Postna ta l D evelopm en ta l Expression of Phosphoprote in s in Synaptosom e

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1 14 1 V o l114,n o Ch inese Jou rnal of B iochem istry and M o lecu lar B io logy Feb (, ),,, (PND ) 3 d 7 d 21 d,, ; PND 21d, :,,, Postna ta l D evelopm en ta l Expression of Phosphoprote in s in Synaptosom e Yan L i2jun W ei Q un (D ep artm ent of B iology, B eij ing N orm al U niversity, B eij ing ) Abstract T he regu lation of the pho spho rylation state of specific sub strate p ro tein s by p ro2 tein k inases and pho sphatases p lays a p ivo tal ro le in neu ronal function ṡ In the develop ing m amm alian b rain, these pho sphop ro tein s are im po rtan t fo r the fo rm ation of synap ses du ring the po stnatal period T herefo re, the elucidation of the changes of these sub strates invo lving the differen t phasing of synap se fo rm ation is an essen tial step tow ard understanding the ner2 vou s system function To detect these pho sphop ro tein s, po stnatal developm en tal exp ression of pho sphop ro tein s w ere quan titatively assayed in rat cereb ral co rtex synap to som e Pho spho2 p ro tein s exp ression show ed developm en tal changes in rat cereb ral co rtex synap to som e on po stnatal days (PND ) 3, 7, 21 and adu lṫ 32 P labelled pho sphop ro tein s appeared in h igh level du ring the periods co rrdsponding to m ajo r synap se fo rm ation Pho spho rylation of sub strate p ro tein s began to decrease un til PND 21 It has been suggested that the Ca 2+ gcalm odu lin2de2 penden t p ro tein pho sphatase 2B, o r calcineu rin (CaN ) are h igh ly exp ressed du ring the m ajo r period of synap se fo rm ation CaN endogenou s sub strates w ere detected in rat cereb ral co rtex synap to som e on PND 3, PND 7, PND 21 and adu lt u sing an ti2can po lyco lonal an tibody, gradi2 en t SD S2PA GE and au to radiagraphy Fu rther studies w ere undertaken to exam ine w hether the exp ression levels of CaN sub strate p ro tein s w ou ld change fo llow ing the developm en tal pattern Key words: Pho sphop ro tein,d evelopm en t, Calcineu rin, Synap to som e, Endogenou s sub strate 3 ( ) : , :

2 , [ 1 3 ], (L T P) (L TD ), CaM K g PKC PKA L T P (calcineu rin, CaN ) L T P,,, L T P, L T P, [ 4 ], CaN, W istar, (CaM ) (cam P) (PS) PM A (pho rbo l2122m yristate 132acetate) PM SF (penyim ethylsu lfonyl fluo ride) CHA PS Sigm a Am pho linerange ( ) Pharm cia, Χ2 32 P2A T P 112 ; CaN 2Sepharo se 4B CaN IgG, EL ISA, CaN d 7 d 21 d 3 6 [ 5, Po lli ],, 3 d 7 d 21 d, 8 12 ;, 4 6, 1 3 H (20 mm o lgl H EPES, ph 7 2, 0 25 m o lgl, 1 mm o lgl ED TA, 100 Λm o lgl leu2 pep tin 100 Λm o lgl PM SF), 4, g 5 m in,, g 30m in H, 114 Kohhar [ 6 ] T ash [ 7 ] 80 mm o lgl T ris2hc l, ph 7 4, 20 mm o lgl M gso 4, 014 mm o lgl ED TA, 10 mm o lgl Β2, 20 Λm o lgl A T P, 3 Λm o lgl PM A, Λm o lgl PS, 20 Λm o lgl cam P, 6 mm o lgl CaC l2, 40 ΛggL CaM, 6 ΛC i[χ2 32 P ]2A T P, 30 5 m in, 32 P, 715% 15% SD S2PA GE, 715% 15% SD S2PA GE, 100 Λg ;, 200 Λg 115, 4, CaN, 32 P, CaN 116 X M TV 1881CCD ( ) 104

2 211 SD S2PA GE (F ig 1A ), PND 3 PND 7 PND 21, 8 11 11 7 T ab le 1, 32 P T ab le 1 F ig 1 Gradient SD S2PA GE auto radiograph analysis on, PND 21, 32 p ro tein pho spho rylation in rat cerebral co

3 2 211 SD S2PA GE (F ig 1A ), PND 3 PND 7 PND 21, T ab le 1, 32 P T ab le 1 F ig 1 Gradient SD S2PA GE auto radiograph analysis on, PND 21, 32 p ro tein pho spho rylation in rat cerebral co rtex Synap to som e extracts of PND 3, PND 7, PND 21 and adult w ere incubated in the absence (A ) o r p resence (B ) of anti2 CaN antibody overnight kd 30kD 24kD 21kD, 35 kd ;, PND 21,, F ig 2, PND 21 Table 1 Q uantitative comparison of synap to som e pho sphop ro teins fo r PND 3, PND 7, PND 21 and adult rat Pho sphop ro teins fo r mo lecular w eigh t (kd )of all k inds Samp le PND PND PND A dult D ata dep icted show relative pho sphop ro tein contents fo r mo lecular w eigh t of all k inds T hese results rep resents a sin2 gle experim ent but rep licate gave comparable results 212 CaN CaN,, SD S2PA GE (F ig 1), PND 3, 9 CaN, PND 7, 7 ; PND 21, 7 ;, 6 ; CaN, 32 P, T ab le 2, 21 kd 24 kd 35 kd (PND 7) 24 kd (PND 21), 2 3 T ab le

F ig 2 P ro tein pho spho rylation auto radiograph in rat cerebral co rtex synap to som es of PND 21 (a) and adult (b) analyzed by 22D 2PA GE A rrow heads show pho sphop ro teins in (a) and (b) T he

4 F ig 2 P ro tein pho spho rylation auto radiograph in rat cerebral co rtex synap to som es of PND 21 (a) and adult (b) analyzed by 22D 2PA GE A rrow heads show pho sphop ro teins in (a) and (b) T he pho sphop ro teins fo r PND 21 co rresponding to adult show using the sam e digit in (a) and (b), 112 kd 87 kd kd 62 kd, PND 21,, PND 7, PND 7, 96 kd 82 kd 41kD 24kD, ; 30 kd PND 7 PND 21,,, [ 425 PND 21 ],, CaN Ca 2+ W einberger [ 8 ] cam P (cam P2 PK) cam P2PK 6 15 d, cam P2PK CaN cam P2PK, cam P2PK, CaN Ca 2+ gcam g, Ca 2+ g, CaM C (PKC) Ca 2+ gcam, SD S2PA GE 14 6 PND 7 PND 21, 4, 3 PND 3 106

5 SD S2PA GE, PND 3 PND 7 PND 21, CaN, CaN, Table 2 Q uantitative comparison of the effect of anticalcineurin on p ro tein pho spho rylation in developm ental and adult rat CaN endogenous sunbtrates fo r mo lecular w eigh t (kd )of all k inds Samp le PND PND PND A duit D ata dep icted show CaN endogenous substrates relative contents fo r mo lecular w eigh t of all k indṡ T hese results rep2 resents a single experim ent but rep licate gave comparable results References 1 M ulkey R M, H erron C E,M alenkn R C A n essential ro le fo r p ro tein pho sphatases in h ippocampal long2term dep ression S cience, 1993, 261: Funauch im, H aruta H, T sumo to T Effects of an inh ibito r fo r calcium gcalmodulin2dependent p ro tein pho sphatase, cal2 cineurin, on inducto in of long2term po tentiation in rat visual co rtex N eu rosci R es, 1994, 19: To rii N, Kam ish ita, T O tsu Y, T sumo to T A n inh ibito r fo r calcineurin, FK 506, block s induction of long2term dep res2 sion in rat visual co rtex N eu rosci lett, 1995, 185: 1 4 4,, (Yan L i2jun, L i D a2h ai,w ei Q un Subcellular localization of calcineurin activity in developm ental and adult rat cerebral co rtex Chin B iochem J ), 1995, 11: Po lli J W, B illingsley M L, Kincaid R L Exp ression of the calmodulin2dependent p ro tein pho sphatase, calcineruin rat brain: developm ental patterns and the ro le of nigro striatal innervato in D ev B rain R es, 1991, 63: Kochhar A Saitoh T and Zivin J A Sp inal co rd ischem ia reduces calcium gcalmodulin2dependent p ro tein k inase activity B rain R es, 1991, 542: T ash J S, Krink sm, Patel J,M eans R L, Klee C B,M eans A R Identification, charaterization, and functional co rrelation of calmodulin2dependent p ro tein pho sphatase in sperm J Cell B iol, 1991, 106: W einberger R P, Ro stas J A P D evelopm ental changes in p ro tein pho spho rylation in ch icken fo rebrain I cam P2stim ulat2 ed pho spho rylation D eve B rain R es, 1988, 43:

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