Tlhophiso ea DeltaFosB botsitso ka phosphorylation (2006)

J Neurosci. 2006 May 10;26(19):5131-42.

Ulery PG, Rudenko G, Nestler EJ.

mohloli o moholo

Lefapha la Psychiatry, Setsi sa Neuroscience ea Motheo, Setsi sa Bongaka sa Texas Southwestern Medical, Dallas, Texas 75390-9070, USA.

inahaneloang

The transaction factor DeltaFosB (eo hape e bitsoang FosB2 kapa FosB [sebakanyana]) ke mokena-lipakeng oa bohlokoa oa polasetiki ea nako e telele e kentsoeng bokong ka ho pepeseha mefuta e fapa-fapaneng ea maikutlo a susumetsang a kelello, ho kenyeletsa le lithethefatsi tsa tlhekefetso, khatello ea maikutlo le ho ts'oaroa ka motlakase. . Karolo e ikhethileng ea DeltaFosB ke hore, hang ha e susumetsoa, ​​e phehella bokong ka nako e telele ho sa hlokahale hore e be teng. Mekhoa e sebelisang botsitso bona, leha ho le joalo, e ntse e sa tsejoe. Mona, re bonts'a hore DeltaFosB ke ntho e tsitsitseng ea mongolo, e nang le halofo ea bophelo ba hoo e ka bang 10 h moetlong oa sele. Ntle le moo, re bonts'a hore DeltaFosB ke phosphoprotein bokong le hore phosphorylation ea serine e setseng e bolokiloeng haholo (Ser27) e DeltaFosB e e sireletsa tšilafatsong ea "proteinasomal". Re fana ka lintlha tse 'maloa tse fanang ka maikutlo a hore phosphorylation e tsamaisoa ke kesiin kinase 2. Liphumano tsena ke bopaki ba pele ba hore DeltaFosB e kentsoe phosphorylated 'me e bonts'a phosphorylation e kenya letsoho ho tsitsitseng ha eona, e leng motheo oa bokhoni ba eona ba ho ikamahanya le maemo a phetoho e sa feleng kelellong.

Selelekela

Ntho ea transcription factor ΔFosB, eo hape e bitsoang FosB2 kapa FosB [form e khuts'oane], ke mofuta oa C terminus-truncated splice oa mofuta oa pele oa mofuta fosbe (Dobrazanski et al., 1991; Nakabeppu le Nathans, 1991; Yen et al., 1991). Joalo ka FosB e bolelele bo felletseng, ΔFosB e na le sebaka sa mantlha se kopanyang sa DNA le "leucine zipper" eo ka eona e thellang le liprotheine tsa Jun ho theha activator protheine-1 (AP-1) factor factor, e laolang polelo ea mefuta e mengata (Morgan le Curran, 1995; Rylski le Kaczmarek, 2004). Leha e se na karolo ea sebaka sa transactivation se fumanoang termeng ea C ea FosB, ts'ebetso ea ΔFosB e sebetsa ka bobeli e le motšehetsi ea matla oa ho ngola le ho hatella liseleng tse bokeletsoeng le bokong (Dobrazanski et al., 1991; Nakabeppu le Nathans, 1991; Chen le al., 1997; McClung le Nestler, 2003; Kumar et al., 2005).

ΔFosB e hlohlelletsoa maemong a phahameng ka ho khetheha ka bokong kamora bokuli bo sa foleng, empa eseng bo le thata, ho pepesetsoa maikutlo a fapaneng a kelello a amang kelello, ho kenyelletsa khatello ea maikutlo, maqeba a itseng, lithethefatsi tsa antipsychotic le antidepressant, lithethefatsi tsa tlhekefetso le moputso oa tlhaho (Tšepo et al., 1994b; Hiroi le Graybiel, 1996; Moratalla et al., 1996; Bing et al., 1997; Mandelzys et al., 1997; Kelz et al., 1999; Werme et al., 2002; Andersson et al., 2003; Colby et al., 2003; Peakman et al., 2003; Perrotti et al., 2004; Zachariou et al., 2006). Ho kenella ha ΔFosB ho amana ka kotloloho le ts'usumetso ea tšebetso ea tse 'maloa tsa tsena kelellong. Ho phehella ha ΔFosB leha ho se na ts'usumetso e eketsehileng ho e khetholla ho lintlha tsohle tse ngotsweng tsa lelapa la Fos, tse khothalletsoang ka potlako ho arabela sepheo se matla, ho bola ho khutlela maemong a basal ka mor'a lihora tse 'maloa,' me ka kakaretso se bonts'a ho lakatsa kamora ho hlohlelletsa ho sa feleng ((Hope et al., 1992; Daunais et al., 1993; Persico et al., 1993; Hiroi le Graybiel, 1996; Perrotti et al., 2004). Sena se etsa hore ΔFosB e be mokhethoa o khahlisang oa ho phetola liphetoho tse ling tsa nako e telele puong ea gene e tlatselletsang phetoho e tsitsitseng ea neuronal e bakoang ke tšusumetso e itseng e sa foleng.

Hobane ho ba teng ha nako e telele ha ΔFosB ho etsahala ho sa hlahisoe mRNA ea eona hape (Chen le al., 1995), re re, ho fapana le FosB ea bolelele bo felletseng le liprotheine tsohle tsa lelapa tsa Fos, tse sa tsitsang, ,FosB e kanna ea ba ntho e tsitsitseng ea mongolo e sa tsitsang (Tšepo et al., 1994b; Chen le al., 1997; Nestler et al., 2001; McClung et al., 2004). Ntle le moo, tlhahlobo e sa bonahaleng ea litho tsa boko tse hlobaetsang le tse matlafatsang li khothalelitse hore ΔFosB e fetohe ka mokhoa o hlakileng Mr (boima ba limolek'hule) ho tloha ∼33 kDa e maemong a hlobaetsang ho ea ∼35-37 kDa nakong ea kalafo e sa foleng (Tšepo et al., 1994a; Chen le al., 1995). Hobane ha ho na bopaki ba boteng ba li-mRNA tse ling tse ka kenyelletsang li-isoforms tsena tse fapa-fapaneng, re boetse re nahana hore ΔFosB e fetotsoe ka mokhoa oa posttranslationally le hore mohlomong sena se kenya letsoho botsitso ba eona bo sa tloaelehang. Ho fihlela joale, ha ho tlhahlobo ea biochemical ea phetoho kapa liphetoho tsa posttranslational tsa ΔFosB li tlalehiloe. Morero oa thuto ea hona joale e ne e le ho bona hore na ΔFosB ke phosphoprotein le hore na phosphorylation e bapala karolo ea botsitso ba eona.

Karolo e fetilengKarolo e latelang

Lisebelisoa le mekhoa

Mehala ea lisele tsa Mammalian le li-DNA lia hlaha.

Lisele tsa PC12 (Clontech, Mountainview, CA) li ile tsa qaptjoa ka glucose e phahameng ea DMEM e nang le l-glutamine (L-Gln) mme e tlatselletsoa ka 5% fetal bovine serum (FBS), 10% pere serum (ka bobeli ba tsoang ho Attitrogen, Carlsbad, CA) , 100 U / ml penicillin, le 100 μg / ml streptomycin (ka bobeli ba tsoa Sigma-Aldrich, St. Louis, MO). Lisele tsa HeLa (American Type setso Collection, Manassas, VA) li ile tsa kengoa ka har'a DMEM e nang le tsoekere e kholo e nang le L-Gln mme e tlatselletsoa ka 10% FBS, penicillin, le streptomycin. Mehala ka bobeli ea lisele e ne e bolokiloe ho 37 ° C mochesong oa 5% CO2 sepakapaka.

Bakeng sa phetisetso ea nakoana le DNA, lisele tsa PC12 kapa HeLa li ile tsa jaloa liphutheloaneng tse tšeletseng (tse koahetsoeng ka collagen I bakeng sa lisele tsa PC12) e le hore li ka fihla 90-100% tsatsing le hlahlamang eaba li fetisoa li sebelisa Lipofectamine 2000 (Invitrogen). Litekong tse ling (bona Lifeiga. 1-7), ΔFosB e ile ea bontšoa butle-butle ka liseleng tsa PC12 ka ts'oaetso ka vaerase ea herpes simplex virus (HSV) e khutlang.

Li-DFosB le FosB cDNA li fumanoe ho li-pTetop-construuine tsa rona.Chen le al., 1997), hape e patiloe ka har'a vec ea pcDNA3.1 (Attitrogen). Matšoao ana a pcDNA3.1-ΔFosB / FosB a ne a sebelisoa bakeng sa ho buoa liseleng tsa liphoofolo tse anyesang mme joalo ka template ea li-mutagenesis tse boletsoeng setsing. HSV-ΔFosB ea Recumbin e hlophisitsoe joalo ka ha ho hlalositsoe pele (Neve et al., 1997), 'me tokisetso e ne e na le karolo ea ∼1 × 108 pfu / ml.

Liteko tsa pele.

Hoo e batlang e le 24 h ka mora ts'oaetso / phetisetso, lisele (PC12 kapa HeLa) ka har'a lipoleiti tse nang le metsi a tšeletseng li ile tsa hlatsuoa makhetlo a mabeli ho isa ho a mararo ka 2 ml ea PBS mme ea kenngoa ka 37 ° C bakeng sa ∼1 h ho 2 ml ea Cys / Met-free DMEM (Invitrogen) e tlatselletsoa ka 5% e holofalitsoeng FBS (Hyclone, Logan, UT) ho tlatsa matamo a kenang ka hare ho Met le Cys. Qetellong ea nako ena ea "tlala", lithethefatsi (haeba lisele li ne li lokela ho phekoloa) li ne li eketsoa, ​​ebe lisele li ngolloa (pulse) le 12-25 μCi of 35S Protein Labeling Remix (PerkinElmer, Wellesley, MA) bakeng sa ∼1 h ho 37 ° C ho ngola liprotheine tsohle tse nchafalitsoeng. Joale radiolabel e ile ea tlosoa ka ho hlatsoa lisele makhetlo a mabeli ho isa ho a mararo ka 2 ml ea PBS, le 35Ho ile ha lateloa liprotheine tse ngotsoeng ka lebitso la (ho lelekisa) ka ho khutlisetsa tse bohareng ka "batang" (nonradioactive) tse kenyelletsoeng le 5% FBS le ho kotula lisele ka linako tse fapaneng. Ho phekola lisele ho ne ho lula ho lelekisoa nako eohle. Lipalo tsohle tsa liteko tsena li bonts'a lipalo tse fapaneng tsa liprotheine tse fapaneng ho ntlafatsa lipapiso tsa litefiso tsa bona tsa phallo.

Liphoofolo le kalafo e tsitsitseng ea electroconvulsive.

Likhoto tsa banna tsa Adult Sprague Dawley (200-300 g; Charles River Laboratories, Kingston, RI) li ne li tšoaroa hang ka letsatsi ka ts'abo ea electroconvulsive (ECS) bakeng sa 7-9 d. ECS e entsoe joalokaha ho hlalositsoe pele (Tšepo et al., 1994a) sebelisa Ugo Basile (Comerio VA, Italy) Yuniti ea ECS e nang le litlhophiso tse latelang: maqhubu, 100 pulses / s; pulse e na, 0.5 ms; nako ea ho ts'oha, 1.0 s; mme hajoale, 75 mA. Liphoofolo tsa taolo ea Sham li ile tsa tšoaroa ka tsela e tšoanang le ho sebelisa li-electrodes tsa tsebe-letho ntle le motlakase o mong le o mong.

32 Ho ngola limolek'hule.

Bakeng sa ho ngolisoa ha methapo ea boko, likhoto li ile tsa hlakoloa, masapo a senyeha kapele, 'me lingoloa tsa bokapele tsa 300 μm li ile tsa hlophisoa ka microslicer ea DSK (Ted Pella, Redding, CA). Likhechana tsena li ne li kentsoe kahare moqomong oa polasetiki ho 2 ml ea CSF (ACSF) e neng e haelloa ke phosphate ebile e bolokiloe 30 ° C tlasa motlakase o sa khaotseng le O.2/ CO2 motsoako (Hemmings et al., 1989). Li -lices (lislices tse peli ka tube e le 'ngoe) li ne li ngotsoe ka 1.3 mCi bakeng sa 8-10 h ho ba teng kapa ho ba sieo ha okadaic acid (100 ng / ml). Qetellong ea ts'ebetso ena, likhakeletsi li ile tsa ts'oaroa bonyane makhetlo a mararo ka ACSF e batang 'me ea homogenized ka sonication ho 250 μl ea radioimmunoprecipation assay (RIPA) buffer [PBS, pH 7.4, 150 mm NaCl, 1% (v / v ) Igepal, 0.5% (w / v) sodium deoxycholate, 0.1% (w / v) SDS, 1 mm EDTA] e tlatselitsoe pele e sebelisoa le SDS ho fihlela 0.6%, proteinase inhibitor cocktail ea lisele tsa mammalian (e sebelisitsoeng ho 5 μl / ml; Sigma-Aldrich), phosphatase inhibitor cocktails I le II (e sebelisitsoeng ho 1: 100; Sigma-Aldrich), 1 mm PMSF, le 2% glycerol. Li-Homogenates li ile tsa belisoa bakeng sa 15 min le ho hlakisoa ke centrifugation ho 15,000 × g bakeng sa 15 min. Ts'ebetso ea liprotheine ho li supernatant tse hlahelletseng e ile ea hlahlojoa ho sebelisoa mofuta oa protheine oa BCA (Pierce, Holmdel, NJ).

bakeng sa 32Ho ngola lisele tsa cultured, ∼24 h kamora ts'oaetso / phetisetso, lisele li ile tsa hlatsuoa makhetlo a mabeli ho isa ho a mararo ka phofo e se nang phosphate mme ea kenella setsing sena bakeng sa ∼1 h. Kamora nako ena ea tlala, 0.2-0.3 mCi ea 32Ph3PO4 (PerkinElmer) li ne li eketsoa selibeng se seng le se seng, 'me lisele li ne li ngotsoe 4-12 h ho latela mofuta oa teko (bona Figs. 1-7 bakeng sa litlhaloso). Lisele li ile tsa hlatsuoa makhetlo a mararo ka PBS 'me tsa tšeloa leqebeng bakeng sa metsotso ea 15 ka 50 μl ea Buffer e tlatselitsoeng. Lysates li ile tsa bokelloa ka ho hula mme tsa fetisoa makhetlo a 10 ka nale ea 25 ga ho kuta Shebile, e phehiloeng bakeng sa 10 min, le centrifuged ho 15,000 rpm bakeng sa 15-30 min ho 4 ° C. Li-lysates tse hlakisitsoeng (li-supernatants) li fetiselitsoe mothong o mocha ebe ho etsoa ts'ebetso ea protheine ea BCA (Pierce). Lipalo tsohle tsa liteko tsena li bonts'a liprotheine tse tšoanang tsa mofuta oa hlaha (WT) le S27A ΔFosB ho ntlafatsa bapisoa ba maemo a phosphorylation.

Mekhoa ea phekolo ea lik'hemik'hale le setso

Okadaic acid (OA; Sigma-Aldrich) e ile ea qhibiliha ka har'a ethanol mme e sebelisoa ho kopanya ea ho qetela ea 100 ng / ml. 5,6-Dichloro-1-β-d-ribofuranosyl-benzimidazole (DRB; Biomol, Plymouth Kopano, PA) e ile ea qhaloa ho dimethyl sulfoxide (DMSO; Sigma-Aldrich) mme e sebelisitsoe moetlong oa sele ka kopanong ea ho qetela ea 50 μm. Spermine (Sigma-Aldrich) e ile ea qhibiliha ka metsing mme ea sebelisoa khopolong ea ho qetela ea 200 μm. Calphostin-C (Biomol) e felisitsoe ho DMSO mme e sebelisoa ho 0.2 μm, athe phorbol 12-myristate 13-acetate (PMA; Promega, Madison, WI) e felisitsoe ho DMSO mme e sebelisoa ho 0.1 μm. myristoylated-autocamtide-2-inhibitory peptide (m-AIP; Biomol) e ile ea qhibiliha ka metsing mme ea sebelisoa ho kopanya ea ho qetela ea 1 le 10 μm. Protheine e nang le protheine ea kinase inhibitors e pharalletseng H-7 le H-8 (Biomol) e ile ea qhibiliha ka metsing 'me ea sebelisoa ho kopanya ea ho qetela ea 150 le 200 μm, ka ho latellana. MG132 (Calbiochem, San Diego, CA) le epoxomicin (Peptides International, Louisville, KY) ka bobeli li qhibilihile ho DMSO mme li sebelisitsoe kopanong ea ho qetela ea 12.5 le 7.5 μm, ka ho latellana. Litekong tsohle, DMSO (koloi) e ile ea eketsoa liseleng ha ho hlokahala ho boloka palo ea DMSO khafetsa lipakeng tsa kalafo. Bakeng sa 32Liteko tsa ho ngola lipilisi, meriana ena e ile ea eketsoa hang-hang pele ho ngolisoa 'me ea bolokoa bakeng sa nako ea ho ngola. Bakeng sa liteko tsa ho hula pelo, lithethefatsi li ile tsa eketsoa ka nako ea Cys / Met "tlala", ea bolokoa nakong ea ho ngola, ebe e eketsoa hape ka ho lelekisa bohareng. Li-inhibitors tsa proteasome li ne li qopetsoe 3-4 h tsohle ho pholletsa le monyetla oa ho lefella phetoho e potlakileng ea li-peptide tsena.

ImmFosB immunoprecipitation, immunoblotting, le autoradiography.

Bakeng sa immunoprecipitations, li-lysates li ne li tlotsitsoe 1: 5 e nang le RIPA e hlakileng ho theola khatello ea SDS ho 0.1% pele o ka etsa ka immunoprecipitation (IP). Ho fokotsa ho tlama ha nonspecific, lysates li ile tsa hlakisoa pele ka ho kenya immunoprecipuling le nonimmune IgG le protheine G-Sepharose (Sigma-Aldrich) bonyane 4 h. ΔFosB e ile ea thibeloa ho tloha ka har'a lysates tse tlositsoeng e sebelisa poli ea polyclonal antibody e hlokomelang epitope e kahare e fumanehang ka bobeli FosB le ΔFosB (SC-48G; Santa Cruz Biotechnology, Santa Cruz, CA) ho 0.5-1 μg IgG ka 10 μg ea (50-300 μg ea protheine e felletseng) ka kakaretso ea 0.5 ml. Li-IP li ne li kopantsoe ka bonolo ho 4 ° C ho rotor bonyane 8 h ebe 15 μl ea protheine G-Sepharose e eketsoa mme li-IP li ile tsa kopanngoa bonyane 4 h. Li-IP li ile tsa susumetsoa ke ho hlohlona ho 3000 × g bakeng sa 3-5 min ho 4 ° C, e hlatsoitse makhetlo a mararo ka 0.5 ml ea RIPA e batang le makhetlo a mabeli ka PBS e batang e nang le 0.1% Tween 20. Li-IP li ile tsa khutlisoa hape ho 0.5 ml ea PBS e batang, tsa fetisoa, tsa pululeloa ka mothapo o mocha, 'me liprotheine tse emisitsoeng ka nako eo tsa natefeloa ke ho eketsoa ha 15-25 μl ea 2 × ho fokotsa mohlala oa protheine ea Laemmli. Protocol ena ea IP e hlahisitse tlhahlobo e tobileng le e sebetsang ea "virtuFosB" kaofela lysate. Liprotheine tse immunoprecipended li ile tsa kenngoa ho SDS-PAGE ka ho kenya IP kaofela ka 12.5% Tris-HCl Criterion gel (Bio-Rad, Hercules, CA), 'me ea fetisetsoa ho PVDF kapa nitrocellulose. Kamora ho fetisoa, membrane e ne e omisitsoe ke moea mme 32P- le 35Li-protein tsa S-radiolabeled li ile tsa bonoa ke autoradiography e sebelisang filimi ea autak ea Rekak (Rochester, NY), hammoho le phosphorimaging ba sebelisa Phofu (Amersham Bioscience, Piscataway, NJ) PhosphorImager. Kakaretso (unphosphorylated and phosphorylated) ΔFosB ka lysates tsa sele kapa li-homogenates tsa boko e fumanoe ka ho ts'oaroa ha protheine ea immunoprecipended (e sebelisa sebopeho se tšoanang se sebelisitsoeng ho bona protheine 32Protheine ea P-labels), kapa liprotheine tse lekanang tsa lysate / homogenate li kenngoa ho SDS-PAGE ebe li fetisetsoa ho PVDF kapa nitrocellulose. Membrane e ne e koetsoe pele ka ho e kenella ka 1% (w / v) lebese le sa omang (Bio-Rad) ho PBS e tlatselletsoang ka 0.1% (v / v) Pakeng tsa 20 (Sigma) ea 1 h ho 25 ° C. Membrane e ile ea emisoa bosiu bo le bong ho 4 ° C ka antiw-anti-FosB polyclonal antibody e entsoeng khahlanong le amino acid 1-16 ea FosB / ΔFosB (e sebelisitsoeng ho 1: 10,000). Kamora ho kenella kahare, li-membrane li ne li hlatsuoa makhetlo a mane bakeng sa 5 min ka thibelo ea buffer, ebe li kenngoa ho 25 ° C bakeng sa ∼1 h ka poli e khahlanong le mmutla IgG e kopantsoeng le horseradish peroxidase (e sebelisitsoeng ho 1: 5000 ho thibela buffer, ho tloha Vector Laboratories, Burlingame, CA). Li-membranes li ile tsa hlatsuoa makhetlo a mararo bakeng sa 10 min ka thibelo ea buffer le nako e le 'ngoe bakeng sa 5 min ka PBS. Li-protein tsa ΔFosB ka kakaretso li bonts'itsoe ka filimi ea Kodak MR ka ntlafatso ea chemiluminescence (Pierce) le / kapa ka ho lemoha fluorescence e sebelisang li-ECL-Plus reagents (Amersham Bioscience) le Storm PhosphorImager.

Ho hloekisoa le ho hloekisoa ha "recombinant ΔFosB" liseleng tsa likokoanyana.

ΔFosB e ile ea hatelloa ka matla liseleng tsa likokoanyana tsa Sf9 ha e le protheine ea N terminus hexa-His tagged (N-His (6) ΔFosB) e sebelisa sistimi ea bac-to-Bac baculovirus expression (Invitrogen) mme e latela litaelo tsa moetsi. Ka bokhutšoanyane, ΔFosB cDNA (masala 2-237) e etelletsoeng pele ke tumellano ea lebitso la N-terminal MGHHHHHHAG e ile ea patoa ka har'a veter ea pFASTBacTM1, e neng e sebelisoa ho hlahisa baculovirus e khutlang. Lisele tsa Sf9 li ne li tšoaelitsoe ke vaerase e khutlang hape, 'me N-His (6) ΔFosB e ile ea hloekisoa lysates tsa sele ka mehato e' maloa ea chromatographic ho kenyelletsa "chromatography" ea ts'ebeliso e sebelisang kholomo ea nickel (Qiagen, Valencia, CA), phapanyetsano ea anion e sebelisa kholomo ea mono-Q (Amersham Li-bioscience), le ho khetholloa ka boholo li sebelisa kh'hara-filtration khala (Amersham Biosciences).

In vitro lithuto tsa phosphorylation.

In vitro karabelo ea phosphorylation bakeng sa thupelo ea nako le tlhahlobo ea stoichiometry li entsoe ka bongata ba 30 μl e nang le 10 μm substrate (N-His (6) ΔFosB kapa substrate e ntle ea taolo), 250 μm ATP, le 1 μCi / μl [γ-32P] ATP, buffer e fanoeng ke moetsi oa kinase, le e 'ngoe ea li-kinase tse latelang: CK2 (20 ng / μl; Holimo, Charlottesville, VA), CaMKII (10 ng / μl; Upstate), PKC (1.6 ng / μl; Calbiochem) kapa p70S6K (2.5 mU / μl; Upstate). Boitšoaro ba nako ea lithuto bo ile ba etsoa ka ho tlosa li-aliquots tsa 5 μl ho tharollo ea karabelo linthong tse boletsoeng nako le ho eketsa palo e lekanang ea 4 × ho fokotsa sampole ea protheine ea Laemmli. Litekanyetso tsa Michaelis-Menten kinetic bakeng sa karabelo ea CK2 li ne li behiloe tlas'a maemo a tsitsitseng a maemo a boemo ba naha. Liphetoho tsena li entsoe XXUMX min ka bongata ba 15 μl e nang le enzyme ea 10 ng / μl, 2 μm ATP, 250 μCi / μl [γ-32P] ATP, le likhakanyo tsa N-His (6) tsa 2.5-30 μm. Liphetoho tsohle li entsoe ka 30 ° C ka bateng ea metsi. Kamora ho SDS-PAGE le ho hlatsoa li-gel ka Bio-Safe Coomassie (Bio-Rad), gel e ile ea omisoa, 'me 32Ho kenyelletsoa ha P-phosphate ho ile ha hlahlojoa ke tlhahlobo ea phosphorimaging (bona ka tlase, Khakanyo ea data, lipalo le lipalo).

'Mapa oa phosphopeptide o likarolo li peli le tlhaiso ea phosphoamino acid.

Ka bobeli liphatlalatso tsena li entsoe ka mokhoa o hlalositsoeng ke Ploegh le Dunn (2000). Ka bokhutšoanyane, likhechana tse ommeng tsa gel li na 32E ngotsoe ΔFosB (ebang e tsoa ho a vitro karabelo kapa ho tsoa ho li-immunoprecipitates tsa lisele tse nang le metabolism e nang le metabolism), li ne li pepesoa, li entsoe ka metsi hape, li hlatsuoa, 'me li kenngoe mohono oa tryptic. Sesebelisoa se phahameng se nang le lihlahisoa tsa tšilo ea tryptic se ile sa hlatsuoa ka makhetlo a 'maloa' me lyophilate ea itlhatsoa makhetlo a 'maloa' me ea qala ho sebelisoa hape ka 10 μl ea buffer ea electrophoresis, pH 1.9. Sampole (3 μl) e ile ea bonoa holim'a seluloa sa cellulose-layer chromatography (TLC) (Analtech, Newark, DE) mme sa aroloa ka tekanyo e le 'ngoe ka electrophoresis le boholo bo bong ka ho nyoloha TLC. Limmapa tse hlahisang phosphopeptide li ile tsa bonoa ke autoradiography le phosphorimaging. Bakeng sa tlhahlobo ea phosphoamino acid, 2 μl ea digests tsa tryptic tse neng li boetse li entsoe ka motlakase oa electrophoresis li ile tsa hlakisoa hape ke HCl hydrolysis ho 105 ° C bakeng sa 25 min ho 3 m HCl tlasa N.2 sepakapaka. Boitšoaro bo ile ba emisoa ke ho hlapela ka makhetlo a ts'eletseng ka metsing, mme motsoako o ile oa kopantsoe. Lyophilate e ile ea qaptjoa hape ho 5 μl ea buroper ea electrophoresis, pH 1.9, 'me ea bonoa polelong ea TLC ea cellulose hammoho le litekanyetso tsa phospho-Ser, -Thr, le -Tyr. Electrophoresis e entsoe ka nako e fetang halofo ea bolelele ba poleiti ea TLC e sebelisa buffer ea electrophoresis, pH 1.9, 'me joale poleiti e fetisetsoa ka pH 3.5 buffer, mme electrophoresis e ile ea etsoa ho phethoa. Litekanyetso tsa phosphoamino acid li ile tsa bonoa ka ho fafatsa poleiti ea TLC ka 1% (v / v) tharollo ea ninhydrin ho acetone, le 32Mehlala ea li-amino acid e nang le lenane-leseling e ne e bonoa ke autoradiography le phosphorimaging ka bobeli.

siRNA-induction CK2α ea koenyeha.

Re sebelisitse mokhoa oa ho kena-kenana le RNA ho khetha ho theola maemo a CK2 (Di Maira et al., 2005). Lisele tsa PC12 li ile tsa jaloa ho li-pllagen I tse apereng hantle lisente tse tšeletseng ho fihlela ∼70-80% confluence ka letsatsi le hlahlamang, ha li ne li fetisoa butle-butle ka 20 nm (mahloriso a ho qetela) a sa ts'oaneng le siRNA kapa siRNA e lebisitsoeng ho mRNA ea catalytic α subunit ea Rat CK2, e sebelisa 5 μl ea moemeli oa phetiso SilentFectin (Bio-Rad) mme o latela litaelo tsa moetsi. Hoo e ka bang 24 h hamorao, lisele li ile tsa fetisoa butle-butle ka ΔFosB plasmid, joalo ka ha ho boletsoe kaholimo. Hoo e ka bang 24 h hamorao (∼48 h kamora phetiso ea siRNA), lisele li ile tsa beoa 32P metabolic labeling kapa pulse-chase analysis e le e hlalositsoeng ka holimo. Tse latelang tse 'nè siRNAs CK2α li ne li sebelisoa le liphello tse tšoanang: 5'P-CAAACUAUAAUCGUACAUCUU3', 5'P-UCAAUCAU-GACAUUAUGCGUU3 ', 5'P-UAGUCAUAUAAAUCUUCCGUU3', 5'P-AAAUCCCUG ACAUCAUAUUUU3 '(Dharmacon, Lafayette, CO). Joaloka taolo e mpe, re ne re sebelisa Silencer taolo e mpe #3 siRNA e tsoang Ambion (Austin, TX). Tekanyo ea CK2 ea ho kokota e ile ea beoa leihlo ka ho sebelisa immunoblotting e sebelisang anti-CK2 polyclonal antibody (catalog # 06-873 from Upstate) bosiu bo le bong ho 1: 1000 dilution. β-Tubulin e sebelisitsoe e le taolo ea ho jarolla mme e fumanoe ka monoclonal antibody (lethathamo la # 05-661 ho tloha Upstate) bosiu bo le bong ho 1: 20,000 dilution.

Mutagenesis e lebisitsoeng sebakeng.

Phetoho ea Ser27 ho Ala kapa ho Asp e phethiloe ho sebelisoa sesebelisoa sa phetoho se potlakileng sa Site-Directed Mutageneis (Stratagene, La Jolla, CA) le ho latela litaelo tsa moetsi. Ho hlahisa phetoho ea Ser27 ka har'a protheine ea ΔFosB, ho sebelisitsoe li-primers tse latelang tsa mutažis. Ser27 to Ala: (reverse primer) 5'GCCGAAGGAGTCCACCGAAGCCAGGTACTGAGACTCGGCGGGGGG3 ′. Ser27 ho Asp (pele primer) 5'CCCTCCGCCGAGTCTCAGTACCTGGATTCGGTGGACTCCTTCGGC3 ′. Likahare tse fetotsoeng li ngotsoe ka mongolo o motenya, 'me kh'outhe ea Ser27 e thathamisitsoe.

Bioinformatics.

Liwebosaete tse ka bang phosphorylation le kinases tsa ΔFosB li ile tsa batloa ka ho tlisa tatellano ea protheine ea mouse ho li-database tse ikhethileng ho kenyelletsa ProSite (http://www.expasy.org/prosite/), PredictProtein (Rost et al., 2004), le NetPhosK (Blom et al., 2004).

Khakanyo ea data, lipalo le lipalo.

Palo ea liprotheine tse teng ka hara pVDF kapa nitrocellulose membrane e ile ea emisoa ho sebelisoa Storm PhosphorImager le software e tsamaeang le ImageQuant (Amersham Biosciences / Molecular Dynamics). Thutong ea sele le lithuto tsa "phosphorylation" ea sele, litekanyetso li fumanoe bakeng sa 32Liprotheine tse ngotsoeng tse ngotsoeng ka nako eo li ile tsa aroloa ke boleng bo fumanoeng bakeng sa ΔFosB kaofela, 'me tsa hlahisoa e le karo-karolelano. Ka har'a a vitro lithuto tsa phosphorylation, palo ea 32P-labels ΔFosB ka molek'hule oa ΔFosB (stoichiometry) e baliloe joalo ka ha ho hlalositsoe pele (Sahin et al., 2004). Litekanyo tsohle li ile tsa nkuoa kahare ho mola oa sesebelisoa se sebelisitsoeng. Litekanyetso tsa Kinetic li ile tsa baloa li sebelisa mofuta oa Michaelis-Menten, ka nako eo V = VMax[S] / ([S]+KM) le VMax = k2[EKakaretso]. Half-lifet1/2) ea ΔFosB le FosB li hakantsoe ho tsoa molemong oa ho hula (ho sebelisa mohopolo o sa amaneng hantle le lintlha tsa data) mme li tsamaisana le nako eo protheine e setseng e leng 50% ea pele. Lipalo tsohle, liphetho tse bontšitsoeng li emela bonyane liteko tse peli ho isa ho tse tharo tse ikemetseng. Ka lits'oants'o tsohle, data e bontsitsoeng ke litekanyetso tsa ± SEMs (3 ≤ n ≤16). Bohlokoa ba lipalo ba liphapang bo ile ba hlahlojoa ho sebelisoa bo sa lefelloeng t teko, e lokiselitsoeng lipapiso tse ngata, le asteris e bontša p ≤ 0.05.

Karolo e fetilengKarolo e latelang

Results

ΔFosB ke ntlha e tsitsitseng ea mongolo e tsitsitseng

Leha re kile ra nahana esale pele hore ΔFosB ke ntho e tsitsitseng ea mongolo (Nestler et al., 2001), tlhahlobo e tobileng ea boemo ba protheine ha e so fihle ho fihlela joale. Ho araba potso ena, re entse liteko tsa "pulse-chase using lisele tsa PC12", tse sebelisitsoeng haholo joalo ka mohala o kang oa neuron, moo ΔFosB e ileng ea hlahisoa butle ka ts'oaetso ka ts'oaetso ea vaerase ea herpes rahisix (HSV-ΔFosB). Liprotheine tse sa tsoa etsoa li ne li ngotsoe ka metabolism le 35S-Met / Cys, le mokhoa oa ho nyenyefatsa oa 35E ngotsoe ΔFosB (35S-ΔFosB) e ile ea beoa leihlo ka ho e kenya ts'ebetsong lysates tsa sele tse fumanoeng libakeng tse fapaneng ka mor'a ho tlosoa ha li-amino acid tsa radiolabeled. Ho hlahlojoa ha li-immunoprecipitates ke SDS-PAGE le autoradiography ho senotse hore halofo ea bophelo ba ΔFosB liseleng tsa PC12 ke ∼10 h (Feie. 1). Liphumano tsena li bonts'a hore halofo ea bophelo ba ΔFosB e phahame ho feta ea lintlha tse sa hlokeng ho ngoloa (sheba Puisano), ho kenyelletsoa le bolelele bo felletseng ba FosB, eo halofo ea bophelo ba bona moetlong oa sele e tlalehiloeng e le ∼90 min (Dobrazanski et al., 1991; Carle et al. 2004). Ntle le moo, ho bohlokoa ho hlokomela hore ho senyeha ha ΔFosB ha ho tšoane le tšilafalo ea boleng ba pele, empa ho e-na le hoo, ho qalile ka sekhahla sa ho fokola butle butle. Sena se supa ho ba teng ha mefuta e fetang e le 'ngoe ea ΔFosB le / kapa tsela e fetang e le' ngoe ea mokhoa oa ho senya.

Setšoantšo sa 1.

Sheba tlhahiso e kholoanyane:

Setšoantšo sa 1.

ΔFosB ke ntlha e tsitsitseng ea mongolo e tsitsitseng. Bophelo ba halofo ea ΔFosB ke ∼10 h ho setso sa sele. ΔFosB e bontšitsoe liseleng tsa PC12 ka ts'oaetso ea HSV-ΔFosB kapa phetisetso ea nakoana le ΔFosB e nang le plasmid, 'me lisele li ile tsa etsoa tlhahlobo ea pulse-chase joalo ka ha e hlalositsoe ho Lisebelisoa le Mekhoa. Liphetho tse lekanang li fumanoe ho sa tsotellohe mokhoa o sebelisitsoeng ho overexpress ΔFosB. Palo e bonts'a nako ea nako (le moemeli oa "autoradiogram") oa tšilafalo ea ΔFosB. Lintlha tse reriloeng ke boholo ba ± SEM ea bonyane lintlha tsa 15 tsa motho ka mong tse fumanoeng ho tsoa bonyane liteko tse hlano tse ikemetseng. Bakeng sa papiso, ho supiloe halofo ea bophelo ba bolelele bo felletseng ba FosB.

ΔFosB ke phosphoprotein bokong

Re fane ka maikutlo a hore ntlafatso ea posttranslational ea ΔFosB e ka kenya letsoho ho bonts'a botsitso ba eona. Hobane phosphorylation e bonts'itsoe ho fetola ts'ebetso ea lintlha tse ngotsoeng ka mekhoa e mengata, ho kenyelletsa botsitso ba bona (bakeng sa tlhahlobo, bona Desterro et al., 2000; Whitmarsh le Davis, 2000), re fuputse hore na ΔFosB ke phosphoprotein. Ho fihlela sena, polelo ea ΔFosB e kentsoe bokong ba ratoa ho sebelisa ECS e sa foleng, kalafo e tsejoang ho susumetsa maemo a phahameng a ΔFosB, haholo ka pele ho cortex (Tšepo et al., 1994a). Letsatsi le le leng kamora kalafo ea ho qetela ea ECS, ha maemo a ΔFosB a lula a le holimo, ho ne ho lokisitsoe lingoloa tse tšesaane tsa "cortical" mme li ngotsoe ka metabolism ka 32P-orthophosphate. Siling e tšoanang e ne e se radiolabeled, mme tsena li ne li sebelisetsoa ho bona maemo ohle a ΔFosB. Kamora ho enta ka popelong ka anti-FosB / ΔFosB e khethehileng le karohano ea liprotheine tse anngoeng ke SDS-PAGE, phosphorylated 32P-e ngotsoeng ΔFosB e fumanoe ke autoradiography, athe ΔFosB e fumanoe ka ho enteloa ka mokhoa oa immunoblotting. Tlhahlobo ena e senoletse hore ΔFosB e kentsoe phosphorylated bokong, joalo ka ha ho pakoa ke ntho e itseng 32Sehlopha sa lab35 kDa se teng ka har'a sampole ea boko e tšoaroang ka mokhoa o sa foleng, empa ha se bonahale ho taolo e sebetsanoang le sham (Feie. 2A). Sena se lumellana le 'nete ea hore, ha ho ba le ts'usumetso e sa feleng, maemo a basel a ΔFosB a tlase haholo. Bohlokoa ba karabelo ea immunoprecipitation bo bontšoa ke ho haella ha lets'oao ho "nonimmune IgG precipitate".

Setšoantšo sa 2.

Sheba tlhahiso e kholoanyane:

Setšoantšo sa 2.

ΔFosB ke phosphoprotein bokong. A, ΔFosB e phosphorylated bokong. Endo native ΔFosB e kentsoe bokong ba ratoa ke kalafo ea ECS e sa foleng joalo ka ha e hlalositsoe ho Lisebelisoa le Mekhoa. Linoko tse tsoang pele tsa cortical li ne li ngotsoe ka mokhoa oa metabolism 32Ph3PO4 ka lihora tse 'maloa. Kamora ho homogenization ha slices, osFosB e ile ea emisoa, 'me ea sebelisoa ke phosphorylated ΔFosB (32P-ΔFosB) e fumanoe ke autoradiography (karolo e kaholimo). Kakaretso ea ΔFosB ho li-immunoprecipitates tse sa sebetseng e fumanoe ke immunoblotting (konopo e ka tlase). Joalo ka taolo e mpe, immunoprecipitate ea phoofolo e tšoeroeng ke sham le Igim e seng tsona. B, Litsi tsa phosphorylation ea baeti le li-kinase tse nang le lintlha tse boletsoeng esale pele tsa tatelano ea "proteinFosB" li fumanoe ke tlhahlobo ea bioinformatic. Libaka tsa baemeli li na le lintlha tse phahameng tsa ho bolela esale pele li totobatsoa ka tatellano ea liprotheine mme li thathamisitsoe ka har'a tafole. Sebaka sa "DNA-binding" ea mantlha le leucine li ngotsoe ka sebete ka tatellano ea protheine. C, D, Phosphorylation ea ΔFosB e eketsoa ke Ser / Thr phosphatase inhibitor OA. C, 32Maemo a P-ΔFosB ho li-immunoprecipitates ho tloha ho lisekoere tsa cortical ea pele e ngotsoeng e le sieo (Ctr) kapa boteng ba 100 ng / ml OA (graph le holimo) li fumanoe ke autoradiography. Phanele e ka tlase e bonts'a ΔFosB e felletseng ho li-immunoprecipitates tse tsoang melaaneng e sa tsoakoang ke immunoblotting. D, Lisele tsa PC12 li ne li tšoaelitsoe ke HSV-ΔFosB kapa HSV-LacZ (veter) mme e ngotsoe ka metabolism ka 32Ph3PO4 ka ho ba sieo (Ctr) kapa boteng ba 100 ng / ml OA. 32Maemo a P-ΔFosB ho li-immunoprecipitates a ile a fumanoa ke autoradiography (graph, holimo konopo). Liphanele tse ka tlase li bonts'a BFosB e fumanoeng lysates tsa sele ka ho enta.

Mohato oa pele oa ho hlakisa hore na ke mang (s) le sebaka sa marang-rang se amehang posphorylation ea ΔFosB, re ile ra beha tlhahlamano ea eona ea amino acid tlhahlobisong e 'ngoe ea biiinformatic. Sena se senotse hore, leha ΔFosB e sena libaka tsa khetho ea Tyr phosphorylation, e na le libaka tsa tumellano tsa Ser / Thr kinase, ho kenyeletsoa libaka tsa CaMKII tse tharo, libaka tse tharo tsa CK2, le libaka tse peli tsa PKC tse nang le lintlha tse ngata tsa polelo ea phosphorylation.Feie. 2B). Haeba, joalo ka ha ho boletsoe esale pele ka bioinformatics, ΔFosB e sebelisoa feela phosphoryl ho Ser kapa Thr mabaki, phosphorylation e lokela ho fetoloa haholo ke ts'ebetso ea phosphatases ea Ser / Thr. Ho leka taba ena, re ile ra ngola linama tse ka pele-pele tsa cortical 32P-orthophosphate boteng kapa ho ba sieo ha OA, inhibitor ea Ser / Thr protein phosphatase inhibitor. Joalokaha ho bontšitsoe Setšoantšo sa 2C, OA e bakile keketseho e kholo (∼2.5-fold) ho 32P-ΔFosB. E boetse e bakile keketseho e nyane ea maemong ohle a ΔFosB, e lumellanang le litlaleho tse fetileng tse hokahanyang litlamorao tsa phello ea OA le bokhoni ba eona ba ho susumetsa liphatsa tsa lefutso tsa pele ho kenyeletsa liprotheine tsa Fos (Miller et al., 1998; Choe et al., 2004). Sephetho ka kakaretso ke keketseho e kholo ea kakaretso (∼60%) maemong a phosphorylated ΔFosB.

Joale re ile ra etsa lipatlisiso hore na, liseleng tsa PC12, tse fumanehang habonolo ho leqhekeng, ΔFosB e bontsitse mokhoa oa phosphorylation o ts'oanang le o bonoang bokong. Re bontšitse ΔFosB ka liseleng tsa PC12 ka ts'oaetso le HSV-ΔFosB mme re ile ra ngola lisele ka metabolism 32P-orthophosphate boteng kapa ho ba sieo ha OA. Polelo e atlehileng le immunoprecipation ea protheine e nang le tšoaetso ea HSV--FosB e bonts'oa ke boteng ba bobeli ba immunoblot (Feie. 2D, karolo e ka tlase) le autoradiograph (karolo e kaholimo) ea sehlopha sa band35 kDa, e sieo ka liseleng tse nang le tšoaetso ea vector. Joalokaha ho hlokometsoe bokong, OA e bakile keketseho e nyane ea maemo ohle a ΔFosB empa keketseho e phahameng haholo (e habeli habeli) 32P-ΔFosB, e hlahisang keketseho e kholo ea (∼50%) ea phosphorylation ea ΔFosB. Ho feta moo, ho latela tumellano ea bioinformatic, kalafo ea lisele tsa PC12 e nang le Thib phosphatase inhibitor ha ea ka ea tlisa liphetoho tse kholo ho 32Maemo a P-ΔFosB (ya data e sa bontswang) Hammoho, liphuputso tsena li senotse hore ΔFosB e kentsoe phosphorylated ho Ser le / kapa masala a Thr bokong le liseleng tsa PC12 mme e netefalitse ntho ea morao-rao hore ke sistimi e ntle ea sele ea ho ithuta lithuto tsa phosphorylation le litšila tsa ΔFosB.

CK2 empa eseng PKC kapa phosphorylates ea CaMKII ΔFosB a vitro

Joalokaha ho hlalositsoe kaholimo, tlhahlobo ea tatellano ea ΔFosB amino acid e senotse lintlha tse phahameng tsa ho bolela esale pele bakeng sa libaka tsa CK2, PKC, le CaMKII phosphorylation. Ho fumana hore na ke efe ea li-kinase tsena e ka etsang phosphorylate ΔFosB, re tsamaisitse letoto la a vitro phosphorylation athari e sebelisang li -asesase tse hloekisitsoeng le recFosB e hloekisitsoeng. Joalokaha ho bontšitsoe Setšoantšo sa 3A, ea li-kinase tse tharo tsa mokhethoa, ke CK2 feela ea phosphorylated ΔFosB ka tsela e ikhethang. Ntle le moo, ho ile ha lekoa li-kinase tse ling tse 'maloa (mohlala, GSK3 le p70S6K) empa tsa hloleha ho qaqisa phosphorylate ΔFosB (ya data e sa bonts'ang). Tšobotsi e 'ngoe ea karabelo ea CK2 ka tlhahlobo ea nako ea lithuto e senotse hore kinase ena e ka kenyelletsa ho kenyelletsoa ha ∼0.5 mol ea phosphate ka molekeli oa ΔFosB (Feie. 3B). Taba ea hore CK2 e ka phosphorylate ΔFosB a vitro ho stoichiometry e kholo (∼50%) e bonts'a karabelo e loketseng ea 'mele. Ka mor'a moo re ithutile kinetics ea karabelo ena ka ho kenya CK2 moo ho nang le palo e ntseng e eketseha ea ΔFosB, 'me re ikemiselitse hore CK2 phosphorylates ΔFosB V, max ea 5.8 pmol · min-1 · Μg-1 ea enzyme, a KM ea 18.4 μm, le a kkatse of 0.2 / s (Feie. 3C). Boleng bo fumanoeng bakeng sa paramente tsena tsa kinetic bo tšehetsa ka ho eketsehileng bohlokoa ba 'mele ba karabelo ena. Mohlala, KM ea CK2 ea DARPP32, e 'ngoe ea likarolo tse tsebahalang ka ho fetisisa, ke 3.4 μm, le kkatse ke ∼0.3 / s (Girault et al., 1989); the KM bakeng sa ATP e tsoa ∼10-40 μm (Cochet et al., 1983; Silva-Neto et al., 2002), le hore na li-casein li tsoa ∼10-50 μm (Meggio et al., 1977; Pyerin et al., 1987). Ka kopanelo, lintlha tsena li bonts'a hore ΔFosB ke karoloana ea 'nete ea fide ea CK2 a vitro.

Setšoantšo sa 3.

Sheba tlhahiso e kholoanyane:

Setšoantšo sa 3.

CK2 empa eseng PKC kapa phosphorylates ea CaMKII ΔFosB a vitro. Autoradiograph (konopo e kaholimo) e bonts'a sehlahisoa sa phosphorylated, mme gel ea Coomassie-stained (konopo e ka tlase) e bonts'a kakaretso ea ΔFosB e teng karabong. A, ΔFosB e hloekisitsoeng e ile ea kenngoa a vitro phosphorylation ke li-kinases tsa likhetho tse fapaneng (tse tharo li bontšoa). B, Tlhahlobo ea nako le litlhahlobo tsa stoichiometric li bonts'a hore CK2 e ka etsa phosphorylate ΔFosB a vitro ka stoichiometry ea ∼50%. C, Tlhatlhobo ea Kinetic ea karabelo ea CK2 e senotse hore ΔFosB ke fides ea nnete a vitro substrate. Theararization ea karabelo e bontšoa ka morero oa ho pheta-pheta habeli (tlase), empa litekanyetso tsa kinetic li ne li nkiloe ho Michaelis-Menten curve (holimo).

CK2 e hlophisa phosphorylation le botsitso ba ΔFosB liseleng tse sebetsang

Ho lekola botebo ba 'mele ba CK2-mediated phosphorylation ea ΔFosB, re tsamaisitse' mapa oa phosphopeptide re sebelisa a vitro phosphorylated le PC12-cell-phosphorylated ΔFosB. Kamora SDS-PAGE le gel elution ea 32P-ΔFosB (ho tloha ho a vitro karabelo kapa ho tsoa immunoprecipitate ea 32Lisele tse nang le lipilisi), protheine ena e ile ea monngoa ka trypsin, 'me phosphopeptides e ileng ea hlaha e ile ea aroloa ka likarolo tse peli. Tlhahlobo ena e senotse hore phosphopeptide ea mantlha e tsoang karabelong ea CK2 e kopantsoe le e 'ngoe ea li-phosphopeptides tse peli tse kholo ho tsoa ho ΔFosB phosphorylated lisele tsa PC12 (Feie. 4A), athe phosphopeptides e hlahisitsoeng ke PKC kapa CaMKII (data e sa bontsitsoeng) ha e a ka ea etsa joalo. Ho bile le phosphopeptide ea bobeli ea ΔFosB 'mapeng e tsoang liseleng tsa PC12, empa ka lebaka la ho se khonehe ha li-kinase leha e le life tseo re li lekileng ho hlahisa phosphopeptide e tšoanang a vitro, ha re tsebe hona joale hore na phosphopeptide ena ea bobeli e na le sebaka sa phosphorylation se fapaneng le phosphopeptide kapa hore na e emela peptide e ikhethang e nang le sebaka sa phosphorylation.

Setšoantšo sa 4.

Sheba tlhahiso e kholoanyane:

Setšoantšo sa 4.

CK2 e hlophisa phosphorylation le botsitso ba ΔFosB liseleng tse sebetsang. A, CK2 empa eseng PKC e bonahala e le phosphorylate ΔFosB liseleng. Limmapa tsa phosphopeptide tse likarolo li peli tsa ofFosB phosphorylated ke CK2 kapa PKC a vitro kapa ka lisele tse sa sebetseng tsa PC12. Metsu e bonts'a ho phahama ha peptide ea CK2-phosphorylated empa eseng PKC-phosphorylated e nang le e 'ngoe ea li-phosphopeptides tsa ΔFosB tse fumanoeng liseleng tsa PC12. B, Phosphorylation ea ΔFosB liseleng tsa PC12 e eketseha ka ho phekola lisele tse nang le popelo ea CK2 activator spermine (SP) mme e fokotsehile ka kalafo ka CK2 inhibitor DRB. Ka lehlakoreng le leng, phosphorylation ea ΔFosB liseleng tsa PC12 ha ea ka ea angoa ke kalafo ka ho sebetsa ke PKC activator (PMA) kapa PKC-inhibitor calphostin-C (Calph) e ikhethang. Ho bontšoa setšoantšo sa "autoradiogram" se kaholimo (konopo e kaholimo) le immunoblot. C-F, Phello ea ts'ebetso ea CK2 ho botsitso ba ΔFosB Lipalo li bonts'a nako ea nako (le moemeli oa "autoradiogram") oa tšilafalo ea ΔFosB. Lisele tsa PC12 tse hlalosang nakoana ΔFosB li ile tsa etsoa liteko tsa pulse-chase tse entsoeng ntle le ho ba teng ha CK2 inhibitor DRB (C), semela sa CK2 activator spermine (D), kapa senotlolo sa inase se pharalletseng H-8 (E), e neng e sebelisetsoa ho laola litlamorao tse senang maikutlo tsa DRB. F, Phello ea ho kokobela subunit e thusang ea CK2 ka botsitso ba ΔFosB Lisele tsa PC12 li ile tsa fetisetsoa ka neRN (Ctr) kapa siRNA e lebisang rat CK2cy le 24 h hamorao li fetisitsoe ka ΔFosB plasmid. Iphanele e kaholimo e bonts'a li-immunoblots tsa lysate tsa sele kaofela li bonts'a phello ea CK2 siRNA ho CK2 le liprotheine tsa ΔFosB. Immunoblot e lumellanang ea β-tubulin e bontšoa e le taolo ea ho laolla.

Ho nts'etsapele tsoelo-pele ea boleng ba CK2 joalo ka ΔFosB kinase, re sebelisitse lisele tsa PC12 tse hlahisang withFosB ka lithethefatsi tse peli tse fetolang ts'ebetso ea CK2. Joalokaha ho bontšitsoe Setšoantšo sa 4B, Phosphorylation ea ΔFosB e ile ea fokotseha ka kalafo ea lisele tsa PC12 ka selefounu e amohelang CK2 inhibitor DRB (Meggio et al., 1990; Szyszka et al., 1995), le ho eketseha ka kalafo ka semela sa polyamine, se tsejoang e le activator se matla sa CK2 (Cochet le Chambaz, 1983; Meggio et al., 1983). Ka lehlakoreng le leng, kalafo ea lisele tsa PC12 tse nang le PKC inhibitor calphostin-C (Kobayashi et al., 1989; Tamaoki et al., 1990) kapa activator ea PKC PMA (Schmidt le Hecker, 1975; Beh et al., 1989) ha ea ka ea baka phetoho ea bohlokoa phosphorylation ea ΔFosB. Tabeng ea PMA, keketseho e nyane (mme eseng e kholo) ea 32P-ΔFosB e ka baloa ka keketseho ea maemo ohle a ΔFosB a bakiloeng ke lithethefatsi tsena; ha e le hantle ho tlalehiloe hore li-phorbol esters li hlahisa polelo ea liprotheine tse 'maloa tsa lelapa tsa Fos, ho kenyeletsoa FosB (Yoza et al., 1992; Suh et al., 2004). Ntle le moo, kalafo ea lisele tse nang le mofuta o khethehileng oa inthanete ea CaMKII inhibitor m-AIP (Ishida le Fujisawa, 1995; Stevens et al., 1999) hape ha ea ka ea fella ka ho fokotseha ha ΔFosB phosphorylation (data e sa bontsitsoeng). Ka bobeli, liphetho tsena li tsamaisana le tsa rona a vitro fumana le ho bonts'a hore liseleng tse nang le ΔFosB e kanna ea fosisoa ke CK2 empa eseng PKC kapa CaMKII. Taba ea hore inhibition ea CK2 ha e thibele phosphorylation ea indicatesFosB ka botlalo e bonts'a libaka tsa "phosphorylation" tse eketsehileng protheine.

Ka mor'a moo re ile ra hlahloba hore na CK2 e bapala karolo ea phetoho ea ΔFosB ebe ka tsela e tsitsitseng. Ho fihlela sena, re entse liteko tsa "pulse-chase liteko" tse sebelisang lisele tsa PC12 tse hlahisoang ke CK2 inhibitor kapa activator ea CK2 e sebelisitsoeng thutong ea phosphorylation. Joalokaha ho bontšitsoe Setšoantšo sa 4C, kalafo ea lisele tse nang le CK2 inhibitor DRB e bile le phello e kholo molemong oa phetoho ea ΔFosB, e bonahatsoang ke phetoho ea sebopeho sa tšilafalo ea eona, ho tloha biphasic ho isa sekhutlong se haufi le sa ho bola ho pepesang. Ka lehlakoreng le leng, boteng ba semela sa CK2 activator se hlahisitse phokotseho e kholo ea sekhahla sa ho senyeha sa ΔFosB, se lebisitseng ho bokelleng ha proteni ka lihora tsa pele tsa ho lelekisa (Feie. 4D).

Joalo ka ha ho na le li-activator tse ngata tsa kinase le li-inhibitors, spermine le DRB li ka ba le litlamorao tsa metabolic kantle ho phetoho ea mosebetsi oa CK2. Ebile, DRB e bontšitsoe ho thibela transcript factor IIH (TFIIH) -associated kinase (Yankulov et al., 1995), e hlahisang thibelo ea sengoloa sa RNA polymerase II-Mediated. Hobane phello ena e ka ama botsitso ba ΔFosB, re ile ra sekaseka phello ea kakaretso e kholo ea kinase inhibitor H-8 (Hidaka le Kobayashi, 1992) ho ΔFosB Turnover at a mahloriso (200 μm) e tsejoang ka ho thibela kinase e amanang le TFIIH empa eseng CK2 (Yankulov et al., 1995). Joalokaha ho bontšitsoe ho Setšoantšo sa 4E, kalafo ea H-8 ha ea ka ea ama sekhahla sa phallo ea ΔFosB. Liphetho tse ts'oanang li fumanoe ka H-7, e 'ngoe e' ngoe e bonoang kinase inhibitor, e sebelisitsoeng sebakeng sa mahloriso se thibelang kinase e amanang le TFIIH empa eseng CK2 (data e sa bonts'ang). Lintlha tsena li boetse li ts'ehetsa tlhaloso ea hore phokotso ea botsitso ba ΔFosB e bakoang ke DRB e kanna ea hlahisoa ke ho thibela CK2.

Ho ntšetsa pele karolo ea CK2 ho phetoho ea ΔFosB, re fuputse liphetho tsa ho kokobela CK2 ka siRNA. Re entse liteko tsena re sebelisa lisele tsa PC12 tse ileng tsa fetisoa ka lekhetlo la pele ka taolo (e sa reroang) siRNA kapa siRNA e shebileng mRNA ea semelo sa katishene sa rat CK2 (CK2α) le 24 h hamorao li fetisitsoe ka ΔFosB. Joalokaha ho bontšitsoe Setšoantšo sa 4F, phetisetso le CK2cy siRNA hantle mme ka kotloloho e theotse maemo a liprotheine tsa CK2cy ntle le ho ama maemo a ΔFosB (phanele e kaholimo). Tlhahlobo ea Pulse-chase e senotse hore ho koenya CK2 ho bakile keketseho e kholo ea sekhahla sa phetoho sa ΔFosB joalo ka ha ho bonts'oa ke ho senyeha ha protheine ka potlako. Taba ea ho thibela ts'ebetso ea CK2 (ebang ke ka kalafo ea DRB kapa siRNA) e eketsa phetoho ea ΔFosB mme e fella ka ho nyamela ha karolo ea butle ea sekhahla sa biphasic curve, athe ts'ebetso ea CK2 e ntlafatsa mohato o tlase oa khohlo, mohopolo oa hore CK2 e bapala karolo ea ho laola botsitso ba ΔFosB

CK2 phosphorylates ΔFosB ho Ser27

Ho qala ho khetholla sebaka (s) ho ΔFosB phosphorylated ke CK2, re ile ra etsa tlhahlobo ea phospho-amino acid ea recombinant ΔFosB phosphorylated ke CK2 a vitro le ΔFosB phosphorylated liseleng tsa PC12. Liteko tsena li senotse hore maemong ana ka bobeli, phospho-amino acid e fumanoeng feela e ne e le phospho-Ser (Feie. 5A). Ho fumana hona, hammoho le stoichiometry e fumanoeng bakeng sa CK2 a vitro phosphorylation reaction (∼50%) (Feie. 3B) le boteng ba sebaka se le seng sa bohlokoa 'mapeng oa CK2 phosphopeptide (Feie. 4A), e fana ka maikutlo a hore CK2 phosphorylation ea ΔFosB mohlomong e lekantsoe ke masala a le mong a serine. Qeto ena e lumellana le tlhahlobo ea sebaka sa tumellano ea phosphorylation (Feie. 2B), e boletseng serine e le 'ngoe ea mokhethoa, ke hore, Ser27, bakeng sa CK2. Tlhahlobo ea taxonomic e senotse hore Ser27 e bolokiloe haholo ka ho iphetola ha litho tsa lelapa la Fos (Feie. 5B), ho fana ka maikutlo a hore e ka ba le ts'ebetso ea bohlokoa ea 'mele.

Setšoantšo sa 5.

Sheba tlhahiso e kholoanyane:

Setšoantšo sa 5.

Phosphorylates ea CK2 ΔFosB ho Ser27. A, Phosphoamino acid e CK2-phosphorylated (a vitro) le PC12 cell-phosphorylated ΔFosB e bonts'ang, maemong ana ka bobeli, phosphorylated e kholo ke serine. B, Tlhatlhobo ea mefuta ea sefapano ea tatellano ea FosB / ΔFosB amino acid e senotse paballo e phahameng ea Ser27 har'a litho tsa lelapa la Fos ho tloha ho motho ho ea ho zebrafish (setšoantšo sa lefifi). Leha ho le joalo, masala a acidic a maemong + 3, a hlokahalang bakeng sa sebaka sa tumellano ea CK2, ha a bolokiloe (leseli le khanyang). C, Lisele tsa HeLa li ne li fetotsoe ka nakoana le mofuta oa hlaha oa ΔFosB (WT) kapa ΔFosB e nang le phetoho ea lintlha ho nkela Ser27 le Ala (S27A). Lisele li ne li ngotsoe ka metabolism le 32Ph3PO4 le ho phekoloa ka koloi kapa ka spermine (SP) ho kenya ts'ebetso CK2. Autoradiogram ea moemeli (konopo e kaholimo) le immunoblot (konopo e ka tlase) ea li-immunoprecipitates tse fumanoeng li bonts'itsoe. The immunoprecipitate ea lisele tse fetisitsoeng ka mokhoa o feteletseng (vector) e bonts'itsoe.

Ho fumana hore na Ser27 e kentsoe phosphorylated ho ΔFosB, re fetoletse masala ana ho Ala, mme ra sekaseka litlamorao maemong a phosphorylation. Ho fihlela sena, re sebelisitse lisele tsa HeLa (tse ka fetisoang ka katleho e phahameng) ho hlahisa WT kapa S27A mutant ΔFosB butle-butle. Hoo e ka bang 24 h kamora phetiso, lisele li ile tsa ngolloa ka metabolism le 32P-orthophosphate le lysate tsa seleng eohle li ile tsa etsoa. Ho latela ts'ireletso le SDS-PAGE, 32P-ΔFosB e fumanoe ke autoradiography le kakaretso ea ΔFosB ka ho kenya immunoblotting. Joalokaha ho bontšitsoe Setšoantšo sa 5C (phanele e ka tlase), ΔFosB ha ea ka ea fumanoa liseleng tse fetisitsoeng ka veter, athe lisele li fetisitsoe ka WT kapa S27A mutant ΔFosB e hlahisitse protheine ka katleho. Re fumane hore phetoho ea S27A e bakile phokotso ea bohlokoa (∼30%) 32Boleng ba P-ΔFosB (Feie. 5C, holimo le graph), e bonts'a hore liseleng tse phelang, ΔFosB e kentsoe phosphorylated ho Ser27. Boitekong ba ho fumana hore na liseleng tsa Ser27 ehlile li kentsoe ke CK2, re bapisitse bokhoni ba semelo sa CK2 activator spermine ho fetola phosphorylation ea WT le S27A ΔFosB. Joalokaha re ne re hlokometse pejana liseleng tsa PC12 (Feie. 4B), kalafo ea lisele tsa HeLa tse nang le spermine e matlafalitseng phosphorylation ea protheine ea WT. Taba ea hore phello ena e fokotsehile haholo ke phetoho ea S27A (Feie. 5C) e tšehetsa toloko ea hore Ser27 ho ΔFosB ke karoloana ea 'mele ea CK2.

Phosphorylation ea Ser27 e laola botsitso ba ΔFosB

Kaha o thehile hore botsitso ba ΔFosB bo fokotsehile ha CK2 e sa thibeloe le ho ntlafatsoa ha CK2 e sebetsoa (Feie. 4), le hore CK2 phosphorylates ΔFosB ho Ser27 (Feie. 5), re boletse esale pele hore ho thibela phosphorylation ea sebaka sena sa marang-rang ho lokela ho senya protheine. Liteko tsa Pulse-chase tse entsoeng le lisele tsa HeLa butle-butle li hlahisa WT kapa S27A mutant ΔFosB li senoletse hore, joalo ka ha ho boletsoe esale pele, phetoho ea S27A e bakile keketseho e kholo ea tekanyo ea ho fokotsoa ha ΔFosB, le phokotso e lekanang le halofo ea bophelo ba protheine. (Feie. 6A). Ka mor'a moo re ile ra etsa lipatlisiso tsa hore na mochine ona oa taolo o hlahella le ka seleng ea "neuron" e kang PC12. Liteko tsena li senotse hore, joalo ka ha re bone liseleng tsa HeLa, phetoho ea ntlha ea S27A e baka phokotseho e kholo ea halofo ea bophelo ba ΔFosB liseleng tsa PC12 (ho tloha ∼11 ho ∼4 h) (Feie. 6B). Taba ea hore ts'oenyeho ena e ts'oana le e hlahisoang ke CK2 inhibition kapa knock-down (Feie. 4) e fana ka tšehetso e eketsehileng bakeng sa mohopolo oa hore CK2-Mediated phosphorylation ea Ser27 e laola botsitso ba ΔFosB. Bopaki bo eketsehileng bakeng sa karolo ea taolo ea phosphorylation ea Ser27 ho phetoho ea protheine ea ΔFosB e fumanoe ka phosphomimetic Ser27 ho Asp mutation (S27D). Phetoho ea S27D e nkoa e le phosphomimetic, hobane e beha sehlopha se seholo se qosoang (carboxyl) ho amino acid 27 mme ka tsela e itseng se etsisa phosphorylation ea Ser27. Joalokaha ho bontšitsoe Setšoantšo sa 6C, phetoho ea S27D e bakile phello e fapaneng ea phetoho ea S27A mme ea baka protheine e tsitsitseng haholo ho feta protheine ea WT. Ka mokhoa o ts'oanang le ho phello e fumanoeng kamora ts'ebetso ea CK2 (Feie. 4D), phetoho ea S27D e hlahisitse khokahano mme ka hona e ile ea eketsa maemo a ΔFosB nakong ea lihora tsa pele tsa ho lelekisa.

Setšoantšo sa 6.

Sheba tlhahiso e kholoanyane:

Setšoantšo sa 6.

Phosphorylation ea Ser27 e laola botsitso ba ΔFosB. A, C, Ts'ebetso ea Pulse-chase e bonts'a phello ea phosphorylation ea Ser27 ho sekhahla sa phallo ea ΔFosB. Sebaka sa ho senya le ho hakanyetsoa ha halofo ea bophelo ba mofuta oa hlaha oa ΔFosB (WT), Ser27 ho Ala mutant (S27A), le phosphomimetic Ser27 ho Asp mutant (S27D) li bonts'itsoe. Liphumano tse tšoanang li fumanoe liseleng tsa HeLa (A) le lisele tsa PC12 (B, C).

Phosphorylation ea Ser27 e tsitsisa ΔFosB ka ho thibela ho senyeha ha eona ea protheine

Ho qala ho hlakisa mochine o sebelisang phosphorylation ea Ser27 ΔFosB, re ile ra hlahloba bokhoni ba protheine ea thibelo ea proteinasome MG132 (Palombella et al., 1994; Tsubuki et al., 1996) le epoxomicin (Hanada et al., 1992; Kim et al., 1999) ho fetola sekhahla sa ho senyeha ha WT le S27A mutant ΔFosB. Liteko tsa Pulse-chase li entsoe ka ho sebelisa lisele tsa PC12 tse tšoaelitsoeng ke HSV e sa kopaneng e hlalosang WT kapa S27A ΔFosB mme li alafshoa ka DMSO kapa e 'ngoe ea li-inhibitors tse peli tsa protein. Liteko tsena li senotse hore le ha tekanyo ea ho senyeha ea protheine ea WT ha e na taba le boteng ba e 'ngoe ea li-inhibitors tse peli tsa protheine (Feie. 7A), ea S27A mutant e tsotella lithethefatsi tsena (Feie. 7B). Sena se bonts'a hore, ho fapana le protheine ea WT, S27A mutant ke sepheo sa ho senyeha hoa protheine. Ka sebele, kalafo ea lisele tse nang le MG132 kapa epoxomicin e khutlisitse ka botlalo phello ea phetoho ea S27A ka sekhahla sa ho senyeha sa ΔFosB, se pakoang ke keketseho ea halofo ea bophelo ba S27A mutant ho tloha ∼4 h ho M∼9 le ho ∼ 132 h bakeng sa epoxomicin (Feie. 7B). Ka kopanelo, liphumano tsena li bonts'a hore phosphorylation ea ΔFosB ho Ser27 e sireletsa protheine ho senyeha ha proteinasomal ka hona e bohlokoa bakeng sa botsitso ba eona bo sa tloaelehang.

Setšoantšo sa 7.

Sheba tlhahiso e kholoanyane:

Setšoantšo sa 7.

Phosphorylation ea Ser27 e tsitsisa ΔFosB ka ho thibela ho senyeha ha eona ea protheine. A, B, Tlhahlobo ea Pulse-lelekisa le lisele tse nang le tšoaetso ea HSV PC12 tse bonts'ang boemo ba boleng bo litšila le halofo ea bophelo ba mofuta oa hlaha oa ΔFosB (A) kapa S27A ΔFosB (B) ka ho ba sieo kapa ho ba le e 'ngoe ea li-inhibitors tse peli tsa protheine [MG132 le epoxomicin (Epoxo)]. Hlokomela 'nete ea hore ha ho na lithethefatsi tse nang le tšusumetso e kholo phetohong ea mofuta oa hlaha oa ΔFosB, athe kalafo ea lisele tse nang le protheine inhibitor e hlahisitse botsitso ba S27A ΔFosB. C, Mohlala bakeng sa karolo ea phosphorylation ea Ser27 matleng a toFosB ho ikopanya le polasetiki ea nako e telele ea kelello. Hang ha e tsitsitsoe, karolo ea ΔFosB e tsitsitse bokong ke CK2-mediated phosphorylation ea S27. Sena se fella ka hore se bokellane, 'me seo se fella ka liphetoho tsa nako e telele polelong ea liphatsa tsa lefutso. Liphetoho tsena tse tsitsitseng polelong ea liphatsa tsa lefutso li kenya letsoho ho tloaelo e tsitsitseng ea boitšoaro. Ka lehlakoreng le leng, dephosphorylation ea S27 ke lipapatso tsa Ser / Thr le PP1 le / kapa PP2A e etsa hore protheine e sebetse hantle le ts'ebetso ea ona ke mochini oa proteasomal.

Karolo e fetilengKarolo e latelang

Puisano

Thutong e teng hona joale, re bonts'a hore ΔFosB e phela halofo ea bophelo ba ∼10 h moetlong oa sele, e etsang hore e ts'oarehe ha e bapisoa le bolelele bo felletseng ba FosB le lintlha tse ling tse ngata tse sa sebetseng, tseo halofo ea eona e phelang moetlong oa sele e ka bang mokhuts'oanyane ka metsotso e seng mekae mme ka seoelo se feta 3 h (Hann le Eisenman, 1984; Dobrazanski et al., 1991; Roberts le Whitelaw, 1999; Ferrara et al., 2003; Hirata et al., 2004). Ntle le moo, re fumana hore ΔFosB e kentsoe phosphorylated bokong le hore phosphorylation ea eona e amehile ka PP1 / PP2A inhibitor okadaic acid. Lithuto tsa rona tsa setso sa lisele li bonts'a hore botsitso ba ΔFosB bo ntlafatsoa ke CK2, ka ts'ebetso e phahameng ea CK2 e tiisa protheine. Kamora nako, liphumano tsa rona li fana ka maikutlo a hore CK2 phosphorylates ΔFosB ho N terminus serine (S27) e bolokiloeng haholo 'me e bonts'a phosphorylation ea S27 e sireletsa degrFosB ho senyeha hoa proteinasomal. Ka hona re fana ka tlhahiso ea hore na phosphorylation ea ΔFosB ho S27 ke CK2 ke mokhoa o bohlokoa oa taolo ea phetoho ea ΔFosB (Feie. 7C). Phosphorylation-Mediated stabilization ea ΔFosB e bohlokoa ka mokhoa o sebetsang, hobane maemo a eketsehang a ΔFosB ka ho khetheha likarolo tsa boko a bontšitsoe hore a laole ka kotloloho mefuta e mengata ea li-neuronal Ka vivo le ho fana ka litlamorao tse matla tsa boitsoaro mefuteng ea liphoofolo ea mafu a 'maloa a amanang le neuropsychiatric (sheba Selelekela).

Leha phosphorylation e le mokhoa o potlakileng hape o feto-fetohang oa ho laola tšebetso ea mongolo oa lintlha tse itseng tsa mongolo, joalo ka CREB (cAMP reaction element binding protein) (Bohmann, 1990), ho fetola ho senyeha hoa lintlha tsa mongolo ho fana ka mofuta o matla haholo (o sa fetoheng habonolo) oa molawana (Desterro et al., 2000). Lintho tse hatisitsoeng tseo ts'ebetso ea tsona e sebetsang e laoloang ho boemo ba litšila tsa bona li kenyelletsa NF VerB (Desterro et al., 2000), c-Myc (Sears et al., 1999), le c-Fos (Ferrara et al., 2003), hara ba bang. Maemong a mangata phosphorylation ke molaoli oa mantlha oa botsitso ba ntho e ngotsoeng, joalo ka ha ho bonts'itsoe c-Fos (Okazaki le Sagata, 1995; Tsurumi et al., 1995), Li-antigen-1 tse amanang le Fos tse amanang le Fos-1 (Vial le Marshall, 2003), Fra-2 (Manabe et al., 2001), c-Jun (Fuchs et al., 1996), JunB (Fuchs et al., 1997), ATF2 (Fuchs et al., 2000), le p53 (Buschmann et al., 2001). Ka hona lithuto tsa rona li eketsa ΔFosB lenaneng lena la lintho tse hatisitsoeng tseo ts'ebetso ea tsona e laoloang ka botsitso ba botsitso ba phosphorylated.

CK2 ke ser /ase kinase e fumanehang hohle le e sebetsang ka matla e nang le likaroloana tse 300 tse boletsoeng ho tsebahala ho fihlela joale 'me li ameha lits'ebetsong tse ngata tsa bioloji, ho kenyeletsoa lefu la sele le pholoho (Litchfield, 2003; Unger et al., 2004), likarabo tsa khatello ea kelello (Yanagawa et al., 1997; Kato et al., 2003), le tokiso ea DNA le ho nchafatsoa ha chromatin (Barz et al., 2003; Krohn et al., 2003). Karolo e fetang karolo ea boraro ea likarolo tse tlatsetsang tsa CK2 ke liprotheine tse kenyellelitsoeng ho tsamaiso ea polelo ea gene (Meggio le Pinna, 2003). Ebile, CK2 e bontšitsoe e le kinase e tsebahalang ea nyutlelie (Krek et al., 1992) (bakeng sa tlhahlobo, bona Yu et al., 2001) le ho sebelisana le likarolo tsa bZIP tsa lintlha tse 'maloa tsa mongolo (Yamaguchi et al., 1998). CK2-Mediated phosphorylation e boetse e bontšitsoe ho lokisa tšilafalo (ekaba ea e ntlafatsa kapa ea e fokotsa) ea liprotheine tse ngata, ho kenyelletsa IkB (Schwarz et al., 1996), PTEN (Torres le Pulido, 2001), lenne connexin (Yin et al., 2000), protheine e amanang le chromatin e amanang le HMG1 (Wisnviewski et al., 1999), le lintlha tse 'maloa tsa mongolo o kang HMGB (Stemmer et al., 2002), Myf-5 (Mariha et al., 1997), le c-Myc (Channavajhala le Seldin, 2002). CK2 e na le boko bo bongata haholo (Alcazar et al., 1988; Girault et al., 1990), mme ts'ebetso ea eona e bile le tšusumetso likarolong tse ngata tsa ts'ebetso ea boko, ho kenyelletsa ho pholoha ha neuronal (Boehning et al., 2003), phapang (Nuthall et al., 2004), ts'ebetso ea mocha oa ion (Jones le Yakel, 2003; Bildl et al., 2004), le bokhoni ba nako e telele le neuronal plasticity (Diaz-Nido et al., 1992; Lieberman le Mody, 1999; Reikhardt et al., 2003).

Leha ho na le bopaki bona bo ntseng bo eketseha ba karolo ea CK2 taolong ea tšebetso ea methapo ea kutlo, ha ho tsejoe letho ka hore na ke eng e laolang tšebetso ea eona. CK2 e nahanoa hore e sebetsa ka mokhoa o hlophisehileng, ka taolo ea bokhoni ba eona ba ho hlophisa likarolo tse ikhethileng tse itšetlehileng haholo ka liphetoho phetohong ea lehae ea eona ea methapo (mohlala, cytosol vs nucleus) (Ahmed le Tawfic, 1994; Yu et al., 1999). Tlhahisoleseling ena e phahamisa potso ea bohlokoa mabapi le hore na ho hlokahala lipontšo life ho kenya letsoho ho bokellana ha ΔFosB bokong kamora ho hlohlona ho sa feleng ((Feie. 7C). Tlhokahalo e le 'ngoe ke ho pheta-pheta hoa fosB mofuta oa tlhahiso ea ΔFosB mRNA (Chen le al., 1995). Lintlha tsa rona li bontša hore CK2 phosphorylation ea ΔFosB e bohlokoa bakeng sa botsitso ba eona, e fana ka maikutlo a hore letšoao la bobeli le ka hlokahala bakeng sa litlamorao tsa nako e telele tsa ΔFosB mabapi le polelo ea gene, e leng, lets'oao le tsosang phosphorylation ea protheine ka CK2. Sena se ka kenyelletsa ts'ebetso ea CK2 ka mochine o itseng o sa tsejoeng kapa ho fetisetsoa ha eona mochophorong. Ntle le moo, phosphorylation ea CK2 ea mayFosB e kanna ea fetoha, joalo ka ha protheine ea ΔFosB e fetoleloa ho arabela ts'usumetso e 'ngoe le e' ngoe, karolo ea eona e ba phosphorylated ebe ka tsela eo e tsitsipane, e le hore ka ts'usumetso e phetoang e bokellana maemong a holimo manonyeletsong a amehang.

Liphumano tsa rona li bonts'a hore CK2 le S27 mohlomong ha se tsona feela sebaka le sebaka se ikarabellang bakeng sa phosphorylation ea ΔFosB, hobane ha ho thibelo ea CK2 kapa phetoho ea S27A ha e khone ho thibela phosphorylation ea ΔFosB ka botlalo. Ka leihlo le ts'oanang, taba ea hore phetoho ea S27A e baka phokotso ea 30% phospho-ΔFosB e tiisa hore S27 ke sebaka se seholo sa phosphorylation ho protheine. Leha ho le joalo, re ntse re etsa lipatlisiso ka li-kinase tse ling tse behang le libaka tsa phosphorylation ho ΔFosB. Qetellong, ho tla ba bohlokoa le ho sekaseka phosphorylation ea S27 le libaka tse ling tsa phosphorylation ho ΔFosB bokong Ka vivo kamora mefuta e fapaneng ea ts'usumetso e sa foleng, ho etsa mohlala, ka tšebeliso ea matla a mangata a ho thibela mahlo kapa a phosphospecific antibodies.

Joalokaha ho boletsoe kaholimo, S27 ho ΔFosB e bolokiloe haholo ho iphetola ha lintho le har'a liprotheine tse ling tsa lelapa la Fos. Leha ho le joalo, sebaka sa tumellano sa CK2 ha se: joalo ka ha ho bonts'itsoe Setšoantšo sa 5B, FosB / ΔFosB feela (le Zebrafish xenolog), empa eseng c-Fos kapa Fra-2, ba nang le masala a acidic ho + 3, senotlolo sa senotlolo sa CK2 phosphorylation (Marin et al., 1986; Meggio et al., 1994). Kahoo, ho haella ha phosphorylation ea CK2 ho S27 ho ka hlalosa lebaka leo liprotheine tse ling tsa malapa tsa Fos li sa tsitsang joaloka ΔFosB. Leha ho le joalo, sena ha se hlalose hore na hobaneng FosB e bolelele bo felletseng, e nang le sebaka sa tumellano se tšoanang sa CK2 joalo ka ΔFosB, ha e ts'oaroe ka mokhoa o tšoanang. Ha re tsebe hore na FosB e bolelele bo felletseng e fositsoe ke CK2 ho masala ana a bolokiloeng. Litlaleho tsa FosB feela (Skinner et al., 1997) le c-Fos (Okazaki le Sagata, 1995; Chen le al., 1996) phosphorylation e hlalosa libaka tsa C-terminal tsa liprotheine tsena, tse sieo ho ΔFosB. Phosphorylation e ka hlahang ea li-FosB tse bolelele bo phethahetseng le liprotheine tse ling tsa malapa tsa Fos ka CK2 e hloka hore ho etsoe lipatlisiso tse tobileng. Leha ho le joalo, le haeba e le phosphorylated, liprotheine tse ling tsa lelapa la Fos li tsejoa li na le li-motifs ho C ea bona C e lebisang liprotheine bakeng sa ho senyeha kapele (Papavassiliou et al., 1992; Jariel-Encontre et al., 1997; Acquaviva et al., 2002). Mohlala, ho bontšitsoe hore masala a ∼21 a leng teng pheletsong ea C ea liprotheine tsohle tsa lelapa tsa Fos, empa a le sieo ho ΔFosB, o sebetsa joalo ka domain name ea c-Fos (Acquaviva et al., 2001). Re fumane hore le ha tatellano ena ka tatellano e tsitsisa bolelele ba FosB e felletseng (Carle et al., 2004), ho ba sieo ha domain name ho ΔFosB ha ho na boikarabello ba ho tsitsa ha eona. Ho fapana le moo, ho kopana ha ho ba sieo hoa domain ena ea C terminus le phosphorylation ea Ser27 ho bonahala e ikarabella ka botlalo phapusing e ka bang makhetlo a mahlano ea botsitso lipakeng tsa ΔFosB le FosB.

Leha ho senyeha ha liprotheine tsa lelapa la Fos ho rarahane ebile ho sa utloisisoe ka botlalo, ho senyeha hoa protheine ho bonahala e le tsela e kholo e amehang (Salvat et al., 1999; Acquaviva et al., 2002; Ferrara et al., 2003). Lintlha tse hlahisitsoeng mona, tseo ho tsona li-protheineal inhibitors li sa fetoleng boemo ba ho fifala ha ΔFosB, li re, ho fapana le liprotheine tse ling tsa lelapa la Fos, ΔFosB e qoba protheine ea 26S, 'me sena se bapala karolo ea mantlha botsitsong ba eona. Ka hona re fana ka maikutlo a moralo oo ka oona botsitso bo ntlafalitsoeng ba ΔFosB bo hlahisoang ke ho kopanngoa ha lintlha tse peli tsa bohlokoa: (1) ho ba sieo ha sebaka sa C-terminal destabilizing domain and (2) phosphorylation ea S27 ka CK2.

Ka kakaretso, thuto ea hona joale e fana ka leseli la ho latela hore na ke hobaneng ΔFosB, sehlahisoa sa mofuta oa pele oa mofuta fosB, ha e tšoane le litho tsohle tsa lelapa la Fos, protheine e telele e phetseng nako e telele. Le ha liprotheine tse ling tsa lelapa la Fos ho nahanoa hore li nka nako e le hore li tsamaisane ka potlako le ka nakoanaMorgan le Curran, 1995), botsitso bo lekanang ba ΔFosB e fana ka eona bokhoni ba ho ikamahanya le liphetoho tse sa feleng tsa mongolo o hlahisoang ke ts'usumetso e sa feleng. Sena se ts'ehetsa pono ea hore ΔFosB e sebetsa joalo ka phetoho ea limolek'hule bokong, butle-butle e fetole likarabo tse matla hore e be liphetoho tse sa fetoheng. Ho tsebahatsa phosphorylation ea Ser27 e le mokhoa oa mantlha oa botsitso ba ΔFosB e bula mekhoa e mecha ea nts'etsopele ea mekhoa ea ho laola ts'ebetso ea ΔFosB 'me ka tsela ena e fetola litlamorao tsa eona tsa nako e telele ho li-neural le boits'oaro bo botle.

Karolo e fetilengKarolo e latelang

Mongolo o botlaaseng ba leqephe

  • E amohetse November 21, 2005.
  • Nchafatso e amohetse ka February 21, 2006.
  • E amohetse April 2, 2006.
  • Mosebetsi ona o tšehelitsoe ke National Alliance for Research on Schizophrenia and Depression Young Inarcherator Award to PGU, National Institute on Drug Abuse (NIDA) National Research Service Award to PGU, and grants from the National Institute of Mental Health and NIDA to EJN We leboha Dr James Bibb bakeng sa thuso ea hae le a vitro Phosphorylation e khothatsa, Dr. Ming-Hu Han bakeng sa thuso ea hae ka ho hlophisoa ha maseke a bokong a sebelisitsoeng bakeng sa ho ngola metabolic, le Dr. Rachael Neve bakeng sa thuso ea hae ka ho kenya li-HSV tse kopantseng.
  • Aterese ea hajoale ea G. Rudenko: Life Sciences Institute le Lefapha la Pharmacology, Univesithi ea Michigan, 210 Washtenaw Avenue # 3163A, Ann Arbor, MI 48109-2216.
  • Litaba tsa mangolo li lokela ho lebisoa ho Eric J. Nestler, 5323 Harry Hines Boulevard, Dallas, TX 75390-9070. Email: [imeile e sirelelitsoe]

Karolo e fetileng

 

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