at of mechanisms MAPK phosphorylation inhibition the CK1, neurofilament and axonal by pathway to of proteolysis MAPK axons contributed concentration into the transport likely by neurofilament sufficient also of Promotion maintaining MAPK neurofilaments. by and CDK5 bundling by proteolysis rather PP1 GSK3 of of activation inhibition by than by this within mediated neurites phosphorylation CK1 although extensive axonal perikarya, of into accumulation CK1 the transport pathway, restricting neurofilament MAPK of the promoted segregation Like by also restrict neurites. to axonal GSK3 neurofilaments essential within bundling was of those perikarya within downregulation of activity MAPK-pathway-mediated phosphorylation CDK5. prior kinases GSK3 proteases required these encompassing phosphatases. events that of and series systematic highlighted hierarchical our a inhibition However, regulated and establishment phase. and the stationary overexpression to the contributed of kinases maintenance these within of were all neurofilaments axons, once that indicating neurofilaments, GSK3 CK1, that indicated 2006). al., et (Xie branching oprsno F–FHwti xnlnuie n bundles and neurites axonal within GFP–NF-H of Comparison h iae tde eenhv utpesbtae beyond substrates multiple have herein studied kinases The dephosphorylate not did we that is analyses these of limitation A b idcdnuoiaetbnln ihnaos These axons. within bundling neurofilament -induced b nadto,ihbto fneurofilament of inhibition addition, In . b b mdae erflmn bundling neurofilament -mediated n D5alicesdbnln of bundling increased all CDK5 and b b ntr otiue oetbihetadmitnneo the of maintenance and establishment to phase. the contributes stationary that turn which neurites, conclusion transport in axonal within the overall increases phospho-neurofilaments and activities the of kinase amount divergent diminish proteolytic these on of not impact impact collective of does their it modulation of systems, and some (Moran kinases by the cells mediate if neurofilaments even these herein Notably, in 2008). studied al., kinesin) phosphatases et same Dubey upon 2005; the dependent al., tau et by also of transport CDK5 is axonal and alter (which not pathway did that herein demonstration MAPK utilized methodologies prior the This our transport. of by axonal manipulation overall however, in reduced, disruptions is from likelihood part derived in distribution were least neurofilament manipulations at in phosphatase possibility alterations al., and/or the the et kinase of following exclude (Morfini some least itself completely at kinesin cannot that on therefore impact We an 2002). directly removes also have herein 2011; indirectly examined so Lee, kinases and the and phosphorylation doing 2009), (Shea al., pool et in Kushkuley transporting whereas and the from bundling, neurofilaments Similarly, neurofilament proteolytic promotes levels. of increased to contribute activity neurofilament further would suppressed which themselves, also enzymes activities phosphatase edt le ciiyo atcptn iae rta fteoverall the of that or kinases system. participating transport of activity alter to need the on without and development long-lasting during activities polarity a macrostructure establish supportive can phosphatase filaments intermediate how of and class demonstrate Lee, this herein kinase and demonstrated (Shea as of individual dynamics neurofilament the interplay more of occurred, The lifetime be has the 2013). support synaptogenesis for once place structural in which, stable remain axons, long-term, in than for nowhere crucial need Perhaps 1994). the and (Fuchs, tissues can cells of to formation strength the phase. to correspondingmediate mechanical stationary neuron their provide the of filaments nor developing repair Intermediate kinases, or a establishment these for for of phosphatases, requirement activity apparent the axons,modulate no within phase is stationary there the form essentially to phase self-assembly neurofilaments undergo phosphorylated stationary However, extensively pathways. the that neuronal given essential of multiple on assembly impact would foster and to activity transport their neurofilament modulate to requirement any Any homeostasis. at neurofilaments out and axon. worn can elongating, the or that along still damaged system locus proximal replace maintenance are a and of regions into repair transition maturation distal stabilization allows while ultimately and transport allows regions same dynamics, differentiation axonal the these neuronal Using simplifies axons. throughout within activity battery systems continued This to transport itself allows system maturation. this of transport Notably, the axonal maturation. in axonal alteration for foster any stationary for essential the need to the establish is avoids neurofilaments to which pool indirectly some transporting cytoskeleton, allowing but the by from transport, withdraw transport axonal with neurofilament interferes with interfere 0 ea oiesrmaddfeetae ih1m iuyy cyclic dibutyryl mM 1 with differentiated and serum containing bovine DMEM in fetal cultured 10% were cells neuroblastoma NB2a/d1 Mouse Differentiation METHODS AND MATERIALS -emnlnuoiaetpopoyainde o directly not does phosphorylation neurofilament C-terminal h iae tde eensaepvtlrlsi neuronal in roles pivotal share herein studied kinases The ora fCl cec 21)17 0447 doi:10.1242/jcs.153346 4064–4077 127, (2014) Science Cell of Journal

Journal of Cell Science Ksklye l,20;Yb ta. 01) rti ocnrto was concentration scientific). Protein (Thermo 2001a). assay interface BCA al., sucrose a et the by Yabe at determined neurofilaments 2009; recovered al., ‘individual’ were et bundles) (Kushkuley cushion; within sucrose contained (not the through sedimented D5atvtrp5(arc ta. 99] diinlclue eetreated were the cultures (20 of roscovitine Additional form inhibitors 1999)]. pharmacological active al., the constitutively with et and (Patrick truncated p35 [the pathway) activator p25 MAPK CDK5 and the 1999) of al., activator et upstream (Li (an MKK1 active constitutively 2004). al., A et enzymes. (Chan conditions same these the under attained with digested plasmid § template prepared cloned then the primers, the into in included were sites consensus which for BamHI, CAAAGTGAACACGGATGCT-3 n etiue t15,000 sucrose, M at 1 containing centrifuged buffer same the and over layered were homogenates Wagner 2009; 2003). al., al., et (Kushkuley et previously described as Rhodamine with F–FHsdam) F-agdsdam nwihtesrn residues serine the which in sidearms wild-type domain, GFP-tagged rod sidearms), the GFP–NF-H lacking (i.e. sidearm CA) NF-H isolated the Carlsbad, GFP-tagged (GFP–NF-H), NF-H (Invitrogen, full-length GFP-tagged Lipofectamine expressing constructs using with transfected were Cells constructs neurofilament of Expression these transport’. of ‘axonal neurites as axonal to along referred and writing, is of cells into clarity and neurofilaments simplicity of For 1999). translocation al., et (Yabe (dbcAMP) AMP ARTICLE RESEARCH iho ihu uiidknss(uhue ta. 09 o t37 at h 2 for 2009) al., incubated et (Kushkuley were kinases above purified without described or as with obtained neurofilaments Individual manipulations neurofilament Cell-free mM 1 containing 6.8, Use pH and MES, Care 0.1M Animal MgCl in Institutional in resuspended our adult out were of carried from Committee) were approval procedures the neurofilaments animal with (all cord accordance genders both Spinal of fraction. mice C57BL6 defined Triton-soluble was the supernatant resulting as the 1% 50 and and containing cytoskeleton, PMSF 6.8, Triton-insoluble mM pH 1 Tris-HCl, (15,000 EDTA, centrifuged mM mM 5 50 X-100, in Triton homogenized were Cells Fractionation Li CK1- mouse UK), College, mouse active constitutively expressing plasmid GSK3 a with transfected were Cells activities kinase of Manipulation was motif 5 KSP primers: last of ACTTCACTTC-3 the set following and the AccI using between PCR sequence by (Chen the NF-H amplified a of 2000), domain is rod al., there the of that et region C-terminal Given the in construct. site NF-H AccI unique GFP-tagged full-length above C-terminalthe distal-most (GFP–NF-H the acids lacking Lee amino NF-H 2003; rat 187 GFP-tagged al., and 2011), et al., (Ackerley et states nonphosphorylated or phosphorylated permanently mimic residues to (GFP–NF-Hala) aspartate residues to alanine mutated or (GFP–NF-Hasp) were sites consensus CDK5 C-terminal the in 60 ih0.1 with h iaecntut(hne l,20;Se ta. 2004). al., et Shea with 2004; (1 co-transfected al., were et utilized (Chan GFP construct displaying were kinase cells the GFP–NF-H that insuring the versus of respectively); times two constructs than more GFP–NF-H, kinase and kinases kinases, with a of co-transfection ERK1/2 manipulation For impact of pathway 1997; downstream MAPK alteration al., the to is that refer et neurofilaments we Given Meijer on 2001). 1995; MKK1 al., of et al., manipulation Sarno et 2004; Dudley al., 1983; et Rena al., et (Cheng respectively + 0 rnfcinefcec o F-agdnuoiaetcntut is constructs neurofilament GFP-tagged for efficiency transfection 70% ooti rcin nihdi ude erflmns additional neurofilaments, bundled in enriched fractions obtain To m ) hc r cieaantCK,MK,GSK3 MKK1, CDK5, against active are which M), 1 M,D46(100 D4476 mM), (10 2 b MET,1m MFad50 and PMSF mM 1 EGTA, mM 1 , agnru itfo hi ilr nttt fPyhar,King’s Psychiatry, of Institute Miller, Chris from gift generous (a m g/ m D5adisatvtrp5 R12ado GSK3 and/or ERK1/2 p35, activator its and CDK5 l 9 mlfe C rget eedgse ihAc and AccI with digested were fragments PCR Amplified . g 5mn.Tersligple a eie sthe as defined was pellet resulting The min). 15 ; a dntdC1hri,Oiee okil,MD), Rockville, Origene, herein, CK1 (denoted D m g 8) GFP–NF-H 187). )adttarmbnoraoe(tBBT; tetrabromobenzotriazole and M) o 5mn ude neurofilaments Bundled min. 15 for 9 n 5 and onsfrsmlct fwriting. of simplicity for points t 9 -CTGAGGATCCTAAGGGG- m /lluetnadlabeled and leupeptin g/ml D m 8 a rprdfrom prepared was 187 ) D85 (10 PD98059 M), m /lluetnand leupeptin g/ml b 9 ,CK1andCK2 m -AGAGTCGC- n 0.5 and g m M), m ˚ C b g Ca ta. 04 hne l,2005). quantified al., were et bundle Chan for the 2004; segment to al., adjacent neurite et GFP-tagged (Chan area of central the of the incorporation of intensity within that of GFP and the extent bundle bundles, the the neurofilament monitor (Chan axonal neurite into To each in neurofilaments within 2004). intensity fragment cone. GFP central al., growth of the as et that and dividing by (defined by fragment hillock distal determined the segments the was excluding rate equivalent translocation distal), three The and into central divided proximal, were condition) otiig01 we-0fr1hte nuae vrih t4 at overnight incubated were saline then Membranes Tris-buffered h in 1 electrophoresis. fluoride for sodium for Tween-20 mM 0.1% 5 urea containing and M BSA 5% 8 with blocked fraction with bundled solubilized The nitrocellulose. gel was SDS onto to transferred subjected and protein, total electrophoresis to according normalized were Samples analysis immunoblot and Electrophoresis e9(hc nciae GSK3 inactivates (which Ser9 GSK3 assays phosphatase and Kinase each ( axonal shaft neurites within the axonal axonal GFP of studies, the excluding additional within of In GFP shaft 2001b). percentage total segments, al., was the the et The to (Yabe equivalent and relative neurites transport. cone, calculated ten was growth axonal axonal segment into the of and along divided hillock index and was an into neurites as constructs Translocation quantified ImageJ. neurofilament using quantified of was distribution distribution Neurofilament neurofilament intracellular of Monitoring antibodies. secondary serum. goat normal 2% 4 and at BSA overnight 1% incubated containing then blocked PBS and were min/rinse), in Cultures (5 min PBS in 30 4% times min for two with 10 rinsed for fixed temperature, 7.4) room pH were at (PBS, coverslips saline phosphate-buffered poly-L-Lysine-treated in paraformaldehyde on grown Cells Immunofluorescence ob oprdwr lcrpoee ntesm e,taserdonto transferred gel, an same the simultaneously. samples from All visualized on 1999). and electrophoresed signal al., nitrocellulose were et background Yabe compared from 2001a; be subtracted al., the to was et lane (Yabe J; digitized identical species the reactive Image in each in area using quantified sized, identically replicas were adjacent, (Promega, of species kit with substrate Immunoreactive images NBT/BCIP incubated room 1999). WI). a at then Shea, using Madison, h buffer developed 1 and and same for temperature (Jung the antibodies neurofilaments secondary with (R39) against alkaline-phosphatase-conjugated washed state directed were phosphorylation antibody Membranes nonphospho- an of (Invitrogen)], and regardless RMO-24 (SMI-32) and SMI- MA) epitopes and Dedham, SMI-34 UK), antibodies (Covance; London, 31 Psychiatry, and of of Institute Invitrogen) gift Anderton, (generous Brian (1:1000, [RT97 phospho-epitopes GFP neurofilament against directed against directed antibodies 4 utrswr isdtretmswt B,icbtdfr3 i at min 30 for incubated PBS, with times three 1:100 37 rinsed RMO- or and were R39, SMI-32 Cultures against SMI-31, or SMI34, 24. RT97, antibody against anti-GFP antibodies of of dilution dilutions 1:500 a and BSA ihatbde ietdaantttlGSK3 total total Ser9, probed against at and phosphorylated directed nitrocellulose antibodies onto transferred with electrophoresis, to subjected rwtotGSK3 without or substrate UsaeBohmcl,Lk lcd Y.Tepretg of 2009). al., al., et ends, (Kushkuley percentage their et neurofilaments at multiple (Kushkuley apart neurofilaments of splay detection The microscopy other facilitating typically neurofilaments fluorescence than aligned NY). using Closely more 2009). by or Placid, quantified three were with Lake aligned neurofilaments Biochemicals, (Upstate uui nioyD1.Gvnthat Given anti- control, loading DM1A. a as antibody and, Signaling), (Cell tubulin Thr41 and Ser37 Ser33, ˚ nPScnann %BAad130dltoso appropriate of dilutions 1:300 and BSA 1% containing PBS in C b ciiywsasydb oioigpopoyaino GSK3 of phosphorylation monitoring by assayed was activity ora fCl cec 21)17 0447 doi:10.1242/jcs.153346 4064–4077 127, (2014) Science Cell of Journal b ctnna e3,Sr7adTr1 ooeae fclswith cells of Homogenates Thr41. and Ser37 Ser33, at -catenin b vrxrsino 0m Li mM 10 or overexpression b ctnn and -catenin, b n hshrlto fteGSK3 the of phosphorylation and ) . b ctnni erddonce degraded is -catenin 0clsfrec condition each for cells 20 b ˚ ctnnpopoyae at phosphorylated -catenin b nPScnann 1% containing PBS in C GSK3 , + b ramn were treatment hthdbeen had that . 5freach for 25 ˚ with C 4075 b at b

Journal of Cell Science ue,M,Caduy . aiu .adSe,T B. T. Shea, and H. Kabiru, P., Chaudhury, M., Dubey, ig . i,W,Lu .C,Yn,J . e,D . i,J,Brhlmuz G., Bartholomeusz, J., Xia, F., D. Lee, Y., J. Yang, C., J. Liu, W., Xia, Q., Ding, B. T. Shea, and J. DeFuria, J. Larner, and S. Creacy, K., Cheng, hn,H .adLus .F. C. Louis, and L. H. Cheng, N. Hirokawa, B. T. and Shea, Z. Zhang, and T., D. Nakata, Ortiz, J., F., Chen, A. Pimenta, T., J. Yabe, K., W. Chan, hn .K,Dcesn . ri,D,Pmna .F,Mrn .M,Mtl J., Motil, M., C. Moran, F., A. Pimenta, L. D., Ortiz, M. A., Garcia, Dickerson, K., and W. P. Chan, J. Julien, S., Millecamps, M., D. Barry, C. C. Miller, and V. Anderson, N., P. Leigh, T., S. al-Sarraj, P., N. and Bajaj, S. C. Strack, C. S., Miller, and Pelech, P. N. H., Bajaj, Zhang, Jr, L., R. Coffee, G., D. Adams, aei wihcnas erfetdi total in reflected be also can (which catenin GSK3 by phosphorylated ARTICLE RESEARCH 4076 H., B. Anderton, J., Brownlees, J., A. Grierson, H., P., B. Thornhill, Anderton, S., P., Ackerley, Thornhill, J., Brownlees, J., A. Grierson, S., Ackerley, Federation. References Science National the by supported was work This manuscript. Funding the S.L., wrote figures. and final experiments the the prepared designed T.B.S. T.B.S. and and S.L. H.P. experiments. all performed S.L. contributions Author interests. competing no declare authors The interests Competing a eetdi h ah h e a re n xoe oXryfilm. X-ray to exposed and dried was gel The radioactivity wash. no the until in pyrophosphate detected sodium was 1% and acid trichloroacetic C,p .,cnann 0m MgCl Tris- mM mM 10 10 in containing gel the 7.5, of pH incubation HCl, by out carried was phosphorylation [ .5 we 0 h e a qiirtdi 0m rsHl H7.5, pH Tris-HCl, mM MgCl 10 in mM equilibrated 10 was gel containing The 40. 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Li, Y., Pan, Y., Li, JNK of roles GSK-3beta. neurofilaments: and phosphorylated of accumulation perikaryal induces synthase. glycogen of activation Specific II. Biochem. adipocytes. rat isolated 286. oano erflmn rti- N-)frstecosrde n regulates and crossbridges the forms (NF-H) protein-H neurofilament of domain a in incorporation cytoskeletal manner. and discontinuous transport axonal undergo can Neurofilaments 4642. hshrlto fteln ie elconnexin49. cell fiber lens the of phosphorylation formation. bundle neurofilament ndr .J,Mlk . at .C n ha .B. T. transport. Shea, axonal and neurofilament C. regulates H. kinase Pant, protein K., Malik, J., S. Snyder, disease. and turnover transport, 313 neurofilament in movements neurones motor affected in present is ALS. and in accumulations (ALS) neurofilament sclerosis label lateral that amyotrophic antibodies in for epitopes (NF-H) generate heavy to neurofilament chain phosphorylates (CDK-5) kinase-5 dependent Cyclin synthase cells. glycogen transfected and in kinase-5 cyclin-dependent kinase-3alpha by fragments side-arm chain holoenzymes. 2A 42644-42654. phosphatase serine/threonine protein by E. B. Wadzinski, ieampopoyainrgltsaoa rnpr fneurofilaments. of transport C. axonal C. Biol. regulates Miller, phosphorylation and arm E. C. side Shaw, N., P. C. Leigh, C. neurons. Miller, transfected and in E. neurofilaments C. of Shaw, transport N., P. Leigh, - K ciiywsmntrdb aenglzmlg CegadLouis, and (Cheng zymology gel casein by monitored was activity CK1 32 ]T o tro eprtr olwdb ahn ih5% with washing by followed temperature room at h 2 for P]ATP 2110-2120. , 161 rg erpyhpamcl il Psychiatry Biol. Neuropsychopharmacol. Prog. 489-495. , 56 183-189. , b ri Res. Brain activity. 20) oiierglto fRf-E12EK/ signaling Raf1-MEK1/2-ERK1/2 of regulation Positive (2005). ˚ nfu hne fteaoebfe containing buffer above the of changes four in C elMtl Cytoskeleton Motil. Cell 20) r soitswt n rmsGK3eafor GSK-3beta primes and with associates Erk (2005). b 20) rei niisnuoiaettasotand transport neurofilament inhibits Arsenic (2007). 2 1181 Dn ta. 05,adces nphospho- in decrease a 2005), al., et (Ding 19) noeoscsi iaeIctlzsthe catalyzes I kinase casein Endogenous (1999). 19) hshrlto fnuoiaetheavy- neurofilament of Phosphorylation (1997). o 0mna omtmeaue Casein temperature. room at min 30 for .Cl Sci. Cell J. 74-82. , .Neurochem. 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Culbert, E., Daniel, Di T., Hagen, Y. Goldberg, E. W. Mushynski, and I. B. Giasson, Wong, L., Rivas, K., M. Saqcena, C., Lora, F., Parisi, S., Riccardo, M., Galletti, E. Fuchs, acoi .B,Ot,C,Cnh,I .adMun and I. I. Concha, C., Otth, B., R. Nairn, Maccioni, Z., Yan, J., A. DeMaggio, A., Nishi, A., J. Bibb, J., Ule, H., X. Ma, F., Liu, C. H. Pant, and P. Grant, J., Gu, Veeranna, S., B. Li, R. R. Desrosiers, and M. Lamarre, G. Shaw, and S. G. Bennett, A., B. Hollander, S. G. Bennett, and A. and B. Hollander, E. R. Barry, C., Wirbelauer, R., C. Thoma, J., Lisztwan, A., Hergovich, er,S .adKlie,S D. S. Killilea, and P. S. Henry, J. A. Harwood, R. A. Saltiel, and J. A. Bridges, J., S. Decker, L., Pang, T., D. Dudley, ik .T,Gat . iaa .adPn,H C. H. Pant, and H. Hidaka, P., Grant, T., W. Link, and F. Letournel, F., J. Leterrier, J., Eyer, S., Lee, K., W. Chan, J., Kushkuley, V. Timmerman, and D. Bouhy, A., Holmgren, eaaay . i .S n at .C. H. Pant, and S. B. Li, S., Kesavapany, B. T. Shea, and C. Jung, E. W. Mushynski, and P. J. Julien, atb ipaigknsncro erflmnsatnaetumdae neurite tau-mediated attenuate neurofilaments cargo: instability. kinesin displacing by part erflmn rtisb rti iaeF gyoe ytaekns 3). kinase synthase (glycogen FA kinase Chem. protein Biol. J. by proteins neurofilament metabolism. neurofilament of 1534-1546. regulation the and (Cdk5) slices. in brain neurofilaments rat of active accumulation metabolically and phosphorylation induces 2A phosphatase otr erflmn-erflmn soitosta opt ihaxonal with compete that associations transport. neurofilament-neurofilament fosters 669-676. hshrlto fnuoiaethaysd-rsb lcgnsynthase glycogen by side-arms heavy kinase-5. cyclin-dependent and neurofilament kinase-3 of phosphorylation subunit. neurofilament heavy Pharmacol. the Biochem. of phosphorylation Neurosci. in J. involved kinases kinases. 1 synthase kinase al. glycogen casein et by and dMyc 3beta D. of kinase Grifoni, degradation F., ubiquitin-dependent for Serras, responsible A., Serra, B., strength. iaeCk.Srcua set,rlsi ergnssadivleetin involvement and neurogenesis in roles aspects, pathology. Alzheimer’s Structural Cdk5. kinase receptors. glutamate metabotropic by USA 1 kinase P. casein Greengard, and C. 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