is properly cited. is properly which permits distribution use, and reproduction medium,in any originalwork provided the anopen articleThis is access underthe terms oftheCreativeCommons License, Attribution Sons Ltd. byFEBS FEBSBio (2017) Open©2017 TheAuthors.Published 10.1002/2211doi: differences to lead between the of thisversion citethisarticle and asVersion Record. Please been throughthe This article acceptedhas been forpublication andundergone fullpeer review buthasnot PICK1 via CKAMP44 PKC phosphorylates Running title P Email: Germany Berlin 10117 1 Charitéplatz Charité A.Shoichet Sarah Prof. * Article :Research Article type C Protein kinase C(PKC) hone +4930450539099 Stella
Accepted ontact information: Article protein [email protected] – - AmreiKunde,
Universitätsmedizin Berlin Universitätsmedizin
copyediting, copyediting, pagination andproofreadingtypesetting, process,which may Neuroscience Research Center / Institute of Biochemistry of /Institute Center Research Neuroscience - Shisa9/ 5463.12261 Charité Charité
Nils Rademacher,Nils Hanna andSarahShoichet Zieger, A.
neuronal scaffold PICK1
CKAMP44 through interactions with
-
Universitätsmedizin Berlin, Germany Berlin, Universitätsmedizin
regulates AMPA receptor
Press and John Wiley andJohn & Press
auxiliary *
Sons Ltd. by FEBS WileyFEBSBio (2017) Open © 2017TheAuthors. Published andJohn& Press ofproteins family including function, receptor modulate that proteins transmembrane brain the in storage receptors synap in changes regulating (α system. nervous central the in transmission T Introduction PKC. of a function novel highlights also and plasticity and transmission C for implications has work This Shisa9/CKAMP44 of phosphorylation regulates thereby and C (PKC), kinase protein the with complex tripartite a of formation facilitates which PICK1, protein neurons. central in plasticity synaptic regulating for important are that proteins scaffold multiple with interacts Shisa9/CKAMP44 receptor AMPA central in the in neurotransmission of transmembrane a is Shisa9/CKAMP44 system. mediators nervous essential are receptors AMPA Synaptic Abstract phorbol PMA, Y2H, Protein; Regulatory AMPAR Transmembrane C; protein kinase density Protein postsynaptic PKC, A; kinase PSD PDZ, kinase guanylate associated Membrane MAGI1, Cysteine CKAMP44, II; kinase protein dependent AMPA, Abbreviations PKC neurobiology, molecular Keywords e ionotropic he -
Acceptedamino Article
α - - amino
3 a - - 5 Dg, n ZO and Dlg1, 95, re regulated is therefore critical for our molecular understanding understanding molecular our for critical therefore is regulated re
hydroxyl
-
3
, - ltmt receptors glutamate
hydroxyl among others among - soitd rti cmlxs Hr w so ta te yooi ti of tail cytosolic the that show we Here complexes. protein associated . - -
5 soitd unlt Kns Ivre 1 MGK Membrane MAGUK, 1; Inverted Kinase Guanylate Associated Recent studies indicate that they are found in complex with additional additional with complex in found are they that indicate studies Recent - methyl postsynaptic proteins, proteins, postsynaptic - 5 - methyl -
; nPIST, neuronal protein interacting specifically with Tc10; Tc10; with specifically interacting protein neuronal nPIST, ; - our understanding of how PICK1 modulates AMPAR modulates PICK1 ofhow understanding our 4 1; PICK1, Protein Interacting with C Kinase 1; PKA, Protein Protein PKA, 1; Kinase C with Interacting Protein PICK1, 1; i strength; tic - . isoxazole 5 SP0, synapse SAP102, 95;
- 4 - isoxazole
We focussed on the interaction with the scaffold scaffold the with interaction the on focussed We are of particular importance for fast synaptic synaptic fast for importance particular of are - propionate) propionate)
Among the Among lcdtn te mechanisms the elucidating AMPAR auxiliary protein, phosphorylation, phosphorylation, protein, AMPAR auxiliary - rpoae CMI, Ca2+/calmodulin CaMKII, propionate; yeast yeast - Knot AMPAR Modulating Protein 44; 44; Protein Modulating AMPAR Knot receptor se ligand se
protein recently found to be present present be to found recently protein - - two hybrid two 12 associated protein 102; TARP, TARP, 102; protein associated - myristate s are are s - gated .
especially important especially e.g. -
13 receptors, receptors,
- - ctt; PSD acetate; the Shisa/CKAMP Shisa/CKAMP the terminal residues. residues. terminal
y hc the which by of information information of the - mediated mediated
AMPA AMPA -
95, 95, for for se se - - Sons Ltd. by FEBS WileyFEBSBio (2017) Open © 2017TheAuthors. Published andJohn& Press AMPA receptor C cytosolic the that PDZ to binds that region surface desen of AMPAR modulation in studied been has auxi AMPAR auxil receptor properties function this for that suggest groups several from this for responsible exclusively interactions t shown tail cytoplasmic the with directly interacting N PSD domain C conserved transmission. Protein) Modulating as to referred also now is proteins Shisa of subgroup function receptor influencing of capable are 8 and modulating properties by transmission synaptic regulates there presence indicate studies recent more Indeed functions. neuronal specific in involved be may proteins these that suggesting Allen (see neurons CNS of subset and the of several that confirmed have analyses for transcript the on focussed brain mouse the in Th Japan. southern in found is that Sphinx) were Shisa o Acceptedumerous Article n fo te el surface cell the from and
- 9 95 /CKAMP , named
that such that , Importantly . xii hg epeso i te brai the in expression high exhibit , h 80 the Dlg1
[ of 3 yooi proteins cytosolic between between ] - l
. In addition, it has now been shown that not only only not that shown been now has it addition, In . ay rtis te AP Tasebae MA Rgltr P Regulatory AMPAR (Transmembrane TARP the proteins, iary terminal sequence typical for proteins that have been shown to bind bind to shown been have that proteins for typical sequence terminal , andZO Shisa ay rtis fr eet eiw, see reviews, recent (for proteins iary
isolated isolated Another property that these these that property Another rtis ee rgnly ecie fr hi rl in role their for described originally were proteins
- function (for review see review (for 90 AA structurally conserved motif originally identified for its presence in in presence its for identified originally motif conserved structurally AA 90
PDZ
, -
after a form of sculpture with a large head (similar to the Egyptian Egyptian the to (similar head large a with sculpture of form a after terminal tail tail terminal family
PDZ domain proteins and AMPA recep AMPA and proteins domain PDZ the latter function function latter the - ligand - most d MA receptors AMPA 1 proteins. 1 proteins.
via this mechanism via this
that domains of specific synaptic scaffold molecules scaffold synaptic specific of domains bsd n their on based ,
depth si
interactions interactions
Shisa [ ti 6
t yass abu PZ oan ad are and domains PDZ harbour synapses at [ s ] 7 euain f AMPA of regulation . ation and also play a critical role in in role play acritical also ation and ] f MA auxiliary AMPAR of ) t : Notably
ransmembrane ransmembrane
.
9 is present in in present is 9 it Since s Since s well is Mouse Mouse te are they , f TARPs of are are , it has become clear in recent years that that years recent in clear become has it , n, e . everal everal s
CKAMP
common
first study on study first important important of and Shisa established that TARPs are are TARPs that established ri Atlas Brain transmembrane receptors, and and receptors, transmembrane Shisa of these proteins also influence also proteins these of AMPAR
each each clearly clearly
AMPA receptor receptor AMPA
fe rfre to referred often [ R transcripts, including including transcripts, 4
, proteins share is the presence of a a of presence the is share proteins
[ 5 blt to ability ufc expression. surface 7 proteins proteins for the CKAMP (Cyst CKAMP the 2 ] , . one has has one 8 ; since then high through high then since ; In light of these new studies, this this studies, new these of light In :
] relies on the C the on relies Shisa Shisa - ). the the interacting proteins interacting http://mouse.brain tor subunits themselves themselves subunits tor Among these these Among trafficking of AMPA receptors receptors AMPA of trafficking can modulate family transcript expression expression transcript family Shisa etitd xrsin n a in expression restricted
AMPA receptor channel channel receptor AMPA AMPAR trafficking to the tothe AMPAR trafficking ed formation head
complexes, and that its its that and complexes, play a decisive role in in role decisive a play
collectively 9 but also also but 9 ei
-
e.g.
terminal cytosolic cytosolic terminal AMPAR synaptic synaptic AMPAR [ ne 9 Cumulative data data Cumulative transmembrane transmembrane novd n the in involved - 11 - -
Knot AMPAR AMPAR Knot map.org rotein Shisa are
] , illustrating illustrating , i capable of of capable
t has been has t
Shisa s AMPA as put the
essential [ ) family family ) 5, 6, 7, 7, 6, 5, 1 are not are
a gating ] in situ in direct direct , and and , ) 6, 7, 7, 6, PDZ PDZ
[ 2 ] ,
Sons Ltd. by FEBS WileyFEBSBio (2017) Open © 2017TheAuthors. Published andJohn& Press domains PDZ and assay Y2H in (summarised a partners in bait as protein the of region of partners binding cytosolic putative determine to order In proteins Shisa Results function. PKC and PICK1 into investigations future by we importantly, of partners interactions. protein this how explore to relevant highly it considered Shisa interactions both Shisa t that shown (Cys conotoxin snail the of set a by mediated It domain. on focus we study, this In of theTARP recent statu phosphorylation the subunit example, for shown, and complexes AMPAR dramatically receptor glutamate It
Accepted Article has PKC 9/ a harbours 9/CKAMP44 9/CKAMP44 interacts with with interacts 9/CKAMP44 further review see review s GluA1 and GluA2 and GluA1 s CKAMP44 , which which ,
a initially was
he C he ɣ influence receptor function, presumably by influencing interactions interactions influencing by presumably function, receptor influence Shisa 2 C In this study study this In been ( highlights a novel function of PKC of function novel a highlights e.g. eosrt ta th that demonstrate - - terminus can dramatically influence its influence dramatically can terminus terminal intracellular domain intracellular terminal with cytosolic scaffolds scaffolds cytosolic with
bind [ 9/CKAMP44 12 subunits themselves subunits
established that phosphorylation of the C the of phosphorylation that established
IK, L1SP7 DLG2/PSD DLG1/SAP97, PICK1, that that six ] ). s
-
Also relevant is the fact that modulating the phosphorylation status status phosphorylation the modulating that fact the is relevant Also ikot to AMPARs and modulate and AMPARs to rpsd that proposed N Fig. cytosolic cytosolic eie eius ihn h C the within residues serine s - Shisa
terminal extracellular cysteines extracellular terminal are phosphorylated phosphorylated are we have have we - influence ikot) that disrupts AMPAR desen AMPAR disrupts that ikot)
1A). Within this list were several several were list this Within 1A). og C long icuig h snpi safl mlcl PICK1. molecule scaffold synaptic the including , 9/CKAMP44 proteins proteins eea synaps several is interaction facilitates facilitates interaction is channel s - emnl yooi tail cytosolic terminal screened for and and for screened ,
its effects effects its and n rglto by regulation and - eitd fet o APR opsto and composition AMPAR on effects mediated , r pcfc cellular specific or also also
a small protein with only one transmembrane transmembrane one only with protein small a is also functional also is
( conductance and opening opening and conductance by the TARP family of auxiliary auxiliary of family TARP the in neurons that will significantly influence influence significantly will that neurons in s
n AMPAR on
e e.g. specific functions at the membrane the at functions specific
- dniid several identified soitd PDZ associated -
- termin localisation and localisation is regulated by other by regulated is
3 DG/A12 DLG4/PSD DLG3/SAP102, 93, PKA, PKC, and C and PKC, PKA, identified cytoplasmic interaction interaction cytoplasmic identified , as as ,
Shisa regulation associated that is is that si - l cytoso al terminal regions of regions terminal ly this region resembles that of of that resembles region this tisation - scaffold prot scaffold mediated transmission transmission mediated
importan 9/CKAMP44, we used this this used we 9/CKAMP44, components rsmby a presumably
[ lic f Shisa9/CKAMP44 of
-
3 containing containing function kinases putative interaction interaction putative ]
. t region of AMPAR AMPAR of region . Like the TARPs, TARPs, the Like . It has since been been since has It a eins harbouring harbouring eins MKII)
rbblt (for probability protein . It has been been has It . proteins .
[ 13 Given that that Given
, and that that and , ionotropic ionotropic target for for target between between - scaffold scaffold - 15 protein protein
More More ] , .
, - can are are
95, 95, we we
Sons Ltd. by FEBS WileyFEBSBio (2017) Open © 2017TheAuthors. Published andJohn& Press PMA using modulated acetate) be could phosphorylation addition, In ( treatment cells, heterologous that PKC demonstrated of target a be might 1) Kinase C with Interacting (Protein regulation. a hypothesis to us led data These on PICK1 influence co in GluA1 also and species amino an contains Shisa9/CKAMP44 of see used constructs deletion of diagram even an a deleted we region, interaction the S1) Shisa9/CKAMP44 in motif Shisa9/CKAMP44 the C PICK1 protein experiments, following the In PKC by The domains Shisa9/CKAMP44 of motif binding PDZ terminal experiments Shisa9/CKAMP44 Shisa associated work recent more in observed likewise Shisa HTRA and LNX2, LNX1, MAGI3, MAGI1, MPP5, n important, n -
Accepted lig a in tails terminal Article as demonstrated in coimmuniprecipitation experiments (Fig. 2B). To further narrow down down narrow further To 2B). (Fig. experiments in coimmuniprecipitation demonstrated as Shisa 9/CKAMP44 9/ CKAMP4
, which activates activates which , further (see (see
9/CKAMP44 interaction with PICK1 with interaction 9/CKAMP44 Fig. PICK1 was named for its role as an interaction partner for the the for partner interaction an as role its for named was PICK1 PDZ domain proteins PICK1, MAGI1, PSD MAGI1, PICK1, proteins domain PDZ - IP experiments IP indicated that the interaction interaction the that indicated
, which has been show been has , which Fig. high affinity interaction that could be subject to temporal or signal or temporal to subject be could that interaction affinity high
2 reduced interaction of PICK1 with Shisa9/CKAMP44 Shisa9/CKAMP44 with PICK1 of interaction reduced 4 C
) S across across , confirming that we indeed observed observed indeed we that confirming , - s ned PZ iad a peitd y eune analysis sequence by predicted as ligand, PDZ a indeed is oaie t snpi sts in sites synaptic to localises and wild Shisa9/CKAMP44 binding. Shisa9/CKAMP44 1 y omuorcptto ( coimmunoprecipitation by ) , highlighting a clear binding specificity a binding clear , highlighting - and IK itrcin s not is interaction PICK1
- that type , PKC Shisa/ -
PDZ which differs from the interaction with interaction the from differs which
the
[ - 17 eitd hshrlto. sn te Phos the Using phosphorylation. mediated Shisa we focussed on focussed we
] inl cascades signal observed . domain interaction domain e CKAMP
In this context, it is interesting that interesting is it context, this In that the binding between PICK1 and Shisa9/CKAMP44 is is Shisa9/CKAMP44 and PICK1 between binding the that 9/CKAMP44 is indeed indeed is 9/CKAMP44
more proximal region proximal more n [ 20
previously [ 5 , Fig. 21 , 16 family family mobility shift shift mobility with with
] cd stretch acid . ,
17 2A) W
MAGUK scaffolds MAGUK ] facilitates Shisa9/CKAMP44 phosphorylation phosphorylation Shisa9/CKAMP44 facilitates e the . . Moreover, while PMA treatment treatment PMA while Moreover, .
. proteins proteins exclusively dependent dependent exclusively
1 W
and the the and therefore asked whether asked therefore to This proximal region of the cytoplasmic tail tail cytoplasmic the of region proximal This )
, suggesting that the C the that suggesting ,
e validated an interaction interaction an validated e interaction interaction rmr neurons primary [ bind 18 Fig. , was abolished abolished was - 19
95, and SAP102 SAP102 and 95,
directly with AMPA with directly that a [ two ]
4 . of the cytosolic tail cytosolic the of
modified modified ] 1 phosphorylation Unexpectedly, w Unexpectedly,
B for PDZ domain 1 and 2 1 and PDZ domain for and most N most is highly conserved between between conserved highly is - of E )
, and confirmed that that confirmed and , relies Shisa
PSD has ( phorbol
following expression in in expression following
this region region this -
( ( terminal MAGUK PDZ PDZ MAGUK terminal following phosphatase phosphatase following - Fig. Fig.
Fig. 9/CKAMP44 9/CKAMP44 95 and SAP102 (Fig. (Fig. SAP102 and 95 exclusively exclusively been n h PDZ the on protein protein
with the full the with - Shisa
terminal region of of region terminal 2B - - - tag 1 12 receptor subunit subunit receptor dependent shift. dependent e observed that that observed e of ; for schematic schematic for ; shown to bind bind to shown
- F and observed observed and
myristate the - 9/CKAMP44 9/CKAMP44 system, we we system, also kinase PKC kinase H - dependent dependent ) on the the on . Further Further . synapse amplified amplified
with the the with - [ . binding binding 3
has an has - length ]
- and and 13 C - - -
Sons Ltd. by FEBS WileyFEBSBio (2017) Open © 2017TheAuthors. Published andJohn& Press Accepted These inducible an towards PICK1 of localisation promotes PICK1 and PKCα between interaction data These (1.388 the in observations of quantification and points PMA with treatment to response in colocalisation in increase FLAG image representative (see endoge of activation following Shisa9/CKAMP44 synaptic with PICK1 protein auxiliary AMPAR primary this synapses of regulation in the in observed PKC endogenous was of importance the activation PKC to ( response neurons hippocampal in Shisa9/CKAMP44 of shift wild following observed Shisa9/CKAMP44 stimulation PMA Article without effectively ( experiments coimmunoprecipitation in complex tripartite a form indeed PKC and PICK1 Shisa9/CKAMP44, that confirmed next We cascades PKC signalling Shisa asubstrate as PKCα purified using target PKC direct ( environment neuronal PKC these observe supporting of phosphorylation total reduced the - relative relative type PKCα ( PKCα type 9/CKAMP44 forms a forms 9/CKAMP44 - CKAMP44 novel novel
. In line with these data, we observe a strong increase in colocalisation of neuronal neuronal of colocalisation in increase strong a observe we data, these with line In . than inactive PKC inactive than the idea the support support phospho ideas are summarised in in summarised are ideas and dynamic dynamic and in
- an Fig. L ih noeos IK pnt) niae apoiaey 1.4 a approximately indicated puncta) PICK1 endogenous with FL
- in vitroin a model a that - eedn cags n h popoyain f hs9CAP4 n a in Shisa9/CKAMP44 of phosphorylation the in changes dependent
Shisa 3B see see Fig. ; lane 3 of Phos of 3 lane ;
Shisa in neurons in ), which is in line with the dramatic phosphorylation phosphorylation dramatic the with line in is which ),
s
9/CKAMP44 signals, signals, 9/CKAMP44 kinase assay kinase complex with PICK1 and PKC and PICK1 with complex Fig. PKC 3C
in proposed earlier proposed to the PICK1 the to 9/ ;
3) Fig. - compare lanes lanes compare CKAMP44 can be regulated by PKC. by regulated be can CKAMP44 mediated regulation of the the of regulation mediated , and we demonstrated demonstrated we and ,
Shisa 3D Fig.
PMA stimulation in the presence of bo of presence the in stimulation PMA . with the the with
). Digital analysis of overlapping signals (synaptic (synaptic signals overlapping of analysis Digital ).
Fig. Shisa We further demonstrate here that that here demonstrate further We ( -
Fig. 9/CKAMP44 tag panel tag 4 - Shisa9/CKAMP44 complex Shisa9/CKAMP44 .
S
2 [ 9/CKAMP44 at syn at 9/CKAMP44 Fig. ) Itrsigy activ Interestingly, 2). 19 E GST 3 ) PKC inhibitors PKC ] . , and
3D bar diagram). 3D bar
). in which activation of PKC stabilises the the stabilises PKC of activation which in - tagged Shisa9/CKAMP44 cytos Shisa9/CKAMP44 tagged A similar phospho similar A
4 ( Fig. of the Phos the of that Shisa9/CKAMP44 is indeed a indeed is Shisa9/CKAMP44 that
that is is that
Shisa 2 C , including including ,
aptic sites aptic regulated and /KM4 C 9/CKAMP44
ated - tag panel), panel), tag
( PMA with PKC nous Importantly, we also also we Importantly, Fig. D +/ - dependent mobility mobility dependent )
-
K bind PKC e.g.
.8; e data see 0.086; in this system, system, this in 3A , by activation of activation by
PKC activation activation PKC which th PICK1 and and PICK1 th
; coIP with or with coIP ; GF109203X confirming confirming - terminus. terminus. enables s o
lic tail tail lic more more - fold
of of at ,
Sons Ltd. by FEBS WileyFEBSBio (2017) Open © 2017TheAuthors. Published andJohn& Press via intracellular process this in participate likewise members protei auxi the Shisa AMPARs synaptic CA1 hippocampal surface into investigations kinet desensitisation AMPAR regulating for important is N the that demonstrated been has it but there date To of status the in phosphorylation changes to leads that ac between interaction the that neurons th that observation subsequent the and PICK1 between interaction explored been has AMPAR of regulation the in PKC and PICK1 both for role a Moreover, P that indicate (PKC) C Kinase Protein and (PICK1) 1 Kinase C with interacting Protein Shisa9/CKAMP44. protein AMPARthe of auxiliary region cytosolic (PDZ a PSD members family play that cell of molecules regulation the scaffold in role established several including proteins, binding Shisa9/CKAMP44 C cytosolic the by mediated CNS the of region interactions terminal molecular the explored have we study, this In Discussion
Acceptedtivity Article
liary proteins liary trafficking of AMPARs AMPARs of trafficking t he formation of a of a formation he - s eis n h ctslc tail cytosolic the on relies ns 9/ ICK1 cooperates with PKC to execute various various execute to PKC with cooperates ICK1 binding strong exhibited PICK1 molecule scaffold the interaction), dependent - , which have so far yielded diverse results, are of particular interest. interest. particular of are results, diverse yielded far so have which , CKAMP44 is expressed is CKAMP44 dependent regulation by intracellular signals. intracellular by regulation dependent – is dynamically regulated by activation of PKC signalling cascades signalling PKC of activation by regulated dynamically is
have an established biochemical connection, and several studies have confirmed confirmed have studies several and connection, biochemical established an have have been been have
icvr ta ti poen is protein this that discovery [
17
region, e overlapping subcellular localisation of localisation subcellular overlapping e [ 19 Shisa [ ] - 3 . It has been shown clearly that this function of TARP family auxiliary auxiliary family TARP of function this that clearly shown been has It . 95, SAP102, and MAGI1. In addition to the synaptic MAGUK proteins proteins MAGUK synaptic the to addition In MAGI1. and SAP102, 95, Shisa , ] hwvr i dnae yu gaue cells granule gyrus dentate in however, ; 26
- 9/ relatively few relatively Shisa 28 - expressed Shisa9/CKAMP44 protein. We have identified several several identified have We protein. Shisa9/CKAMP44 expressed CKAMP44 9/ o h snpi membrane synaptic the to ] these two proteins proteins two these . In the context of these studies, these of context the In . CKAMP44
the 9/ at higher levels higher at CKAMP44
AMPAR receptor auxiliary protein protein auxiliary receptor AMPAR
- [ mediated 9 investigations into investigations - - - 11 - PICK terminal extracellular domain of Shisa9/CKAMP44 Shisa9/CKAMP44 of domain extracellular terminal el communication, cell ] ; it is therefore plausible that CKAMP family family CKAMP that plausible therefore is it ; - deficient neurons did not exhibit a reduction in in reduction a exhibit not did neurons deficient 1
-
, studies on KO animal KO on studies , may be a target of finely tuned temporal or temporal tuned finely of target a be may PKC complex in response to PKC activation PKC activation to response in PKC complex nvl K tre, r of are target, PKC novel a Shisa ouain of modulation
regulatory functions in neurons in functions regulatory Further Further 9/ ics CKAMP44 involv
the function of function the [ PICK1 and Shisa9/CKAMP44 and PICK1 3 molecular molecular , 17 support
our data highlighting a highlighting data our e.g. es both both es ] . AMPAR AMPAR
In the the In
PICK1 and the MAGUK MAGUK the and PICK1 in primary neurons. primary in ing , where where , Shisa9/CKAMP44 Shisa9/CKAMP44 mechanisms. mechanisms.
TARP and CKAMP CKAMP and TARP
context of our data, data, our of context - tissue this idea, w idea, this eitd plasticity mediated Shisa
trafficking to the the to trafficking supports the idea idea the supports –
as their names names their as
interest 9/ suggest that that suggest endogenous CKAMP44
e [ 22 In strong o the to . found
- O and 25 the the
ur ur in in ] n - , .
Sons Ltd. by FEBS WileyFEBSBio (2017) Open © 2017TheAuthors. Published andJohn& Press complexes receptor of trafficking and the for regulated of formation Shisa o Importantly, guided synaptic localisation andsubsequent functional interaction with AMPAR subunits. between separated; spacially and/or temporally are interactions these might protein. N scaffold this to binding in involved the namely region, additional modulation on AMPAR effects its for important also is which domain, intracellular one least at that demonstrate Although 95 applies model similar a that AMPAR of mediators as TARPs of function the for important PSD with interaction between interaction transmembra the to involves that mechanism a propose close region cytosolic proximal more a C the not is it protein TARP trafficking receptor influence can TARPs of regions C most PSD AMPAR with interact to ability its affects the of status TARP phosphorylation the subunits: AMPAR and TARPs both of content synaptic shown been has parallels regulation O ur observations that that observations ur
Accepted- Article family 9/ ɣ ɣ compete for this binding region of Shisa9/CKAMP44 of region binding this for compete
2 C 2 - TARP CKAMP44 [ - terminal region responsi region terminal 30 eitd yatc transmission. synaptic mediated
and Shisa9/ between how TARPs and CKAMP family proteins proteins family CKAMP and TARPs how between
synaptic synaptic
suggests that suggests and interacts differentially interacts and , - Shisa9/ 31 terminus influences its association with the membrane lipid bilayer lipid membrane the with association its influences terminus this this
- ɣ the ] r bevto ta tee are there that observation ur terminal PDZ terminal , 2 can can /nPIST intera /nPIST tripartite tripartite CKAMP44 and PICK1 and CKAMP44
intracellular
CKAMP44 CKAMP44 MAGUKs. pcfcly hn it when specifically ht phosphorylation that modulate modulate - TARP 95 that ensures synaptic localisation. Both of these interactions are are interactions these of Both localisation. synaptic ensures that 95 the it complex is important is complex
may have a have may - ɣ Shisa to binding region of TARP of region binding 2
ction
scaffold the syn the Shisa lal bns o PICK1 to binds clearly n nPIST and ble for interacting with synaptic MAGUKS synaptic with interacting for ble 9/CKAMP44 , which is is which ,
9/ -
with certain scaffold proteins proteins scaffold certain with aptic targeting of AMPAR of targeting aptic early on in the secretory process secretory the in on early
CKAMP44
nPIST transitory role, perhaps comparable to that proposed proposed that to comparable perhaps role, transitory s n a in is during early trafficking could could trafficking early during - - 95
dependent interactions of TARPs can regulate regulate can TARPs of interactions dependent n route en wih s olwd y PDZ a by followed is which , It has also been shown that that shown been also has It This leads to the possibility that PICK1 and G and PICK1 that possibility the to leads This
relevant
[ rmtc fet o te phospho the on effects dramatic 13 (
. also also ope wt PICK1 with complex nrclua dmi is domain intracellular The fact that the that fact The , , which is which , 14
] known as GOPC/CAL as known ɣ tee hne hv ipiain for implications have changes these ; [ to the membrane. Indeed, Indeed, membrane. the to 29 2 that is responsible for the function but but function the for responsible is that 2 at specific time points in the in points time specific at ] .
In addition, addition, In
able to bind PICK1 and and PICK1 bind to able via .
influence receptor trafficking receptor influence An additional possibility is is possibility additional An for example for
trafficking t PDZ its complexe e domain. ne complex complex suggest suggest
n PKC and be - the i the terminal region of its its of region terminal
tre of target a –
followed by PSD by followed -
– , and it is plausible plausible is it and , a binding motif, we we motif, binding can be can nteraction nteraction s , )
- ,
that PDZ in addition to the the to addition in a brief interaction interaction brief a
domain [ – a Golgi a 32
These authors authors These
other cytosolic cytosolic other ]
suggest - . there [ -
euain of regulation 15 independent
In this case, case, this In
dynamically dynamically [ 17 also PSD also ] numerous numerous -
- phospho mediated mediated secretion secretion and also also and enriched enriched between between ]
, is also also , is may be be may s
luA1 luA1 - that that that
95 .
It - - -
Sons Ltd. the by FEBS WileyFEBSBio (2017) Open © 2017TheAuthors. Published andJohn& Press containing constructs The peptide). signal the (including CKAMP44 of acids amino cloning M the standard or DYKDDDDK) acids (amino tag FLAG using the for sequence coding the cloned generated we vector were constructs full For All techniques. mouse 424AA). on based Q9CZN4; are study (Uniprot this in used constructs CKAMP44 The Constructs (N mouse of tail cytoplasmic the using Y2H), yeast The Y2H screen andMethods Materials PICK1 involve that Shisa thereby with association t summary, In for thecalcium sensing signals calcium to response in exocytosis regulated undergo trans that membrane membrane the PIC reticulum endoplasmic the from export their of regulation the in participates subunits receptor glutamate ionotropic has ER the from of subset might observe we that phosphorylation Shisa dependent investigations YC tag (amino acids EQKLISEEDL) behind the coding sequence for the first N first the for sequence coding the behind EQKLISEEDL) acids (amino tag YC Accepted- Article LexA K1 and active PKCα is transient, is PKCα active and K1 9 9/ /CKAMP44
CKAMP44 - as h groundwor the lays bait
excitatory excitatory - - -
Golgi two associated associated receptor
- C (Cter his study study his - illustrate a role for PICK1 in in PICK1 for role a illustrate
hybrid screen was performed by HYBRIGENICS (Hybrigenics ULTImate ULTImate (Hybrigenics HYBRIGENICS by performed was screen hybrid
[ 43 ewr drvd raels n h dnrts f hippocamp of dendrites the in organelles derived network AMPARs -
recently recently . PICK1 and associated and
- neurons. ] length FLAG length - trafficking trafficking , where the transition of assembled assembled of transition the where , free) with an adult mouse brain cDNA library. cDNA brain mouse adult an with free)
PDZ domain scaffolds could take place. take could scaffolds domain PDZ [ PICK1 highlights a novel a highlights 40
- been : interaction interaction 42 via
fr fu for k
] - Interestingly, a Interestingly, . PKC ( e.g. In addition, it has been shown that the interaction between between interaction the that shown been has it addition, In reported interaction with with interaction -
or MYC or AMPAR protein complexes complexes protein AMPAR
complex complex see
te ivsiain into investigations rther and primarily important for initial targeting of PICK1 to to PICK1 of targeting initial for important primarily and n sbeun PKC subsequent and
[
influence
34 [ molecular mechanism mechanism molecular Shisa 39 - tagged CKAMP44 DNA constructs in the pCMV pCMV the in constructs DNA CKAMP44 tagged -
37 in this process. this in ] role for PICK1 in the export of export the in PICK1 for role , both n PKC and ] ; for review see review for ; 9/CKAMP44 (171 9/CKAMP44 h PICK1 the
al pae of phases early basal basal - mediated phosphorylation of other other of phosphorylation mediated (
e.g. - hs9CAP4 complex Shisa9/CKAMP44
AMPAR [ - the 44 mediated via
Moreover, there is evidence evidence is there Moreover,
[ that may contribute to contribute may that 38 see ] -
, further supporting a role a supporting further , 424 AA, cloned into pB27 pB27 into cloned AA, 424 PKC signalling cascades cascades signalling PKC functional functional ] AMPAR trafficking trafficking AMPAR )
.
[ I 27 complexes to other other to complexes t is is t SHISA , Shisa 33 possible possible AMPA ] ) modulation modulation n activity and 9/CKAMP44 9/CKAMP44 9/ - al terminal 29 29 terminal CKAMP44 CKAMP44 receptors receptors neurons neurons that PKC in . It It . the of a -
Sons Ltd. by FEBS WileyFEBSBio (2017) Open © 2017TheAuthors. Published andJohn& Press w particles Lentiviral medium. culture neuron Accepted2 for maintained were neurons cells/cm a with poly coated of coverslips mixture glass onto plated then were neurons mechanically; dissociated and glutamine) mM 0.5 and B27 with supplemented A (Neurobasal subsequent medium culture neuron and DMEM hipp the in trypsin, of (Biochrom) traces remove FBS to DMEM in 10% washing with reaction the stopping After min. 37 at (Lonza) Trypsin/EDTA in digested partially was Tissue (Lonza). DMEM cold were rats 5 from hippocampi Unilever decapitation, Following used. Wistar E18 embryonic cultures, neuronal hippocampal rat primary for neuronal Primary previously described PMA. as µM essentially generated 0.2 were cultures with treatment min 60 by followed GF109203X inhibitor ( experiment inhibitor the COS recommendations. manufacturer’s Transient L mM 2 (Sigma), FBS % 10 COS transfection / treatment Cells con the vector under shuttle pHACE FLAG particles, used virus of we generation For (K368R) Article #21235). DN mutant PKCα deficient huma #21232, clone pEGFP the For #10714). (clone Addgene from obtained was MAGI1c) flag pcDNA3 1432 (mouse, AA) 415 human, Q9NRD5, technology DNA recombinant standard using PSD FLAG previously pCMV2A constructs The 424AA (171 ΔC as EGFP sequences (AA) acid amino following the express CKAMP44 of tail cytoplasmic - - 95 7 cells cells 7 - PSD ), or GST GST or - - C1 2 D1mt n pCMV2A and PDZ12mut pGEX . Cell debris was removed after healthy neurons adhered to the cover slips, and and slips, cover the to adhered neurons healthy after removed was debris Cell . -
1A) pEGFP 415AA),
transfections were done using Lipofectamine 2000 (Invitrogen) according to the the to according (Invitrogen) 2000 Lipofectamine using done were transfections - [ - PKCα 45 95 ( purchased from ATCC from purchased ] - - , PDZ123mut have also been described been also have PDZ123mut fusion protein fusion 6PI - D and - - T osrc i or td, e sd pHACE used we study, our in construct WT lysine (Sigma) and Laminin (Sigma) (Sigma) Laminin and (Sigma) lysine - CKAMP44 n, Uniprot P17252, 672 AA) as a as AA) 672 P17252, Uniprot n, h cntut pCMV2A constructs the Fig. - - C1 FLAG trol of a synapsin of human trol -
s glutamine and penicillin/streptomycin at 37 at penicillin/streptomycin and glutamine 2D
- - : pEGFP : rm ua cDNA human from CKAMP44 ΔC (186 ΔC - 4 weeks with minimal perturbation at 37 at perturbation minimal with weeks 4 ), cells were treated for 120 min with 3.5 µM or 7 µM PKC µM 7 or µM 3.5 with min 120 for treated were cells ), - PSD - FLAG )
were maintained in DMEM (Lonza) supplemented with supplemented (Lonza) DMEM in maintained were - - - 5 n pCMV2A and 95 - 415AA C1 7 cells were treated with 1 µM PMA for 30 min. For For min. 30 for PMA µM 1 with treated were cells 7 - - PSD NC (199 ΔNΔC - CKAMP44 (171 CKAMP44 - - FLAG ) W coe pCMV3A cloned We . ere prepared by the NeuroCure Virus Core Core Virus NeuroCure the by prepared ere 95 . -
10 pups pups 10 - D3u wr cloned were PDZ3mut A osrc epesn FLAG expressing construct A . - CKAMP44 was cloned into a lentiviral lentiviral a into cloned was CKAMP44 - - 1 promotor. PSD -
1A) pGEX 415AA), cloning cloning [ 46 t dniy f prxmtl 10 approximately of density a at ocampal tissue was suspended in in suspended was tissue ocampal - were isolated and collected in ice in collected and isolated were - 95 ] FLAG ; - -
424AA), pEGFP 424AA), PKCalphaDN (Addgene clone clone (Addgene PKCalphaDN the constructs pCMV2A constructs the
- PDZ3 [ 45 template
] - , with minor modifications: modifications: minor with , A12 ee d were SAP102 - PKCalphaWT (Addgene (Addgene PKCalphaWT - SH3 - - MYC 6PI ,
-
and for the kinase the for and
K n pCMV2A and GK
°C with 5% C0 5% with °C °C with 5 % CO % 5 with °C - in similar fashion fashion similar in KM4 (186 CKAMP44 - IK (Un PICK1 - C1 - CKAMP44
escribed escribed °C for 5 5 for °C - - MAGI1 MAGI1 FLAG eithe iprot iprot 2
in in 2 5 ------r .
Sons Ltd. by FEBS WileyFEBSBio (2017) Open © 2017TheAuthors. Published andJohn& Press Accepted Scientific). Thermo MWCO; Column(40K Desalting Zeba Spin a using desalted were glut using the For blot. western and SDS PAGE by analysed were proteins bound the and times three washed were beads agarose centr speed Triton final a generate to added was buffer TN min. 15 for ice on kept were lysates and 1%) of concentration (final lysate the Inhibitors Complete Mini ice in directly placed and removed were brains whole dislocation; cervical by sacrificed were mice C57Bl6 adult experiments, fro or brains mouse adult from lysate GST the GST For (Pierce). agarose glutathione using purified were proteins tagged Triton 1% containing TBS in lysed were cells and IPTG, mM 0.1 with induced was proteins GST of Expression O/N. cultured were constructs BL21 Briefly, recommendations. GST purification andGST protein assays GST Pulldown Article4 at minutes Protein with down 4 at hours 3 for 000 20 at min 10 for centrifugations two by cleared were Mini 0.1 NaCl, mM 100 7.5, pH Tris mM (50 buffer (IP) immunoprecipitation Twenty partners. interaction Shisa9/ Coimmunoprecipitation permit the under reported and German purposes T0280/10 to research according for treated animals were of study use this the on in regulations used animals All min. 15 for PMA µM DIV10 Lois by described as essentially Facility - Complete Inhibitors (Roche)) and lysed with 5 with lysed and (Roche)) Inhibitors Complete agd rtis ee xrse an expressed were proteins tagged - 15 CKAMP44 proteins were overexpressed in COS in overexpressed were proteins CKAMP44 -
. tagged proteins (bound to the glutathione agarose beads) were incubated with with incubated were beads) agarose glutathione the to (bound proteins tagged athione agarose and eluted with 50 mM glutathione in 200 mM Tris (pH 8). They They 8). (pH Tris mM 200 in glutathione mM 50 with eluted and agarose athione and analysed after fixation at DIV20 at fixation after analysed and
ifugation (20 000 000 (20 ifugation °C. Samples were analysed by SDS analysed were °C. Samples
°C with 2 µg of the respective antibody and transfected protein transfected and antibody respective the of µg 2 with °C - G agarose (Roche) for one hour. Beads were washed 3 times for 5 5 for times 3 washed were Beads hour. one for (Roche) agarose G
without EDTA (Roche)) and homogenised. Triton homogenised. and (Roche)) EDTA without - or or post hours four g - X concentration of 0.5%. Cell debris was removed by high high by removed was debris Cell 0.5%. of concentration X . fe tre or icbto a 4 at incubation hours three After ). n vitro in - cold TN buffer (50 mM Tris, pH 7.5, 100 mM NaCl, and and NaCl, mM 100 7.5, pH Tris, mM (50 buffer TN cold . coli E. m transfected COS transfected m
t al et kinase assay, GST fusion proteins were purified purified were proteins fusion GST assay, kinase
rnfre wt te eadn GST regarding the with transformed - . d purified according to manufacturer’s manufacturer’s to according purified d 24.
- [ 47 PAGE and western blot western and PAGE - - X. After lysate clearing at 20 000 000 20 at clearing lysate After X.
transfection cells were harvested in in harvested were cells transfection - PMA treatment of n of treatment PMA 10 strokes using a 30G needle. Lysates Lysates needle. 30G a using strokes 10 ] Piay ern wr tasue at transduced were neurons Primary . - 7 cells together with their candidate candidate their with together cells 7 - 7 cells. For brain lysate pulldown lysate brain For cells. 7 g . For IP, lysates were incubated incubated were lysates IP, For .
eurons w eurons °C, the glutathione glutathione the °C,
- (WB)
- 1 % Triton % 1 Pulldown assays, assays, Pulldown - X was added to added was X . s were pulled pulled were s
as
with 0.2 0.2 with g
- protein protein - , GST , X and and X - - Sons Ltd. by FEBS WileyFEBSBio (2017) Open © 2017TheAuthors. Published andJohn& Press as presented and condition untreated the of mean the to normalised are Data (magenta). Accepted FLAG legend. figure described experiments the for of conditions analysed intere were of Regions neurons Alexa568). hippocampal rat of FLAG of colocalisation segments Dendritic objective. 63X Le a with acquired were Fluoromount with mounted were coverslips PBS, in washes final After solution. blocking in antibodies secondary with incubated subsequently and PBS then were They prim the temperature. with overnight room incubated at hour one for PBS in (BSA) albumin serum bovine Triton 0.2% in permeabilised previously described phosphate in After Colocalisation Analysis / Immunofluorescence FastAP. 37 at hours 2 lysate for dephosphorylated subsequently were The proteins and centrifugation (Roche). EDTA without inhibitors protease Complete Mini were contain (Fermentas) proteins Buffer FastAP 1X the in lysed were proteins, cells Article transfected phosphorylated for The Fermentas). (FastAP; control phosphatase alkaline thermosensitive a using a dephosphorylated As setup. blotting transfer Phos recommendations. manufacturer’s the to bis a in Phos µM 50 Phos with supplemented blot. western subsequent and gel, phosphorylation facilitates Shisa9/ Phos Phos with analysed #P61 (SignalChem GST desalted and Purified assay PKCαkinase In vitro - 2 tag SDS tag - 4 weeks in culture, differentiated neurons were fixed in 4% paraformaldehyde (PFA) (PFA) paraformaldehyde 4% in fixed were neurons differentiated culture, in weeks 4 - CKAMP44 phosphorylation was assessed using the Phos the using assessed was phosphorylation CKAMP44 tris
previously - buffered neutral buffered - buffered saline (PBS) for 10 min and immunofluorescence was pe was immunofluorescence and min 10 for (PBS) saline buffered - PAGE - - tag SDS tag 8) codn t te auatrrs ntutos Smls were Samples instructions. manufacturer’s the to according 18G)
- [
50 CKAMP44 - CKAMP44 [ ica laser ica 49 ] . Number of cells ( cells of Number . -
eedn rtre poen irto truh polyacrylamide a through migration protein retarded dependent ] -
CKAMP44 protein was used for an for used was protein CKAMP44 - - pH gel system either as described previously described as either system gel pH PAGE followed by western blot. western by followed PAGE according to standard IF protocols. Briefly, cells were were cells Briefly, protocols. IF standard to according - X in PBS for 5 min, washed in PBS and blocked with 4% 4% with blocked and PBS in washed min, 5 for PBS in X st (ROIs) of merged images were selected selected were images merged of (ROIs) st n aaye wt te mgJ plug ImageJ the with analysed and - - tag) was established using an optimised protocol with protocol optimised an using established was tag) - FL puncta (cyan) were analysed for colocalised PICK1 PICK1 colocalised for analysed were (cyan) puncta FL scanning confocal microscope (Leica TCS (Leica microscope confocal scanning ary antibodies in the same solution at 4 at solution same the in antibodies ary - FL (cyan, Alexa488) with endogenous PICK1 (magenta, (magenta, PICK1 endogenous with Alexa488) (cyan, FL - a gl lcrpoei (6 electrophoresis gel tag n ) and number of cultures ( cultures of number and )
- tag gels were generally blot generally were gels tag
- G (Southern Biotech). Images Images Biotech). (Southern G in vitro in - tag system (Wako), which which (Wako), system tag - n uca nlzr as Analyzer Puncta in
n 1 Triton 1% ing - PKC N 8% polyacrylamide polyacrylamide 8% s ) are stated in the the in stated are )
[ were °C, washed with with washed °C, 48 - ted using a wet wet a using ted α kinase assay assay kinase α SP5 II) using a using II) SP5 ]
blind to the the to blind or according according or cleared by by cleared rformed as as rformed
°C using using °C - X and and X
Zn 2+
Sons Ltd. by FEBS WileyFEBSBio (2017) Open © 2017TheAuthors. Published andJohn& Press The of Interest Conflict SFB958 and 665 SFB ‘Sonderforschungsbereich’ and (EXC257) Excellence of Cluster NeuroCure DFG the from support additional and (DFG) Forschungsgemeinschaft’ ‘Deutsche the from funding infection. for space lab and viruses providing for Charité the at (VCF) Facility Core Virus NeuroCure the thank also M from assistance technical for grateful very are We Acknowledgements A mouse ab175678), (Abcam, Alexa405 experiments immunofluorescence for BSA/PBS 4% in or WB for milk/PBST 5% in diluted were antibodies studies. coimmunoprecipitation in controls negative for used Unspecificused. wereIg antibodiesmouse Neuromab 028 1:2000 WB AB5622 Millipore Abcam Biotechnology Healthcare GE Signalling Cell 1:5000 AB6673 Antibodies Antibodies 7(two Prism +/ mean -
Accepted11036 or Article W 1: WB , authors declare no competing financial interests. financial no competing declare authors - , FLAG ),
lx58 n anti and Alexa568 AB9485 W 1:5000 WB , -
E (tnad ro o te en. ttsis ee calculate were Statistics mean). the of error (standard SEM ), PKCα (mouse, (mouse, PKCα ), sd n hs td icue F (os, Roche (mouse, GFP include study this in used A 2
75 - . - Mann tailed 000
(1:1000) 11031 andA W 1:10 WB ,
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066 , WB 1:2000 WB , (Sigma HRP 27457701V , A12 mue Neuromab (mouse, SAP102 ), ; guinea pig, Synaptic Systems, 188004 Systems, Synaptic pig, guinea ; ). , LG mue rbi, Sigma rabbit, (mouse, FLAG ), , WB 1:5000 WB , Fr immunofl For . For coimmunoprecipitation experiments 2 µg of the respective respective the of µg 2 experiments coimmunoprecipitation For -
00 Whitney Whitney - -
rabbit 21441 respectivel 21441 ), W 1:5000 WB , ), GluA1 (rabbit, Millipore (rabbit, GluA1 ),
Millipore A8592 actin (rabbit, Sigma A2066, WB 1:3000), 1:3000), WB A2066, Sigma (rabbit, actin - lx48 Lf Tcnlge, ThermoFisher Technologies, (Life Alexa488 anti U ), MYC ),
W 1:5000 WB , test for unpaired data). data). unpaired for test
- 05 uorescence experiments, we used used we experiments, uorescence mouse - 154 - ), CKAMP44 (rabbit, custom made by David’s David’s by made custom (rabbit, CKAMP44 ), HRP (Abcam HRP Gs, as required (SantaCruz SC (SantaCruz required as Gs, y - , WB 1:2000 WB , lx48 n anti and Alexa488 ).
, Y (os, Clontech (mouse, MYC ), 75 Finally, we are grateful for central project project central for grateful are we Finally, -
058 elanie Fuchs and Anke Schönherr Anke and Fuchs elanie
10 ad 72 respectively F7425 and F1804 AB1504
W 1:2000 WB , AB62928 ), PSD ), ),
PICK1 (rabbit, Abcam (rabbit, PICK1
, WB 1:2000 WB , 11814460001 All primary and secondary secondary and primary All - 95 (mouse, Neuromab (mouse, 95 - , WB 1:5000 WB , - Universitätsmedizin Berlin Berlin Universitätsmedizin rabbit ) and vGlut1 (mouse, (mouse, vGlut1 and ) - d using GraphPad GraphPad using d lx58 r anti or Alexa568 - 2025, 2 µg), were µg), 2 2025,
GAPDH (rabbit, (rabbit, GAPDH ), anti 631206 ga, Abcam goat, ;
MAP2 (rabbit, (rabbit, MAP2 ), GST (goat, (goat, GST ), A - - guinea 21200 and and 21200
AB3420 ; rabbit, rabbit, ; , WB WB , . We We . -
pig 75 - - - ,
Sons Ltd. by FEBS WileyFEBSBio (2017) Open © 2017TheAuthors. Published andJohn& Press Accepted provi subunits trafficking. receptor Auxiliary (2013). A. Sumioka, and 7 trafficking receptor AMPA of Regulation (2012). R.L. plasticity. synaptic Huganir, and V. Anggono, 6 at Rao receptors AMPA D.C., 10682. traps Shisa6 Rotaru, (2016). A.B. J.C., Smit, prev and sites postsynaptic and Lodder, D. Choquet, E., H.D., Mansvelder, Normand, L.J., Schmitz, Stroeder, R.V., Klaassen, 5 differentially family CKAMP the of subunits Auxiliary (2015). properties. AMPAreceptor modulate J. Engelhardt, von and H. Stern M., Aslam, A., Schulmann, Y., Sapir, K., Khodosevich, P., Farrow, 4 short brain a CKAMP44: (2010). H. Monyer, and P.H. Seeburg, A.B., Stern K.W., Li, N., Kavenstock, R., Sprengel, V., Mack, J., Engelhardt, von 3 Nature Genome (2007). C.L., A.R. Jones, and Thompson, C., Thaller, T.A. Zwingman, B., Zhang, X.F., Yuan, B.L., C.D., Youngstrom, R.C., Young, M.B., Yaylaoglu, M., Teemer, A., Tam, Wh A., M., Visel, L.R., Varnam, Sutram, S.M., Sunkin, N.R K.R., Sarno, Stumpf, N.N., Stewart, A.J., Sodt, M.J., B.I., S.A., Smith, K.A., Smith, S.C., Smith, C.R., Slaughterbeck, Rowland, Ruiz, H.R., Rockett, J.J., Z.L., S.E., Riley, Parry, Royall, R.B., T.H., Pak, Puchalski, C.C., O.C., Overly, Pearson, G.J., S.D., Orta, Pathak, R., Ng, L.L., Ng, N.F., Mosqueda, Michaels, M.T., L.T., Luong, Y., Lemon, C., Liu, Lau, A.J., K.D., Liang, Larsen, A.R., T.A., Laramee, H J.H., Kidney, Lake, R., K.J., C.L., Kawal, Kuan, P.T., R.H., Karr, Glattfelder, Knapik, R.A., J.M., Johnson, S.N., D.P., Jeung, Gates, M.P., Howell, J.G., Article C., Hohmann, Frensley, T.P., S.R., Fischer, Fliss, R., Fields, B.A., Facer, C., Faber, L.K., Desa Estin, Duncan, G., J.G., Eichele, N.R., A.J., Dougherty, Ebbert, H.W., Dee, B.J., Dong, S., M.J., Donelan, Datta, T.A., Dolbeare, C.N., E., Diep, Dang, T., A., Desta, Czaplinska, A.F., M.C., B.E., Chin, Boe, Crook, T.M., A., Chen, J., L., Bernard, Chen, Chong, A., L., Chen, Bensinger, E.J., M., Byrnes, K.S., Ayres, Brockway, N., M.S., Boguski, Ao, M.J., Hawrylycz, E.S., Lein, 2 Comparative (2006). J.A. Belo, development. early and during 235, 2567 A.C. homologues Shisa Silva, chicken and M., mouse Bento, of expression L., Goncalves, M., Filipe, 1 References m the reviewed authors All manuscript. the wrote SAS and SAK data. analysed SAS and NR SAK, data. the collected and experiments performed HZ and NR SAK, study. the designed SAS and SAK Author Contributions - term synaptic plasticity in the dentate gyrus. gyrus. dentate the in plasticity term synaptic
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d er idn poen n sbtae o poen iae ioae b te es two yeast the by isolated C kinase protein for substrate and protein binding lear
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Nature Nature - Neuron Neuron d Huganir, R.L. (1999). Clustering of AMPA receptors receptors AMPA of Clustering (1999). R.L. Huganir, d Amino PLoS One PLoS One - 95 and stargazin control synaptic AMPA receptor receptor AMPA synaptic control stargazin and 95 Neuron Neuron - 7. - 408, 936 3 - 66, 755
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methyl - - alpha. 34. - Alonso, M., Hjorth, J.J., van van J.J., Hjorth, M., Alonso,
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ar retention of AMPA AMPA of retention ar - ippocampal synapses. synapses. ippocampal 3 - - ciae kns II kinase activated hydroxy
- J Biol Chem Chem Biol J terminal GluR2/3 GluR2/3 terminal
58, 736 - resident PDZ PDZ resident - 5 - methyl - 48.
289, 289, - 4 - - - Sons Ltd. by FEBS WileyFEBSBio (2017) Open © 2017TheAuthors. Published andJohn& Press Germline (2002). D. Baltimore, and E.J. Brown, Science S., Pease, tissue E.J., and transmission Hong, C., Lois, 47 Chem Biol (2013) S.A. Shoichet, and Binding. Ligand by Promoted and Domains PDZ by Mediated V.M. Is Formation Multimer MAGUK Kalscheuer, S.A., Kunde, N., Rademacher, 46 truncation patients. MAPK10/JNK3 unrelated 461 novo two in de disorders of cognitive with Characterisation associated mutations (2013). S.A. Shoichet, and V.M. Kalscheue R., Ullmann, E., Wiedersberg, A., Tzschach, N., Rademacher, S.A., Kunde, 45 trans I: exocytosis. Part regulated neurons. hippocampal cultured Maletic 44 Spatiotemporal 39, 173 (2006). PKC. and N. PICK1 Saito, between interaction and molecular Y. the of Shirai, analysis S., Ohmori, N., Sakai, K., Masukawa, 43 in cargo confine complexity reticulum endoplasmic of dend neuronal zones Local (2012). M.D. Ehlers, Cui PKC 42 and PKA Coordinated (2003). M.D. receptors. of NMDA Ehlers, and T.A. RXR suppresses phosphorylation Blanpied, D.B., Scott, 41 Neurosci re NMDA An M.D.(2001). Ehlers, C.and Zhang, G.T., Swanson, T.A., Blanpied, D.B., Scott, 40 composition. AMPAR synaptic 6504. determine to trafficking GluA2 regulates J.A., Esteban, and S., R. Gatti, Huganir, L., M., Passafaro, Mapelli, M., Alessio, F., E., Raimondi, Anken, Van F., H., Fang, Fanelli, S., Bassani, A., Gurgone, M., Giannandrea, M.L., Mignogna, 39 o regulation for mechanisms Molecular PICK1. (2006). receptor J.G. NMDA Hanley, in 38 PICK1 for role essential An (2008). J.T. dependent bidirectional synaptic plasticity. Isaac, and J C.J. Rah, McBain, K.A., Pelkey, A., Terashima, 37 calcium 85. mediate receptors clustering. NMDA PICK1 dendritic (2006). in R.C. changes bidirectional Carroll, dependent, and B.L. Court, K.G., Sossa, 36 trafficking. synaptic GluA2 transient mediates PICK1 of (2012). J.G. expression Hanley, and J.M. Henley, N., Jaafari, 35
Accepted splicing. alternative and phosphorylation by regulated signal retention ER Article ceptor
- 71.
- - Wang, T., Hanus, C., Cui, T., Helton, T., Bourne, J., Watson, D., Harris, K.M. and and K.M. Harris, D., Watson, J., Bourne, T., Helton, T., Cui, C., Hanus, T., Wang,
Biochem Soc Trans Trans Soc Biochem 81. 295, 868
21, 3063 J Neurosci J Neurosci - 20, 1044 20,
aai, . n Mlnw R (1998). R. Malinow, and M. Savatic, rites. rites. D'Adamo, P. (2015). The intellectual disability protein RAB39B selectively selectively RAB39B protein disability intellectual The (2015). P. D'Adamo, - 72. - 72. - Cell Cell Neuropharmacology Neuropharmacology 54.
J Neurosci J Neurosci 32, 11618
- akn AP rcpos uig glycine during receptors AMPA lacking
148, 309 - pcfc xrsin f transgen of expression specific 34, 931 - 30. 18, 6803 - - 21. mediated ER retention and regulates the regulates and retention ER mediated -
5.
45, 755 45, - C, u, .. Rce KW, olnrde G.L., Collingridge, K.W., Roche, Y.H., Suh, .C., 13. Neuron Neuron
- og network Golgi - 67.
57, 872 Calcium
es delivered by lentiviral vectors. vectors. lentiviral by delivered es - 82. - evoked dendritic exocytosis in in exocytosis dendritic evoked
- eie ognle undergo organelles derived Mol Cell Neurosci Neurosci Cell Mol Acta Histochem Cytochem Cytochem Histochem Acta - nue AP receptor AMPA induced f AMPAR trafficking by by trafficking AMPAR f Hum Genet Genet Hum a Commun Nat
surface delivery delivery surface . Synaptic Synaptic . 31, 574 31, 132, 6, r, J - - -
Sons Ltd. by FEBS WileyFEBSBio (2017) Open © 2017TheAuthors. Published andJohn& Press right. the on shown Overlay is signals. adjacent shows middle) (magenta, H a post middle), G µm. 10 bar Scale below. shown CKAMP44 F left subsequently the C/D/E) on and shown control, is control negative (lysate) Input a blot. as western by (mIgG) detected IgGs mouse with or antibodies, MYC immuno FLAG (B), full tagged SAP102 B cDNA library. 171 A sites 1 Figure Legends Figures immunocytochemistry an synapses: Quantifying number. synapse quantify assay to based (2010). C. Eroglu, and D.M. Ippolito, 50 granules. stress of appearance the regulates Mol Genet Hum and granules RNA neuronal of component X The (2011). V.M. Kalscheuer, S.A., Kunde, 49 profiling. phosphorylation protein foradvanced pHconditions neutral Kinoshita and E. Kinoshita, 48
-
AcceptedE Article -
2, yolsi ti) a a N an as tail), cytoplasmic 424,
. Co FLAG hi primary typical of image Overlay of Confirmation two yeast a of Results precipitated with the antibodies indicated, indicated, antibodies the with precipitated
:
- Coimmunoprecipitation experiments experiments Coimmunoprecipitation -
- - staining of FLAG of staining Novel interaction partners of partners interaction Novel L ca) neuron (cyan); FL length - AI () FLAG (C), MAGI1 -
CKAMP44
- synapti
20, 491620, Shisa Musante, L., Grimme, A., Fischer, U., Muller, E., Wanker, E.E. and and E.E. Wanker, E., Muller, U., Fischer, A., Grimme, L., Musante, c protein. Overlay is shown on the right. the shown on is Overlay c protein.
selected selected /KM4 (FLAG 9/CKAMP44 - L ca, et clclss ih noeos PSD endogenous with colocalises left) (cyan, FL - 31. - CKAMP44
are s - - hybrid screen with with screen hybrid
interaction chromosome - iua E (01. mrvd Phos Improved (2011). E. Kikuta, - PSD
co - LexA - - - stained for MAP2 (magenta); high resolution insert insert resolution high (magenta); MAP2 for stained 5 D or (D) 95 FL (cyan, left) with endogenous presynaptic vGlut1 presynaptic endogenous with left) (cyan, FL J Vis Exp Exp Vis J s - Shisa Shisa , namely those with those namely , /MYC ppocampal neuron neuron ppocampal - linked intellectual disability protein PQBP1 is a a is PQBP1 protein disability intellectual linked (COS 9/CKAMP44 9 - CKAMP44 - i.e. C fusion, performed on a mouse brain brain mouse a on performed fusion, C Shisa
- 7) FLAG
with commercially available FLAG or or FLAG available commercially with f vrxrse FLAG overexpressed of 9/CKAMP44 (mouse, amino acids acids amino (mouse, 9/CKAMP44 - A12 E. rtis were Proteins (E). SAP102 - L tgte wt MYC with together FL)
and localisation at synaptic synaptic at localisation and Scale barµm. 2 Scale
PICK1, MAGI1, PSD MAGI1, PICK1, (DIV21) (DIV21) Proteomics - a SDS tag
(longer exposures in in exposures (longer Scale bar bar 2 µm. Scale xrsig FLAG expressing - - 95 (magenta, (magenta, 95 PAGE under under PAGE
11, 31911, -
r MYC or - - 95 and and 95 PICK1 PICK1 - 23.
- - -
Sons Ltd. by FEBS WileyFEBSBio (2017) Open © 2017TheAuthors. Published andJohn& Press (FLAG Shisa9/CKAMP44 of CKAMP44 expression following experiments Coimmunoprecipitation A neurons Figure antibodies indicated sta panel, (lower below shown is samples Phos PKC E Phos via observable indicatedabove. are concentrations are GF109203X Inhibitor proteins PKC the phosphorylated of application by inhibited and D phosphorylation the shift mobility highlighting thereby control, negative a as used is phosphatase alkaline with treatment phosphatase blot; proteins western via observed and Phosphorylated PAGE min). 30 µM, non from separated (1 are (indicated) PMA by induction following phosphorylated the C using detected and control) (negative mIgG antibodies indicated. Inputs areshown on the or antibody GFP using precipitated C Shisa9/CKAMP44 B tail scale. to drawn are Constructs cytoplasmic the of region terminal ΔC or of tail cytoplasmic the only with Constructs yellow). domain, transmembrane (TM: domain extracellular the in peptide signal tag): respective the of location A Figure
Accepted Articledel are and red in shown are sequences ligand PDZ GST).
(PDZ α - a SDS tag - dependent dependent PKC activation status influences CKAMP44/PICK1/PKCα complex formation: formation: complex CKAMP44/PICK1/PKCα influences status activation PKC An P Full coimmunoprec Comparative of overview Schematic 2 3 opoyain of hosphorylation
. -
-
-
- binding in vitro in Shisa Shisa - FL), PICK1 (MYC PICK1 FL), length length .
- PAGE). Presence of PKCα and kinase substrate substrate kinase and PKCα of Presence PAGE). 9/CKAMP44 9/CKAMP44 9/CKAMP44
- PKCα mobility shift of the phosphorylated substrate GST substrate phosphorylated the of shift mobility eiin variant) deficient Shisa ). -
emnl ains EGFP variants terminal
kinase assay assay kinase /KM4 (FLAG 9/CKAMP44 Shisa - - PICK1), and PKCα (EGFP PKCα and PICK1), interacts with PICK1 with interacts PICK1 complex formation formation complex PICK1 Shisa
Shisa In /KM4 (FLAG 9/CKAMP44 CKAMP44 - of region conserved A .
9/CKAMP44 constructs used in this study (including the the (including study this in used constructs 9/CKAMP44 ptto eprmns f IK (FLAG PICK1 of experiments ipitation hshrltd proteins phosphorylated 9/CKAMP44 9/CKAMP44 followed by Phos by followed i dltd n h cntut CKAMP44 construct the in deleted is ) - dr Lemi SDS Laemmli ndard CKAMP44 left. - - CKAMP44, CKAMP44, L (full FL
are N are
and isphosphorylated by - CKAMP44 - length) - - - tag SDS tag - terminally tagged ( tagged terminally L, xrse i COS in expressed FL), PKCα - and regulation by active by regulation and tag - C or ΔC,
eted in constructs labelled with with labelled constructs in eted - (indicated) ,
SDS - Shisa the tag tag the WT) in COS in WT) - - PAGE analysis highlights a highlights analysis PAGE FL - PAGE
- – PAGE. PKC inhibitor inhibitor PKC PAGE. ) is induced with PMA PMA with induced is ) n COS in /KM4 (blue 9/CKAMP44 ΔNΔC - CKAMP44 (top panel, panel, (top CKAMP44 rsn in present - s C is dependen
via and western blot; blot; western and with either E either with ) Poen were Proteins . - - - terminal to the the to terminal Phos 7 cells indicate indicate cells 7 cells 7 - PICK1) with with PICK1) PKCα - 7 cells, is is cells, 7 the same same the - ce tag
Again . - PKC
ΔNΔC. ΔNΔC. of the the of -
SDS
GFP N ;
in - - -
Sons Ltd. by FEBS WileyFEBSBio (2017) Open © 2017TheAuthors. Published andJohn& Press SEM t compared 716.5) +/ 1.388 of factor the by min) 15 for PMA µM (0.2 treatment PMA after significantly increased colocalisation The condition. treated: FLAG the with quantified was neurons µm. 5 bar Scale right. the on shown are images Merged indicated. are puncta overlapping PICK1 PICK1 endogenous to antibody an antibody and an (magenta), (cyan), antibody FLAG a with Immunofluorescence or panel) Shisa infected were Neurons PKC: of activation after neurons D PAGE. D at harvested FLAG of transduction blot affects C right. the on indicated are control) loading levels protein relevant for controls min); 30 µM, (1 PMA by activated was PKC shift). Phos panel; (top shown is 7) and 3 lanes wild EG either of presence the in cells 7 B status. PKCactivation by modulated EGFP that indicates min) 30 µM, 1 activation, PMA without or (with Comparison control. pulldown negative a as serve (mIgG) IgGs Mouse indicated. as blot, western by detected are proteins fo proteins three the that
Accepted Article FP , left panel). panel). left , The colocalisation of FLAG of colocalisation The . 9 - - -
CKAMP44 PKCα as PKCα /CKAMP44 (FLAG /CKAMP44 vrapn lclsto of localisation Overlapping PMA by PKC of activation neurons, hippocampal primary In overexpressed of Phosphorylation
Shisa n
4 nuos from neurons 48 = - fe atvto o PC y M (. µ, 5 min 15 µM, (0.2 PMA by PKC of activation after /KM4 popoyain tts cmae ae 3 n 4 n h Phos the in 4 and 3 lanes (compare status phosphorylation 9/CKAMP44 N cmae ae 3 n 4 wt o wtot IK (MYC PICK1 without or with 4) and 3 lanes compare DN; V2 o te ih, oto lvl o rltd rtis r osre va SDS via observed are proteins related of levels control right, the on IV22; are shown below (Laemmli below shown are - Shisa oito wt FLAG with sociation FL infected neurons (untreated: (untreated: neurons infected FL utetd ern (. +/ (1.0 neurons untreated o - 9/CKAMP44 proteins were observed by western blot following viral viral following blot western by observed were proteins 9/CKAMP44 CKAMP44 rm a complex. After pull After complex. a rm -
CKAMP44 - to endogenous MAP2 (blue) is shown. shown. is (blue) MAP2 endogenous to
0.086 (*** 0.086 N -
CKAMP44
Puncta Analyzer (ImageJ plugin) in dendritic segments of of segments dendritic in plugin) (ImageJ Analyzer Puncta - = 4 cultures) and normalised to the mean of untreated untreated of mean the to normalised and cultures) 4 = FL, with or without activation by PMA. Neurons were were Neurons PMA. by activation without or with FL, Shisa -
FL) and fixed at DIV 20 DIV at fixed and FL) - ye r kinase or type - - 9/CKAMP44 and PICK1 in primary hippocampal hippocampal primary in PICK1 and 9/CKAMP44 CKAMP44 tag: phosphorylated proteins observed by mobility mobility by observed proteins phosphorylated tag: P Shisa - -
SDS FL and endogenous PICK1 in rat hippocampal hippocampal rat in PICK1 endogenous and FL = 0.0008, two 0.0008, = - 9/CKAMP44 (FLAG 9/CKAMP44
- - 0.074). 0.074). PAGE down FLAG down n
- = 49 neurons from neurons 49 = FL/MYC -
ed Kα (EGFP PKCα dead and western blot western and aa r rpeetd s en ± mean as represented are Data DV 10 (DIV - tailed Mann tailed - - PICK1 protein complex is is complex protein PICK1 24 - CKAMP44 without a treatment (upper (upper treatment a without - 15) 15) (0.2 µM (0.2 - CKAMP44 with virus expressing expressing virus with - N Shisa Whitney Whitney
- ). Antibodies used used Antibodies ). = 4 cultures; PMA cultures; 4 = FL, coprecipitated coprecipitated FL, - lwr panel lower ; , 15 min) 15 , PICK1; compare compare PICK1; - PKCα 9/CKAMP44 - FL) in COS in FL) U
- test, test,
WT and and WT likewise likewise (a ctin: - U tag
). ). = – - -
Sons Ltd. by FEBS WileyFEBSBio (2017) Open © 2017TheAuthors. Published andJohn& Press Accepted Article Shisa9/CKAMP44, S2: Figure PDZ S1: Figure ofsupplementary figures List activation. PKCα enabling thereby bind to able likely is and dimerise can PICK1 Figure 4
-
A model illustrating the the illustrating A model - dependent Shisa9/CKAMP44 protein Shisa9/CKAMP44 dependent - eitd hshrlto of phosphorylation mediated PICK1 and PKC form a protein complex. protein forma PKC and PICK1 Shisa
9/CKAMP44 Shisa 9/CKAMP44 and PKCα at the same time, time, same the at PKCα and 9/CKAMP44 - PICK1 - protein interactions protein Shisa - PKCα protein complex. protein PKCα 9/CKAMP44 upon kinase kinase upon 9/CKAMP44
Sons Ltd. by FEBS WileyFEBSBio (2017) Open © 2017TheAuthors. Published andJohn& Press Accepted Article
Sons Ltd. by FEBS WileyFEBSBio (2017) Open © 2017TheAuthors. Published andJohn& Press Accepted Article
Sons Ltd. by FEBS WileyFEBSBio (2017) Open © 2017TheAuthors. Published andJohn& Press Accepted Article