2.1 Purification of 14 -3 -3
2.2 Reverse Phase Separation of 14-3-3 Isoforms 2.3 Renaturation of 14-3-3 proteins 2 .4 Recom binant 14-3-3 2.5 PKC Purification 2.6 PKC Assay 2.7 PKC Inhibitor Assay 2.8 Circular Dichroism 2.9 Isoelectric Focusing
2 .1 0 Analytical Size Exclusion Chrom atography 2.11 Phospholipid Binding
2.12 Antibody production and W estern Blotting 2.13 Phospholipase k z Assay
2 .1 4 Polymerase Chain Reaction
2.15 Insertion of Maize PKCI into pGEX-2T V ector 2 .1 6 Transform ation of f. coliTB ]
2.1 7 Analysis of Transform ed £ coli TB1 colonies 2 .1 8 Large Scale Plasmid Preparation
2.19 Dideoxy Sequencing of Maize PKCI Gene 2 .2 0 Maize PKCI Protein Purification 2.21 Sequencing of Maize PKCI Protein
2 .2 2 Electrospray Mass S pectrom etry of Maize PKCI 2 .2 3 Crystallization of Maize PKCI
MATERIALS
All re a g e n ts w ere analytical grade, from BDH, Sigma or Boehringer. Protein assay reag en t w as obtained from BioRad. FPLC and h.p.I.c. solvents and H2O were obtained from
Romil.
MARCKS peptide (KKKKRFSFKKSFKLSGFSFKK) and PKC Pi C-terminal p eptide (CRDKRDTSNFDKEFTRQPVELTPTDK) w ere sy nthesized in-house by Mr. P. F letcher (Sequencing and Synthesis Service, N.I.M.R.), using an Applied Biosystem s 4 3 0
A utom ated Peptide Synthesizer. PKC e-peptide (ERMRPRKRQGSVRRRV) w as provided by Dr. P. J. Parker.
DNA oligomers w ere synthesized in-house by Miss J. Mistry (Sequencing and Synthesis Service, N.I.M.R.), using a Beckman Oligo 1 000 DNA Synthesizer.
P h o sp h atid y lch o lin e ( 1 -O -h ex ad ecy l-2 -O -arach id o n y l-sn -g ly cero -
3-phosphocholine) and p-APMSF w ere obtained from Calbiochem. Phosphatidylserine (L- -phosphatidyl-L-serine, dioleoyl, sodium salt) and diacylglycerol
( 1 ,2-dioleoyl-sn-glycerol (C l 8:1, (c is)-9 )) w ere o b ta in ed from Sigma.
Molecular w eight rainbow m arkers range 1 4 ,3 0 0 - 2 0 0 ,0 0 0 Da w ere ob tained from Am ersham . Coomassie brilliant blue R-250 w as from Serva. Isogels pH 3 - 10 w ere obtained from Flowgen Instrum ents Ltd. Immobiline isoelectric focusing apparatus, Immobiline dry plates pH 4 - 7, pH m arkers range 2.5 - 6.5 and 3 .5 - 9.3 and Serva carrier am pholytes pH 3 - 10, w ere obtained from Pharmacia.
p a lm ito y l-2 -a ra c h id o n y l-(a ra c h id o n y l-1 -1 4 C ), (2 GBq/mmol, 1.9 MBq/ml) w ere obtained from New England Nuclear Research Products. 1-stearoyl 2-3H -arachidonyl phosphatidylcholine, 1-acyl 2-i4C-arachidonyl phosphatidylethanolam ine and
a35SdATP (9 .2 5 MBq/25 jil) were obtained from Amersham. Enlightening w as obtained from NEN Research Products. X-ograph blue X-ray film w as obtained from X-graph Ltd. P81 ion-exchange paper and 3MM paper w as obtained from V\fetman.
W estern blot antigens w ere visualized with th e Enhanced Chemiluminescence (ECL) detection system from Ammersham.
Human brain 14-3-3 was a gift from Prof. R. J. Thom pson, S outham pton University. Exol w as obtained from Prof. R. Burgoyne, Liverpool University; and bovine brain recom binant t 14-3-3 was d o nated by Drs Collier and Fu, Harvard Medical School, Boston.
PKC isoform s (bovine a , human Pi and P2, hum an/bovine chimaera of wild ty p e y and
m urine e) w ere ob tained from Dr. P. J. Parker, I.C.R.F.
pMZ2-12 (pUCI 3 plasmid containing th e Mz2-12 (m aize) g ene) w as a kind gift from Gordon Simpson, who cloned th e gene in th e course of screening a lambda g tl 1 cDNA expression library of Zea mays (maize) A 619 endosperm with a cDNA probe for th e hum an protein 70K (Theissen, e t al. 19 8 6 ). Polyclonal antisera raised ag ainst bovine PKCI w as kindly provided by M. P. Walsh.
Molecular biology kits w ere as follows: Ultra pure dNTP s e t (Pharm acia); 1 kb DNA ladder, 2 5 0 n g /2 4 7 .5 \i\ (B ethesda Research Laboratory); GENECLEAN II Kit (BIO 101 Inc.); T 4 DNA Ligase Kit (N orthum bria Biologicals); plasmid SELECT-100 purification
kit (5 Prime - 3 Prime, Inc.); S equenase Version 2 .0 Kit (United S ta te s Biochemical Corp.). DNA manipulations w ere perform ed in f. coli (JM83 hsdR (rk-m|<+); New England Biolabs).
The following solutions w ere prepared in-house by th e media departm en t: L-broth (1 g/m l Bacto try p to n e, 0 .5 g/m l Y east ex tract, 0 .1 7 M NaCI, pH 7); L-agar (1 g /m l Bacto try p to n e, 0 .5 g/m l Y east ex tract, 0 .1 7 M NaCI, 1.5 g /m l Difco agar, pH 7); PBS (0 .1 7 M NaCI, 10.2 mM NazHP04, 4 .9 mM KCI, 2.7 mM KH2PO4, pH 7).
For d atab ase searches and sequence manipulations th e GCG package w as used (Genetic C om puter Group, 199 1).
METHODS
2.1 P u r if ic a tio n o f 1 4 - 3 - 3
14-3-3 w as isolated from sh eep brain by a com bination of anion-exchange and hydrophobic interaction chrom atography, according to th e m ethod of Toker e t al.
( 1 9 9 0 ). SDS-PAGE w as ad ap ted from Laemmli (1 9 7 0 ). SOS polyacrylamide slab gels w ere electrophoresed under reducing or non-reducing conditions. Slab gels (2 0 X 20 cm, 3 mm thickness) were used for analytical work, whilst mini-gels w ere used to check protein purity a t each purification step. All SDS-PAGE w as 12.5% w /v
acrylamide resolving gel overlaid with 5% w /v acrylamide stacking gel, unless w here otherw ise stated .
The concentration of th e purified proteins was estim ated by amino acid analysis (on a Beckman 121MB or a Beckman 6 3 0 0 Analyzer with p ost column ninhydrin d etectio n ), or by th e BioRad protein concentration assay (according to m anufacturers instructions).
14 -3-3 w as purified from hen brain as detailed in Patel e t. al., 1994.
2 .2 R e v e rs e P h a s e S e p a r a tio n o f 1 4 - 3 - 3 Is o fo rm s
Pure 14-3-3 was sep arated into individual isom ers on reverse-p hase h.p.I.c., using A quapore RP300 (C8, 4 .6 X 100 mm; Toker e t al., 1 992), and Bakerbond WP-C4 (4 .6
X 2 5 0 mm and 10 X 25 0 mm) columns. The separations em ployed shallow
H zO /acetonitrile gradients containing trifluoroacetic acid (TEA). The isoforms eluted b etw een approxim ately 49 - 60% v /v acetonitrile.
2 .3 R e n a tu r a tio n o f 1 4 - 3 - 3 P r o te in s
1 4 -3 -3 isoform s elu ted in TFA /acetonitrile (pH ca. 2) from rev erse-ph ase h.p.I.c. w ere n ot resto red to th eir native s ta te , when simply neutralized with 50 mM Tris-CI pH
7.5. This also resulted in very poor recovery of protein, jud ged by tw o criteria, circular dichroism and dimeric stru c tu re (se e below). The following m etho d w as developed to ren ature 14-3-3 isoforms to a s ta te resembling th e native protein.
Fractions of purified y, ôand e isoform s (as identified by specific antibodies, Martin e t al., 1 9 9 3 ) w ere diluted with 5 volum es of Milli-Q distilled H2O and concentrated a t
4 0c to 4 0 0 - 5 0 0 \i\ in Centricon tu b e s (m ass c u t off 1 0 ,0 0 0 Da) using a Beckman GPR centrifuge a t 4 0 0 0 X g. For larger scale renaturation of isoforms an Amicon
ultrafiltration stir cell (1 0 ml) w as used. The co n ce n trate d fractions w ere diluted again to 2 ml with 20 mM Tris-CI pH 7.5 (0 .2 M NaCI), reco n c en trated to 2 0 0 - 3 0 0 \i\ and assayed for PKC inhibition. The renaturation of th e individual isoforms w as verified by circular dichroism analysis (to check th e proteins had refolded to give an a -h e lic a l c o n te n t similar to native protein) and by size exclusion chrom atography on a Superose
12 column ( to confirm th a t th e protein had regained its dimeric stru ctu re ).
2 .4 R e c o m b in a n t 1 4 - 3 - 3
Recom binant proteins w ere expressed in £ coli (Jo n es e t al., 1 9 9 4 ). The epsilon isoform w as expressed as a m altose-binding-protein fusion protein, from th e v ecto r pMALC2 (New England Biolabs). The ra t brain epsilon c-DNA was a kind gift from Dr. P. Roseboom, N.I.H. The carrier protein w as cleaved with Factor Xa, to leave a glycine residue a t th e N-terminus of th e protein.
The T-cell isoform w as expressed as an in tact protein from th e v e c to r pKK233-2 (Pharm acia). The T-cell 14-3-3 c-DNA (hum an) w as a kind gift of Dr. P. J. Nielsen. Purification stra te g ie s were as detailed for th e sh eep brain isoforms.
2 .5 PKC P u r if ic a tio n
Purification of mixed brain PKC w as from fresh sh eep brain, carried o u t by a
modification of Ellis e t al. (1 9 8 7 ). Immediately a fte r slaughter (by m ethod o th e r th an captive bolt or anaesthesia) one whole sh eep brain w as homogenized in 1 L of ice cold Buffer A (0 .2 5 M sucrose, 20 mM Tris-CI, pH 7.5, 2 mM EDTA, 10 mM EGTA, 1 mM DTT, 1 .5 6 6 m g/m l benzamidine, 75 ng/m l PMSF, 10 ng/m l soyabean trypsin
inhibitor, 1 [xg/ml leupeptin, 1 jxg/ml pep statin ) using a Waring commercial blender. The hom ogenate w as centrifuged in a Beckman model J2-21 a t 2 ,3 0 0 X g for 30 min. The soluble fraction w as loaded onto a DEAE-cellulose (W btman) column (1 2 .5 cm2 cro ss sectional area X 15 cm height) equilibrated with buffer B (2 0 mM Tris-CI, pH 7.5, 1 mM EDTA, 1 mM EGTA, 1 mM DTT). PKC w as eluted using a linear NaCI gradient (0 t o 0 .5 M NaCI in 3 8 6 min a t 3.5 ml/min); 15 ml fractions w ere collected.
Fractions b etw een 0.1 and 0 .2 M NaCI w ere assayed for PKC activity and active fractions w ere pooled and sto red a t -70°C, in 40% glycerol (added drop-wise).
I
Recom binant PKC isoform s yyere obtained from P. J. Parker, eith er as purified protein (P i, P2 and Y isoforms); or as frozen, induced cells (a , ô a n d e isoform s) which w ere
purified by a modification of th e m ethods of Oliver and Parker (1 9 9 1 ) and Schaap and Parker ( 1 9 9 0 b ) as follows:
Frozen cells w ere briefly vortexed in th e presen ce of 10 ml extraction buffer (2 0 mM Tris-CI, pH 7.5, 5 mM EDTA, 5 mM DTT, 20 mM NaF, 1% Triton-X-10 0 , 2 5 0 \ig /m \
leupeptin, 0.1 mM PMSF, 10 mM benzamidine), followed by an incubation on ice for 10 min. The Triton-X -1 0 0 insoluble material w as rem oved by centrifugation on a
Beckman L8-M ultracentrifuge a t 1 5 ,0 0 0 X g for 2 0 min a t 4^0. The su p ern atan t was
loaded o n to a Mono Q 5 /5 anion exchange column and protein was eluted with buffer containing (2 0 mM Tris-CI, pH 7.5, 2 mM EDTA, 5 mM DTT, 10 mM NaF, 0.0 2%
T riton-X -10 0 , 5 0 [xg/ml PMSF, 10 mM benzam idine) with a linear (0 to 0 .5 M) NaCI gradient over 50 min. Fractions (1 ml) containing PKC activity w ere pooled and sto red a t - 2 0°C in th e presence of 1 mM DTT and 50% glycerol.
2 .6 PKC A ssay
The PKC assay w as based on Parker e t al. (1 9 8 4 ), ex cep t for th e following :
Total assay volume w as 55 pi, consisting of 12.5 pM (y- 3zp) ATP (2 7 kBq/nm ol ATP), CaClz (0.1 mM), MgClz (1 3 .5 mM), phosphatidylserine ( 0 .0 4 m g/m l), MARCKS peptide (3 6 pM). This synthetic peptide is based on th e phosphorylatable region of th e MARCKS protein - a specific su b stra te for PKC. The sequence of this peptide and site s of phosphorylation are detailed in Am ess e t al. (1 9 9 2 ). The reaction w as initiated by th e addition of ATP and afte r 10 min a t 300C th e reaction was stopped by spotting 4 0 pi of th e assay volume on to phosphocellulose paper (P81 ). Unbound 32R-ATP w as rem oved by thorough washing in 75 mM phosphoric acid. The am ount of 3zp incorporated into MARCKS peptide w as m easured by scintillation counting. PKC activity is defined as 1 nmol of phosphate incorporated into MARCKS peptide in 1 min a t 30°C per ml of PKC, in th e presence of saturating concentrations of phosphatidylserine. The assay was carried o u t in th e presence and absence of phosphatidylserine, and expressed as
phosphatidylserine dep enden t kinase activity, in order to co rrect for PKM (th e proteolysed, cofactor independent form of PKC).
2 .7 PKC In h ib ito r A ss a y
The PKC assay (described above) was modified to analyse th e inhibitor proteins as follows:
To a standard concentration of PKC (with known activity), th e required concentration of inhibitor w as added (to ta l assay volume 55 pi) and inhibitory activity w as expressed as
a p ercen tag e of phosphatidylserine-dependent kinase activity.
2 .7 .1 D is p e rs e d P h o s p h o lip id V e s ic le s T hese w ere prepared as follows:-
5 A/I phosphatidylserine (2 0 m g/m l in chloroform :m ethanol,1:2) w as dried under
nitrogen; 22 7 A/I of 20 mM Tris-CI pH 7.5 w as added, vortexed, and son icated on ice a t 2 ix am plitude, for 3 X 30 sec bursts, with 30 sec re s t intervals, using a MSE Soniprep
150 (fitte d with a 3 mm probe tip). 5 \i\ of th e se dispersed vesicles was added to each assay tube.
2 .7 .2 M ixed M icelle V e s ic le s
This m etho ds w as modified from Hannun e t al., 1985. Mixed micelle vesicles w ere prepared by drying to g e th er, under a stream of nitrogen:-
8 A/I diacylglycerol (1 0 m g/m l in chloroform im ethanol, 1:2) and 15 a/I
phosphatidylserine (2 0 m g/m l in chloroform im ethanol, 1:2). 100 a/I of 0.3% v /v Triton-X-10 0 in 20 mM HEPES pH 7, w as added to th e dried lipid. This solution w as vortexed, followed by a 5 min incubation a t 300C. 5 \i\ of th e s e mixed micelle vesicles was added to each assay tube.
2 .7 .3 P h o rb o l E s te r /D ia c v lq lv c e r o l A s s a y
The required concentration of diacylglycerol (final assay concen tratio n of 6 A/M - 6 0 0 A/M) or phorbol e s te r (PMA; final assay co n centration of 10 nM - 1 0 0 0 nM) w as dried down with 5 a/I phosphatidylserine (2 0 m g/m l) under nitrogen, and prepared as described for phosphatidylserine vesicles. The assay was th e n carried ou t as above.
2 .8 C ir c u la r D ic h ro is m
Martin (D ep artm en t of Physical Biochemistry, N.I.M.R.). Far-UV circular dichroism sp e c tra w ere recorded from 2 6 0 to 185 nm using a Jasco J-6 0 0 sp ectropolarim eter operated with an instrum ent tim e co n stan t of 0.5 sec. The sp ectra were recorded a t 22 °C for protein solutions (2 0 mM Tris, 100 mM NaCI, pH 7 .5 ) a t co n ce n tratio n s in th e range 0.1 to 1.0 m g/m l in 0.1, 0.2, 1 or 2 mm fused silica c u v ettes. The sp ectra shown represent th e average of a t least four scans. All data are reported in te rm s of molar circular dichroism absorption coefficient, Ae, based upon an assum ed mean residue w eight of 111. The residue molar elipticity, [ 0 ] m r w . may be obtained from th e relationship [ 0 ] m r w = Ae x 3 3 0 0 .
The analysis of secondary stru c tu re co n te n t from far-UV CD sp ectra w as perform ed using th e m ethod of Hennessey and Johnson (1 9 8 1 ).
The dissociation c o n sta n t for maize PKCI w as calculated using a non-linear least- squares analysis which assum ed formation of a 1 :1 complex.
2 .9 I s o e le c tr ic F o c u s in g
Isoelectric focusing w as carried o u t using a Multiphor II Isoelectric Focusing Unit (P h a rm a c ia ).
2 .9 .1 A g a ro s e Is o e le c tr ic F o cu sin g
Isogels (Flowgen Instrum ents Ltd.) are a kit form of prepoured isoelectric focusing gels. 2 to 5 [xl of 2 - 10 m g/m l sam ple was loaded per track. Gels w ere pre-focused a t 1 W for 10 min and focused a t 1 0 0 0 V 25 W for 4 0 min. Coomassie Blue Staining w as as detailed in m anufacturer instructions.
2 .9 .2 Im m o b ilin e I s o e le c tr ic F o c u s in g
Immobiline gels (Pharm acia) have ad v an tag es over m ethods, which use carrier am pholytes, as th e y have charge groups fixed to th e polyacrylamide matrix, th ereb y allowing form ation of narrow er pH gradients. Immobiline dry plates, pH 4 to 7, w ere rehy drated in th e immobiline rehydration c a s s e tte , as detailed in m anufacturer instructions. Isoelectric focusing conditions w ere optim ised to cathodic loading of 1.6 nmol 14-3-3 in th e p resen ce of Servalyte carrier am pholytes pH range 3 to 10 (20% v /v , added to sample); electrofocusing w as carried o u t a t 1 mA, 5 W, 5 0 0 V for 4 h followed by 2 5 0 0 V for 14 h. Coomassie Blue Staining w as as detailed in m anufacturer in stru ctio n s.
2 .9 .3 T i tr a ti o n C u rv e
The pH optimum for dissociation of th e dimeric 14-3-3 isoforms w as determ ined on an Isogel. Before loading th e sample, th e carrier am pholytes w ere pre-focused (1 W for 10 min). This established a pH gradient which increases left to right as indicated by th e arrow (figure 4 .2 .1 .4 ). The pre-focused gel w as ro ta te d 9 0 ° and th e sample of native
14-3-3 isoforms w as loaded in a strip equidistant betw een anode and cathode. The gel w as again placed in th e electrophoresis app aratu s and re-focused a t 1 W for 10 min followed by 1 000 V 25 W for 4 0 min. This analysis can also indicate chan ges in oligomeric s ta te of a protein due to exposure of charged groups previously masked.
2 .1 0 A n a ly tic a l S iz e E xclusio n C h r o m a to g r a p h y
Pure 14-3-3 sam ples (1 .9 nmol) w ere applied o n to a Superose 12 column
(Pharm acia), equilibrated to pH 3 (1 0 0 mM NaCI, 50 mM citric acid), pH 4 (1 0 0 mM NaCI, 50 mM formic acid), pH 4 .5 (1 0 0 mM NaCI, 50 mM sodium a c e ta te ), pH 5 (1 0 0 mM NaCI, 50 mM acetic acid), pH 5.5 (1 0 0 mM NaCI, 20 mM ammonium a c e ta te ), pH 7 .5 (1 0 0 mM NaCI, 2 0 mM Tris-CI) respectively. The column w as calibrated a t pH 7.5,
in b etw een each chrom atogram , by eluting molecular m ass stan d ard s as follows: BSA (6 9 kDa), carbonic anhydrase (2 9 kDa); Flow ra te w as 1 ml/min.
2 .1 1 P h o s p h o lip id B in din g
Phospholipid binding assay w as carried o u t by a m ethod adapted from Boustead, 1988. 1.25 mmol phosphatidylserine, phosphatidylcholine, or phosphatidylserine and phosphatidylcholine, w as dried under nitrogen into an eppendorf tu b e , on ice. 1 ml buffer A (2 mM CaClz, 1 0 0 mM KCI, 1 mM EGTA, 2 mM MgCl2, 20 mM HEPES, pH 7 .4 )
was added to th e dried lipid. This solution was sonicated on ice a t 2 ^ am plitude for 3 X 30 sec bursts, with 30 sec re s t intervals. 150 \i\ phospholipid vesicle (1 8 7 .5 nmol) and 10 Jig protein (1 4 -3 -3 or Calcium -binding-protein p 3 3 /p 3 7 ) w ere m ade up to 5 0 0 jil with buffer A. This mixture w as incubated a t room te m p eratu re for 15 min, followed by centrifugation a t 12 ,0 0 0 X g for 10 min. The su p ern ata n t (81 ) w as rem oved