747Biochem. J. (1984) 224, 747-753Printed in Great Britain

Multiple phosphorylation of pp3O, a rat brain polyribosomal protein, sensitiveto polyamines and corticotropin

Louise H. SCHRAMA,* Geert WEEDA, Philippa M. EDWARDS, A. Beate OESTREICHERand Peter SCHOTMAN

Division of Molecular Neurobiology, Institute of Molecular Biology, Rudolf Magnus Institute forPharmacology and Laboratory ofPhysiological Chemistry, State University of Utrecht, Padualaan 8,

3584 CH Utrecht, The Netherlands

(Received 11 June 1984/Accepted 28 August 1984)

A rat brain polyribosomal protein with an apparent M, of 30000, designated pp3O,was further characterized. The protein was identified by its phosphorylation by an

endogenous protein kinase sensitive to both corticotropin and spermine. Two-dimensional separation of a polyribosomal fraction was applied, combining non-

equilibrium pH-gradient-gel electrophoresis in the first and sodium dodecylsulphate/polyacrylamide-gel electrophoresis in the second dimension. In this system,pp3O was separated into at least five defined phosphoprotein spots. Pulse-labellingwith [y-32P]ATP followed by a chase for various time periods with excess unlabelledATP resulted in a shift of the distribution of radioactivity and protein staining alongthe spots towards the anode. This suggests that the various spots of pp3O may

represent multiple phosphorylation states. Limited proteolysis of the five spots withthree different proteinases resulted in the same one-dimensional peptide maps with a

given proteinase, indicating that all five spots represent different forms of a singlephosphoprotein. Inhibition of the overall phosphorylation of pp3O by corticotropin or

spermine was accompanied by a shift in the recovery of labelled phosphate towardsspots nearer the cathode. Immunoblotting with monoclonal antibodies directedagainst ribosomal protein S6 stained only one band, a protein that had an apparent Mrof 34000 and was clearly distinct from pp3O.

Incubation of a free polyribosomal fraction fromrat cerebrum with [y-32P]ATP revealed thephosphorylation of eight phosphoproteins in theMr range 20000-80000 (Schrama et al., 1984b).The protein kinases phosphorylating these pro-teins were not sensitive to either cyclic nucleotidesor Ca2+. The phosphorylation of one of theseproteins, of M, 30000 (pp3O), was selectivelyaffected by the polyamine spermine and by ACTH(Schrama et al., 1984b,c). The ACTH sequencesinhibiting pp30 phosphorylation all containedbasic residues (Schrama et al., 1984c). A possiblerelationship between the modulation of pp3Ophosphorylation and of protein synthesis has beensuggested because of the similarities in sensitivities

Abbreviations used: SDS, sodium dodecyl sulphate;ACTH, adrenocorticotropic hormone (corticotropin);eIF-2, eukaryotic initiation factor 2.

* To whom correspondence and requests for reprintsshould be addressed.

to Mg2+ and spermine and in structure-activityrelationships for ACTH analogues (Schrama et al.,1984a,c).The Mr values ofribosomal proteins S6 and pp3O

are in the same range. S6 has been proposed to playa role in the translation ofmRNA (for reviews seeLeader, 1980; Traugh, 1981).The present paper describes the characteriza-

tion of pp3O as a phosphoprotein with multiplephosphorylation sites and distinct from ribosomalprotein S6.

Materials and methodsChemicals

All chemicals used were of analytical grade.Ribonuclease-free sucrose and papain were ob-tained from Serva (Heidelberg, Germany), sper-mine was from Sigma Chemical Co. (St. Louis,MO, U.S.A.), low-Mr protein calibration kit was

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L. H. Schrama and others

from Pharmacia (Uppsala, Sweden), Ampholineswere from LKB Produkter (Bromma, Sweden),Staphylococcus aureus proteinase V8 was fromMiles Laboratories (Stoke Poges, Slough, Bucks.,U.K.), chymotrypsin A4 was from Boehringer(Mannheim, Germany), dioctyl sodium sulpho-succinate and 3,3',5,5'-tetramethylbenzidine werefrom Merck (Darmstadt, Germany), ACTH-(15-24)-peptide was from Organon International (Oss,The Netherlands), and [y-32P]ATP (2900Ci/mmol)was from New England Nuclear (Boston, MA,U.S.A.). Monoclonal anti-(ribosomal protein S6)antibodies, which react with rat brain S6 (Nielsenet al., 1982), were generously given by Dr. H.Towbin (Ciba-Geigy, Basel, Switzerland), rabbitanti-mouse immunoglobulins coupled with horse-radish peroxidase were from Nordic (Tilburg, TheNetherlands), and pig serum was from the localslaughterhouse.

Preparation of a polyribosomal fractionAll solutions and equipment were sterilized

before use to minimize ribonuclease contamina-tion. Polyribosomes were isolated from a postmito-chondrial supernatant fraction (S20) (Schrama etal., 1984b) from rat cerebrum in a Beckman SW27.1 rotor. Then 11 ml of S20 in 50mM-Tris/aceticacid (pH7.4)/25mM-potassium acetate/2mM-mag-nesium acetate/2mM-dithiothreitol was layeredover 2ml of 1.5M-sucrose and 4ml of 2M-sucrose.Both sucrose solutions were made in 50mM-Tris/acetic acid (pH 7.4)/70mM-potassium acetate/5mM-magnesium acetate/2mM-dithiothreitol. Thediscontinuous gradient was centrifuged in a SorvallOTD-2 centrifuge at 85000g for 20h at 4°C. Thesupernatant and sucrose gradient were discardedand the tube wall was cleaned with a cotton-wooltip. The pellet was rinsed with 50mM-Tris/aceticacid (pH7.4)/25mM-potassium acetate/2mM-mag-nesium acetate/2mM-dithiothreitol and carefullyresuspended with a glass rod in the same buffercontaining 250mM-sucrose, frozen in small por-tions in liquid N2 and stored at -80°C.

Non-equilibrium pH-gradient-gel electrophoresisThis was performed by the method of O'Farrell

et al. (1977). The composition of the gel wasessentially the same as for an isoelectric-focusingslab gel (Zwiers et al., 1980), but it contained adifferent Ampholine mixture: 0.8% (w/v) pH6-8,0.8% (w/v) 8-9.5 and 0.2% (w/v) pH 9-1 1. Poly-merization and sample preparation were as de-scribed by Zwiers et al. (1980) The upper (anode)reservoir was filled with 1 mM-H2SO4 and thelower (cathode) reservoir was filled with 0.1M-NaOH. The gel was run for 16h at 200V, followedby I h at 400V.

SDS/polyacrylamide-gel electrophoresisPhosphoproteins were separated by electrophor-

esis on SDS/12%-polyacrylamide gels; furtherprocessing and autoradiography were performedas described by Van Dijk et al. (1981). Non-equilibrium pH-gradient-gel electrophoresis fol-lowed by SDS/polyacrylamide-gel electrophoresisin the second dimension were performed withsimilar gels except that a comb without slots wasused. Further processing was performed as de-scribed by Zwiers et al. (1980).

Phosphorylation ofpolyribosomesIncubations were carried out at 30°C in polypro-

pylene tubes in a total volume of 25 p1. Phosphoryl-ation was performed in 50mm-Tris/acetic acid(pH 7.4)/100mM-potassium acetate/I mM-magne-sium acetate/2mM-dithiothreitol with variousMgATP and [y-32P]ATP concentrations. Thephosphorylation reaction was terminated by addi-tion of a denaturing solution to give final concen-trations of 62.5mM-Tris/acetic acid, pH6.8, 2%(w/v) SDS, 10% (v/v) glycerol, 0.001% (w/v)Bromophenol Blue and 5% (v/v) 2-mercapto-ethanol. These samples were applied directly togels for SDS/polyacrylamide-gel electrophoresis.For application to gels for non-equilibrium pH-gradient-gel electrophoresis the reaction wasterminated by the addition of EDTA (5mM finalconcn.) and immersion in liquid N2

Silver staining ofproteinsThis was performed by a combination of the

methods described by Oackley et al. (1980) andPoehling & Neuhoff (1981). All solutions weremade in double-distilled water. Gels were fixedwith 50% (v/v) methanol/7% (v/v) acetic acid for10min, then transferred to 10% (v/v) methanol/5%(v/v) acetic acid and incubated for 1-24h, andfinally rinsed for 3 x 10min in 15% (v/v) methanolto remove the acetic acid. After 30min incubationin 5% (v/v) glutaraldehyde, gels were rinsed for20min in 15% (v/v) methanol and then for 10minin water; this methanol/water procedure wasrepeated three times. Silver staining was per-formed for 20min in 1.4% (v/v) NH3/0.04M-NaOH/0.3% (w/v) AgNO3. The gel was rinsedthree times in water. Gels were developed in0.00075% (w/v) citric acid/0.015% (w/v) formalde-hyde. The reaction was stopped in 10% (v/v)methanol/5% (v/v) acetic acid.

Peptide mapping ofpp3OAfter two-dimensional separation of the poly-

ribosomal proteins, the gel was subjected to auto-radiography at -20°C. The phosphoprotein spotsof pp3O were excised on the basis of the autoradio-

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Multiple phosphorylation of polyribosomal protein pp3O

gram, and the accuracy of excision was verified bya second autoradiogram of the gel.The excised protein spots were subjected to

limited proteolysis essentially as described byCleveland et al. (1977), but with the followingmodifications. The gel sections were soaked in125 mM-Tris/HCl buffer, pH6.8, for 30min and puton the bottom of a slot of a 5%-polyacrylamidestacking and 15%-polyacrylamide running gel(2mm thick). Spaces around the gel sections werefilled with lOyl of 20% (v/v) glycerol. Finally, thegel sections were overlayered with 25lul of buffercontaining 5 mM-Tris/HCl, pH 6.8, 3% (w/v) SDS,10% (v/v) glycerol, 0.003% (w/v) BromophenolBlue and a given amount of proteinase. The gel was

+

run at 0mA for 2h and subsequently at 35-40mAuntil the dye front had reached the bottom of thegel.

Immunochemical staining of ribosomal protein S6Electrophoretic transfer of protein from gels to

nitrocellulose filters (0.45 um pore size) was per-formed as described by Nielsen et al. (1982). Onehalf of a symmetrical blot was stained for proteinwith 0.1% (w/v) Amido Black in 45% (v/v)methanol/10% (v/v) acetic acid and destained in90% (v/v) methanol/2% (v/v) acetic acid; the otherhalf of the blot was stained by using immuno-chemical techniques for protein S6 (see below).The incubation with the primary anti-(protein

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Polyribosomes (140pg of protein) were phosphorylated with 100upM-ATP for 15s (a and b) or 120s (c and d). Theautoradiograms after separation on non-equilibrium pH-gradient-gel electrophoresis (NEPHGE)-SDS/polyacryl-amide-gel elecirophoresis (SDS/PAGE) after 15 and 120s are shown in (a) and (c) respectively. Correspondingprotein patterns after silver staining are shown in (b) (15s) and (d) (120s). Identification of the positions of thelabelled spots 1-5 with corresponding spots in the protein-staining pattern, as indicated by the numbered arrows,was checked by developing a second autoradiogram of the gel after selective excision of the protein spots shown in(b) and (d).

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S6) antibody and the secondary antibody (rabbitanti-mouse immunoglobulins) coupled with horse-radish peroxidase was performed as described byNielsen et al. (1982). The peroxidase was detectedby the staining method of Buckel & Zehelein(1981) as follows: 30ml of phosphate/citrate buffer(7.6mM-citric acid/22.5mm-NaH2PO4 adjustedwith HCI to pH5.0) was mixed with lOml ofethanol containing 24mg of tetramethylbenzidineand 80mg of dioctyl sodium sulphosuccinate;H202 (final concn. 0.01%) was added to themixture immediately before use. The reaction wasstopped by transferring the blot to water.

ResultsTwo-dimensional separation

Polyribosomes were phosphorylated for 15 or120s under conditions described in the Materialsand methods section and in the Figure legends.Two-dimensional separation by conventional iso-electric focusing-SDS/polyacrylamide-gel electro-phoresis did not give a clear separation of pp3O(results not shown). The protein pp3O appeared atMr 30000 as a heterogeneous smear from pH 8.5 topH5.2. The same result was obtained when theribosomal proteins were first extracted by themethod of Roberts & Ashby (1978).

After separation by non-equilibrium pH-gradient-gel electrophoresis-SDS/polyacrylamide-gel electrophoresis, with a pH gradient from 6.0 to9.0, pp3O appeared as several clearly separatedspots on the autoradiograms (Figs. la and lc). Fivespots coincided with the protein-staining pattern(Fig. ld) and were numbered 1-5 from cathode toanode. The two most anodic spots (4 and 5) wereonly visible on the protein-staining pattern afterprolonged incubation with ATP (cf. Figs. lb andld).

Effects ofACTH and spermineIn the presence of ACTH-(15-24)-peptide or

spermine, total phosphorylation of pp3O wasdecreased (Fig. 2, inset). Moreover, a shift in thedistribution of label between the spots wasobserved, as quantified by densitometric scanningof the autoradiogram (Fig. 2). Inhibition ofphosphorylation resulted in a decreased amount oflabel in spots 4 and 5, accompanied by an*increased amount in spot 1. The phosphorylationof spots 2 and 3 was decreased by spermine, but ap-parently not affected by ACTH-(15-24)-peptide.

Pulse-chase-labelling ofpp3OAfter incubation of polyribosomes with [y-32p]-

ATP for 15 s, 1 mM-ATP (including 1 mM-Mg2+)was added and incubation was continued for afurther 0, 30, 60 or 120s. The various phospho-

1 2 -~~~~~~~~~~~~

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Fig. 2. Sensitivity ofpp3O phosphorylation to ACTH-(J5-24)-peptide and spermine

Polyribosomes (100 jug of protein) were phosphoryl-ated with 7.5 /iM-ATP for 75s in the presence of100 uM-ACTH-(15-24)-peptide, 1 mM-spermine orbuffer. The autoradiogram obtained after two-dimensional separation of the proteins was scannedby densitometry to quantify incorporation oflabelled phosphate into each of the five spots ofpp3O. The inset shows the total incorporation intopp3O after a control incubation and after incubationwith ACTH-(15-24)-peptide or spermine.

protein spots were excised and quantified byliquid-scintillation counting of radioactivity (Fig.3). The total amount of incorporated 32P remainedequal during the chase period up to 120s (Fig. 3,inset), indicating negligible phosphatase activityand the adequacy of the chase procedure (see alsoSchrama et al., 1984b). However, increasing theincubation time with non-radioactive ATP shiftedthe label from the more cathodic spots 1 and 3towards the anodic spots 4 and 5. The amount oflabel decreased gradually with time in spots 1 and2, whereas a continuous increased was observed inspot 5. A similar shift from the cathode to theanode was also observed in the protein-stainingpattern (results not shown).

Peptide mapping ofpp3OThe five spots of pp3O were subjected to limited

proteolysis with Staphylococcus aureus proteinase,papain or chymotrypsin. Incubation of the radio-actively labelled spots with a given proteinaseresulted in similar breakdown products for all thephosphoprotein spots. The results of proteolysis ofspots 1 and 5 are shown in Fig. 4. Staphylococcusaureus proteinase digested all the phosphorylated

1984

750

Multiple phosphorylation of polyribosomal protein pp3O

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Fig. 3. Pulse-chase-labelling ofpp3OPolyribosomes (140 pg of protein) were labelled for15s with [y-32P]ATP, followed by the addition ofmM-ATP, and incubation was continued for a

further 0, 30, 60 or 120s. After non-equilibrium pH-gradient-gel electrophorebis-SDS/polyacrylamide-gel electrophoresis the five pp3O spots were excisedand the incorporation of labelled phosphate was

quantified by liquid-scintillation counting of radio-activity. The inset shows the total amount of 32pincorporated in pp3O after the various incubationtimes.

Fig. 4. Peptide mapping ofphosphorylated pp30Phosphorylation and separation of pp3O wereperformed as described in Fig. 1 legend for 120s.The five spots of pp3O were excised and subjected tolimited proteolysis on a 5%-polyacrylamide stackingand 15%-polyacrylamide running gel. Peptide mapsare shown of spots 1 and 5 after digestion withStaphylococcus aureus proteinase (1.6pg/slot, a),with papain (0.8pg/slot, b) and with chymotrypsin(1.6 pg/slot, c). The position of the original pp3O isindicated by the arrow. The M, values on the right ofthe Figure indicate the positions of the M, markers.

pp3O, of which the original position is indicated byan arrow (Fig. 4a). The protein pp3O was digestedto give three major peptides (25.4, 23.2 and21.5 kDa) and two minor peptides (17.5 and16.2kDa), which are not visible on the photo-graphic reproduction of the autoradiogram.Papain digested most of pp3O into two major (20.0and 16.5 kDa) and two minor peptides (28.7 and23.6kDa) (Fig. 4b). Exposure to chymotrypsin leftmost of the pp3O unhydrolysed (Fig. 4c), but somewas digested, giving three fragments of 28.2, 25.7and 21.OkDa.

Immunochemical detection of ribosomal protein S6Detection of ribosomal protein S6 was per-

formed with a monoclonal antibody. Phosphoryl-ated polyribosomes were separated on an SDS/polyacrylamide-gel-electrophoresis gel. A nitro-cellulose. blot -from this gel was used for bothprotein staining with Amido Black (Fig. 5a) andimmunochemical detection of S6 (Fig. Sd).- Themonoclonal antibody specifically detected S6, asshown by the staining of a single band in Fig. 5(d).

Autoradiography of the immunostained blot (Fig.5c) and of the protein-stained blot (Fig. Sb)revealed pp3O just above the M, marker carbonicanhydrase (Mr 30000), whereas S6 had an Mr ofapprox. 34000.Immunochemical detection of S6 was also

performed on a blot from a non-equilibrium-pH-gradient-gel-electrophoresis-SDS/polyacrylamide-gel-electrophoresis gel of phosphorylated poly-ribosomes (cf. Fig. 1). The autoradiogram of theblot revealed the spots 1-5 of pp3O, whereas nopositive staining of S6 was observed.

Discussion

This paper describes the characterization of a30000Mr phosphoprotein (pp3O) from free poly-ribosomes of rat brain, sensitive in its phosphoryl-ation to spermine and ACTH-(15-24)-peptide, butnot to cyclic nucleotides or Ca2+/calmodulin(Schrama et al., 1984b,c). Initial attempts toidentify this phophoprotein further according to itsisoelectric point on isoelectric focusing-SDS/poly-

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751

L. H. Schrama and others

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Fig. 5. Immunochemical detection of S6Polyribosomal phosphorylation was performed as described in Fig. 2. After SDS/polyacrylamide-gel electro-phoresis, the protein was blotted on nitrocellulose. The protein-staining pattern and corresponding autoradiogramare shown in lanes (a) and (b) respectively. The immunochemical staining of S6 indicated by an arrow and corre-sponding autoradiogram are shown in lanes (d) and (c) respectively. The Mr value on the left of the Figure indicatesthe position of carbonic anhydrase.

acrylamide-gel electrophoresis were unsuccessful,owing to the low resolution power of the basic partof the isoelectric-focusing gel (O'Farrell, 1975).Neither removal of nucleic acids nor addition ofSDS improved the separation.

Resolution of the phosphorylated polyribosomalfraction on non-equilibrium pH-gradient-gelelectrophoresis-SDS/polyacrylamide-gel electro-phoresis resulted in a clear separation of pp3O intofive discrete spots (Figs. la and lc). The same spotswere also observed by silver stain after prolongedphosphorylation (Fig. ld). The observed hetero-geneity is not due to the presence of nucleic acids,as described by O'Farrell (1975), since removal ofRNA before non-equilibrium pH-gradient-gelelectrophoresis gave the same separation pattern(results not shown).

Heterogeneity of phosphoproteins in a charge-dependent separation system has been describedby others. Phosphorylation of a cyclic AMP-binding protein (Rangel-Aldao et al., 1979) or eIF-2 (Farrell et al., 1978) followed by separationon isoelectric focusing-SDS/polyacrylamide-gelelectrophoresis resulted in a shift of the pl. Adifferent charge-dependent system, using urea/polyacrylamide-gel electrophoresis, run at pH 8.6in the first and pH 4.05 in the second dimension

(Lastick & McConkey, 1976), is used for theseparation of basic ribosomal proteins. In thissystem S6, the major phosphorylated protein in vivoin the 40S ribosomal subunit, appears hetero-geneous (see, e.g., Leader & Coia, 1978; Roberts &Morelos, 1979; Nielsen et al., 1982; Nishimura &Deuel, 1983). In rabbit reticulocytes, phosphoryl-ated in vivo, up to five phosphoprotein spots of S6can be detected, depending on the pH duringincubation with [32p]p; (Perisic & Traugh, 1983).The heterogeneity seen after two-dimensional

separation of S6 is caused by multiple-site phos-phorylation of this protein. Multiple-site phos-phorylation has also been described for the a-sub-unit of eIF-2 (Tuazon et al., 1980) and for thebrain-specific ACTH-sensitive membrane proteinB-50 (H. Zwiers, J. Verhaagen, C. J. van Dongen,P. N. E. de Graan & W. H. Gispen, unpublishedwork).

In both separation systems described above,phosphorylation of proteins produced a shift of thephosphoprotein towards the anode, owing tonegative charging of the protein with phosphate.The heterogeneity of pp3O described in this

paper is, at least partly, caused by the degree ofphosphorylation of the protein. The evidence forthis is as follows: (i) the inhibition of total

1984

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Multiple phosphorylation of polyribosomal protein pp3O 753

phosphorylation with either ACTH-(15-24)-pep-tide or spermine appeared to be associated with alower phosphate labelling of the anodic spots 4 and5, accompanied by an increased labelling of spot 1(Fig. 2); (ii) increasing the degree of phosphoryl-ation by prolonged incubation with ATP resultedin a shift of the distribution of label towards theanodic spot 5, with a concomitant appearance ofprotein staining of spots 4 and 5 (Fig. Id); (iii)pulse-chase-labelling ofpp3O clearly demonstratedthe shift of radioactive phosphate towards theanode with time, in relation to the increased degreeof phosphorylation of pp3O (Fig. 3); (iv) the fivephosphoprotein spots detected by non-equilibriumpH-gradient-gel electrophoresis-SDS/polyacryl-amide-gel electrophoresis are derived from differ-ent forms of a single protein, since proteolysis withthree different proteinases in each case resulted inthe same breakdown products for all spots.

Partial proteolysis of pp3O with three differentenzymes resulted in no detectable qualitativedifferences between the breakdown products ofspots 1 and 5, indicating at least partially randomphosphorylation of pp3O. Extensive proteolysis ofribosomal protein S6 with trypsin after phosphoryl-ation of five sites by proteinase-activated kinase IIindicated partially random and partially sequentialphosphorylation (Perisic & Traugh, 1983). How-ever, under physiological conditions, orderedphosphorylation may be more important, as shownby Martin-Perez & Thomas (1983), after serumstimulation of quiescent 3T3 cells.

Incubation of polyribosomal proteins, afterseparation on SDS/polyacrylamide-gel electro-phoresis, with monoclonal S6 antibodies, clearlyshowed pp3O to be different from S6. S6 had Mrapprox. 34000 (Fig. 5) in our separation system. Itcould not be detected on our non-equilibrium pH-gradient-gel electrophoresis-SDS/polyacrylamide-gel electrophoresis system. This could be becauseS6 ran off the gel under the electrophoreticconditions used, implying a high pI, as is typical ofribosomal proteins.We describe in this paper a first characterization

ofa novel polyribosomal phosphoprotein, pp3O. Itsphosphorylation is sensitive to polyamines, but notto cyclic nucleotides and Ca2+/calmodulin. Itcontains multiple phosphorylation sites, but is notidentical with ribosomal protein S6.

We express our gratitude to Dr. H. M. Greven and Dr.J. W. F. M. Van Nispen (Organon International BV, Oss,The Netherlands) for the gift of ACTH-(15-24)-peptide,to Dr. H. Towbin (Ciba-Geigy, Basel, Switzerland) for

the gift of the monclonal S6 antibody, to Professor Dr.W. H. Gispen and Dr. H. Zwiers for stimulatingdiscussions, to Ms. Frankena and Mr. J. H. Brakkee fortechnical assistance and to Mr. E. D. Kluis for drawingthe Figures. This research was supported by FUNGOGrant no. 13-31-43 of the Netherlands Organization forthe Advancement of Pure Research (ZWO).

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Farrell, P. J., Hunt, T. & Jackson, R. J. (1978) Eur. J.Biochem. 89, 517-521

Lastick, S. M. & McConkey, E. H. (1976) J. Biol. Chem.251, 2867-2875

Leader, D. P. (1980) in Recently Discovered Systemsof Enzyme Regulation by Reversible Phosphorylation(Cohen, P., ed.), pp. 201-233, Elsevier/North-Holland, Amsterdam

Leader, D. P. & Coia, A. A. (1978) FEBS Lett. 90, 270-274

Martin-Perez, J. & Thomas, G. (1983) Proc. Natl. Acad.Sci. U.S.A. 80, 926-930

Nielsen, P. J., Manchester, K. L., Towbin, H., Gordon,J. & Thomas, G. (1982) J. Biol. Chem. 257, 12316-12321

Nishimura, J. & Deuel, T. F. (1983) FEBS Lett. 156,130-133

Oackley, B. R., Kirsch, D. R. & Morris, N. R. (1980)Anal. Biochem. 105, 361-363

O'Farrell, P. H. (1975) J. Biol. Chem. 250, 4007-4021O'Farrell, P. Z., Goodman, H. M. & O'Farrell, P. H.

(1977) Cell 12, 1133-1142Perisic, 0. & Traugh, J. A. (1983) J. Biol. Chem. 258,

13998-14002Poehling, H. M. & Neuhoff, V. (1981) Electrophoresis 2,

141-147Rangel-Aldao, R., Kupiec, J. W. & Rosen, 0. M. (1979)

J. Biol. Chem. 254, 2499-2508Roberts, S. & Ashby, C. D. (1978) J. Biol. Chem. 253,288-296

Roberts, S. & Morelos, B. S. (1979) Biochem. J. 184, 233-244

Schrama, L. H., Edwards, P. M. & Schotman, P. (1984a)J. Neurosci. Res. 11, 67-77

Schrama, L. H., Frankena, H., Edwards, P. M. &Schotman, P. (1984b) Neurochem. Res. 9, 1265-1278

Schrama, L. H., Frankena, H., Edwards, P. M. &Schotman, P. (1984c) J. Neurochem. in the press

Traugh, J. A. (1981) Biochem. Actions Horm. 8, 167-208Tuazon, P. T., Merrick, W. C. & Traugh, J. A. (1980) J.

Biol. Chem. 255, 10954-10958Van Dijk, A. M. A., Benitez-King, G., Schotman, P. &

Gispen, W. H. (1981) Neurochem. Res. 6, 847-861Zwiers, H., Schotman, P. & Gispen, W. H. (1980) J.

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