The EMBO Journal vol.7 no.1 pp.21 -27, 1988

Primary structure of the human, membrane-associatedCa2+-binding protein p68: a novel member of a proteinfamily

Mark R.Crompton, Raymond J.Owens',Nicholas F.Totty2, Stephen E.Moss, MichaelD.Waterfield2 and Michael J.Crumpton

Imperial Cancer Research Fund, PO Box 123, Lincoln's Inn Fields,London, WC2A 3PX and 2Ludwig Institute of Cancer Research, 91Riding House St., London, WIP 8BT, UK

'Present address: Celltech Ltd, 250 Bath Road, Slough, Berks, SLI4DY, UK

Communicated by M.J.Crumpton

cDNA clones encoding human 'p68', a membrane-asso-ciated Ca2+-binding protein, were isolated from a Xgtllexpression library of the human T-leukaemia cell line J6,by using a rabbit antiserum against denatured purifiedlymphocyte p68, and from a liver cDNA library by using32P-labelled p68 cDNA fragments. The amino acidsequence of p68, deduced from the sequences of overlap-ping cDNA clones, is described. The results show thatp68 is closely related to a family of proteins whichincludes intracellular substrates of the EGF receptor andpp6(" tyrosine kinases. The p68 amino acid sequenceis internally repetitive, being constructed from eightrepeats of varying lengths, each of which contains ahighly conserved sequence. Multiple copies of the lattersequence are also present in the related proteins p36, lipo-cortin I and protein II. We discuss how the commonstructural features of these proteins may reflect commonfunctions and, furthermore, how the eight repeat struc-ture of p68 may have evolved. The sequences of indepen-dent cDNAs suggest that alternatively-spliced mRNAscould encode different p68 protein species. This sugges-tion is consistent with the observation that purified p68migrates as a closely-spaced doublet when analysed bySDS-PAGE.Key words: p68/Ca2'-binding protein/cDNA sequence/primary structure/gene duplication

Introduction

p68 is an intracellular Ca2'-binding protein of -68 000mol. wt, that was first identified as a major component ofthe non-ionic detergent-insoluble residue of purified B-

lymphoblastoid cell plasma membranes (Davies et al., 1984;Owens and Crumpton, 1984). Further studies showed it tobe present in other cell types and tissues, such as fibroblastsand liver, but not intestinal epithelium brush border mem-branes. Although it appears to be primarily associated withmembranes and/or the cytoskeleton, there appears to be a

small soluble pool of p68 in the cell cytosol (Owens et al.,1984; Davies and Crumpton, 1985). The protein species.found in the detergent-insoluble residue of plasma mem-

©IRL Press Limited, Oxford, England

branes may, by analogy with erythrocyte ghosts, be com-ponents of a submembranous cytoskeletal network complex(Mescher et al., 1981; Davies et al., 1984; Apgar et al.,1985; Apgar and Mescher, 1986). The association of p68with this complex is Ca2+-dependent and may be mediatedby occupancy of the single high affinity (KD = 1.2 ItM)Ca2'-binding site detected in the soluble protein (Owensand Crumpton, 1984). Transient rises in the intracellular con-centration of free Ca2+ ions have been demonstrated inresponse to a variety of cellular stimuli (Tsien et al., 1984)and such changes are thought to constitute a 'secondmessenger' system acting on Ca2+-receptor proteins (Kret-singer, 1979). The Ca2'-binding characteristics and intra-cellular location of p68 suggest that it may have a'receptor-like' capacity to respond to the changes in Ca2'ion concentrations observed in activated cells.Work in several laboratories has identified what appears

to be a family of Ca2+-regulated proteins, members ofwhich have properties similar to those of p68. Groups ofproteins which bind chromaffin granule membranes in thepresence of Ca2+ (Creutz et al., 1983), which bind tophenothiazine columns in a Ca2+-dependent manner(Moore et al., 1984), or which are released by Ca2+-chelators from detergent-insoluble residues of both smoothmuscle (Raeymaekers et al., 1985) and liver membranes(Geisow et al., 1984; Shadle et al., 1985), all include mono-meric proteins of 67 000-70 000 mol. wt. These groupsof proteins also include a number of polypeptides in the30 000-40 000 mol. wt range, one of which has beenshown to be homologous with p36 (Gerke and Weber, 1984;Geisow et al., 1984; Davies and Crumpton, 1985; Creutzet al., 1987), the major intracellular substrate for the tyrosinekinase activity of the Rous sarcoma virus transforming geneproduct (Erikson and Erikson, 1980; Radke et al., 1980).A structural relationship between these proteins was first sug-gested when an antiserum raised against Torpedo calelec-trin (an electric fish protein) was found to recognize severalCa2+-regulated mammalian proteins (Sudhof et al., 1984).Since then antigenic cross-reactivities between a number ofthese proteins (Smith and Dedman, 1986; Creutz et al.,1987), including p68 (Davies and Crumpton, 1985), havebeen reported. Further, the existence of a family of Ca2+-regulated proteins with homologous amino acid sequenceshas now been unequivocally demonstrated by both aminoacid (Geisow et al., 1986; Weber et al., 1987) and cDNAnucleotide sequencing (Huang et al., 1986; Saris et al., 1986;Wallner et al., 1986). These proteins, namely Torpedocalelectrin, p36 (calpactin I heavy chain, lipocortin II),lipocortin I (calpactin II, p35) and protein II, are all in the30 000-40 000 mol. wt range.

In this report, we describe the primary structure of humanlymphocyte p68 as deduced by cDNA cloning and sequen-cing and demonstrate that p68 is another member of thisfamily of Ca2+-regulated proteins.

21

M.R.Crompton et al.

Table I. Amino acid sequences of purified p68 tryptic peptides

Fraction Acetonitrile Peptide Amino acid sequence(%) no.

A 8 1 IMVSR2 DAISGIG(T)DEK

B 10 PAND(GN)PDADAK4] GELS(FD)FEK

C 15 5 STPEYFAED 32 6 EEDDVV-EDLV

Acetonitrile concentrations refer to the elution of the respective pep-tides from the first HPLC purification step (see Materials andmethods).Amino acid residues in brackets represent tentative assignments and adash represents an unassigned residue.

0

a

E<1mlUfE

.4

ISO

100

so

6 E L S F D F E KNot quantitated

. ./.

1 2 3 4 5 6 7 8 9 10 I11 12Residue number

P A N D 6 N P D A D A K

Fig. 1. Amino acid sequence analyses of the purified p68 trypticpeptide fraction B containing peptides 3 and 4. The solid linesrepresent yields of PTH-amino acids assigned to peptide 3 and thebroken lines represent the yields assigned to peptide 4.

Resultsp68 amino acid sequenceAutomated Edman degradation of - 2 nmol of p68 purifiedfrom either human B-lymphoblastoid cells or tonsil lympho-cytes gave no appreciable levels of a specific cleavage pro-duct in either case, suggesting that the N-terminal residueis blocked. For this reason, - 6 nmol of p68, purified fromB-lymphoblastoid cells as described by Owens and Crump-ton (1984), was cleaved with trypsin and the resultingpeptides separated by reverse-phase HPLC in 0.1 % trifluoro-acetic acid (TFA) (see Materials and methods). Four majorpeaks, identified by absorbance at 208 nm, were subjectedto amino acid sequencing. One peak only (peptide 6) gavea single sequence (Table I); the other peaks (A, B and C;Table I) yielded multiple signals at each cycle. The latterpeaks were further purified by reverse-phase HPLC in10 mM ammonium bicarbonate. Amino acid sequenceanalysis of the products provided three more unique se-quences (peptides 1, 2 and 5), and two unequally-representedsequences which were assigned to a pair of co-purifying pep-tides (peptides 3 and 4) on the basis of yield of residues ateach step of the sequencing cycle (Figure 1).

Anti-(p68) serumThe anti-(p68) serum was raised by injection of SDS-denatured, purified p68 with the specific intention ofgenerating a reagent suitable for the screening of a Xgtl 1expression library. The resulting antiserum visualizedpurified p68, by Western blotting, as a closely-spaced doublet

22

Fig. 2. Reactivity of the rabbit anti-(p68) serum as judged by Westernblotting. Lane 1, SDS-PAGE analysis under reducing conditions ofthe purified p68 used for raising the rabbit antiserum, stained withCoomassie Blue. Lanes 2 and 3, Western blots of J6 T-leukaemia celllysates with rabbit pre-immune serum diluted 1/20 and rabbit anti-(p68) serum diluted 1/100, respectively.

A2lo 4 - ---- 4 --al

4 --al .41- 4- ob44 so - 44

C2.-... ----4-

10.6 -4-

= mRNA+open reading frarne

- cDNA clone

m=m 13 sequencing strategy.-------- .unsequenced cDNA

Fig. 3. p68 cDNA clones and sequencing strategy.

of mol. wt - 68 000 comprising a heavy upper band andfaint lower band (data not shown). These bands were coin-cident in mobility with those revealed by staining purifiedp68 with Coomassie Blue or by Western blotting lysates ofthe human T-leukaemia cell line J6 and visualizing with theanti-(p68) serum (Figure 2). In contrast, the antiserum fail-ed to immunoprecipitate a recognizable band of - 68 000mol. wt from detergent lysates of [35S]methionine-biosyn-thetically-labelled J6 cells or 1251-labelled microsomes.Thus, presumably, the antiserum does not recognize thenative protein.

Isolation of p68 cDNA clonesThe relationships between the cDNA inserts which wereisolated, and the sequencing strategy used to characterizethem, are shown in Figure 3. The rabbit anti-(p68) serumrecognized one plaque in a screen of 4 x I04 plaques ofthe Xgtl 1 T-leukaemia cell J6 library. After two furtherrounds of screening, this phage isolate (namely A2) was clon-ed; digestion of the phage DNA with the restriction en-zyme EcoRI allowed the isolation of an insert of -2 kb.

Structure of Ca2+-binding protein p68

cc__asohhccoGhGACC A 0OCC AA CCa OCA CM 0T oCC AacMT1 Ale Lys Pro Ale Gln Oly Ale Lys

sAC COGOGOC ?CC AIC CA? caC TsC CCa GOC Tm caC CCC aMC CMaG? oCC GM OCT CO AC ACT 0CC a£c AM Oc TT OCc ACT SaC Aac GMo aCC AlA C10 GAClye ACV Sly Ser Ile via Asp PM Pro Gly PM Asp Pro Ass G1s Asp Ale Gin Ale Lou Tye Thr Ale MT Lys Gly Pb Sly Ser Asp Lys Gin Ale Ile Leu AspAlA AIc aeC TC COG MeC aMC aMc CaM AOc CM GcaSIC TOC CaM ac TAC AaM sCC dC TAC SOC aM Sac CC A?? SC? SaT ec AM TA? SMa TTO saO SOC AMctl. 1i. hir Ser Arg SeAsaa Ar9 GIs Aca GIn Glu Tel Cys Ols Ser ?yr Lye Ser Leu Tyr Gly Lye Asp Leu Ile Ale Asp Len Lys Tyr Clu Len lhr Oly Lysm GSA COG se Aas 010 SOC CIO aVG aMG CCA CC? 0CC TA? 10 cas OCC MAA Gaa Ass aaA Gas OCC AsC SCO SOC All OC AC? SaT GAC Aac SC CTC AsT SAGPM Ole Ar, Leu 1i. Vel Gly Leu MT Ar, Pro Pro Ale Tyr Cys Asp Ale Lye Gli 11. Lys Asp Ala II. ger Gly Ile Gly Ihr Asp Glu Lye Cys Lew Ile Glu

AIC ?TS OCT TCC COGce Cacc Mac CMc Asc CAC CMa CIO 510 oCa OCa TAC MA SAT 0CC TAC oac COG SAC CT1 Gac SC? SAC ATC ATC OC SAC acC IC SOC Cac11. Lou Ale Ser Ar, lhr As Gn Gln MT via oln Len Val Ale Ale Tyr Lye Asp Ale Tyr Glu Arg Asp Leu Slu AlaAsp 11. Ile Gly Asp lbr Ser Sly Nis

TIc CM AM a£1 CTS 010 OsC CTO CTC CM SA acC AOM oac Ga Sal SAC osA 010 ac GM SAC C10 01a CM CMa SAT GTC CMa SAC C?A Tac GMa oCA OSO MaaPb GIn Lye MT Lnu Vel Val Len Leu Gln Sly Shir Arg Gin in Asp Asp Vel VelJor Gl Asp Leu Val Sln Gln Asp Val Gln Asp Lnu Tyr Clu Ale Gly GluCTC AMA 100 eGA Aca SaT SMa eCC CaM TSC ATT Tac ATc 1T GSA aaM COC aC AMc CaM CA? CTT CSO 10 GT0 TC SAT GMa TAT Csc AMG ACC acA OSO AaM CCcLon Lye Srp Gly Thr Asp Glu Ale Gln Pte II@ Tyr Ile Len Sly Asn Arg Ser Lys Gln Nis Lnu Ar, Lon Val Phe Asp Clu Tyr Lou Lys Thr Thr Cly Lys Pto

ATT SM 0CC acC ATc CGA SOG GMa CTC ICT OGG SAC TTT GM Ac C?A£a1 C10 0CC 01£ 010 AM 101 ATC COG MC MC CCG SM TAT IT CTcaa aMO C?C TIC1l. Gln Ale Ser 1i. Ar, Gly Gin Leu Ser Gly a Pb. Glu Lys Lnu MT Leu Ale Vel Val Lys Cys Ile Ar, ger Thr Pro Clu Tyr Phe Ale Glu Arg Leu Pb.

AaM SC? a£1 AaM OCc C10 OOG acT COO Gac aaC aCC C10 ATC COC Asc a£1 OsC ICC COT acT GMa T1T SaC AT£ C?C SAC ATT COG SAG ATC TTC COG ACC AMG TATLys Ale MT Lys Gly Leu Sly lhi Ar, Asp Asn Tir LeU Ile Arg 1i. RT Val Ser Agr gor GIn Leu Asp MT Leu Asp Ile Ar, clu Ile Phe Ar, Thr Lye Tyr

GM AaM ICC CTC Tac aMC a£1 ATc AaM aaT SAC aCC TCT GOC GMa Mac Aac AaM aT CTG CTG AMG CTG ICT GOG GSa SAT SAT SAT GCT OCT GOC CaM TTC TTC CCCGln Lye Set Leu Tyr Ser RT 1i. Lys Asn Asp lhr Ser Sly Glu Tyr Lye Lys TAr Leu Leu Lys Leu Ser Gly Gly Asp Asp Asp Ale Ale Gly Gln Pb. Phe Pro

GMA OCA 0CC CaM 0T1 0CC TAT CaM AT1 S MGRA CTT aMT Ca 010 0cC CSA 01a GM CTO AMG eGaACTA?0 COC CCA 0CC AMT SaC TTC aaC CCT SAC GCA SAT OCCGin Ale Ale Sln vTe Alelyr Gln NT Trp GiU Len gor Ale Vel Ale Arg Vel Glu Leu Lye Gly lbr Vel Arg Pro AlaAsn Asp Phe Asn Pro Asp AlaAsp AleMA GCO CTG COO aaA 0CC aT1 AaM eSa C?C ece acT GMc SaM Gac Ach AIC AIC SAT A?C ATC ACc CaC ccc aC aaT GTC CM COG CMa CAG ATC COG CaM ACC TICLye Ale LeU A,9 LyS Ale NT LyS Gly LeU Gly lir Asp Glu Asp lhr Ile 11 Asp tie tIle lir Nis Ar, Ser Asn VTl Gln Ar, Gln Gln Iie Ar, Gln Tir Phe

AaM tCT Cac m SOC COG Gac TTA £10 ACT GMc C1G AaM IC? GMc ATc CT OSa SAC CTG OCA aGG CTG ATT CTG OGG C?C AT£ ATG CCA CCG OCC CAT TAC SAT OCCLye Set Nie Pb. Gly Ar, asp Lou RT lbr Asp LeU Lys ferluGIn 1Ser Sly Asp Lou Ale Arg LeU Ile LeU Gly Leu MET RUT Pro Pro Ale Hie Tyr Asp AleAaM CaM T10 AaM AaM 0CC £a1 GMa 0Da CC SOC acA GSA SMa aaG OCT CTT AT SA ATC CG GCC ACT COO aCC aaT OCT SaM ATC COO OCC ATC aaT SAG OCC TATLye CGi Len Lys Lye Ale RUT Glu Gly Ale Sly Tla Asp Glu Lye Ale Leu II* Glu Ile Leu Ale Tir Ar, Tir Aen Ale Glu Iie Ar, Ale Ile Asn Glu Ale Tyr

AaM GAM Gac TA? Cac Aac ICC Cse GAM SaT SC? C10 AMC ICA Gac acA ICT OCC CM TIC aMG G ATC C?C ATT TCT CTG OCC ACO GGG CAT CGT cG GMa ¢GA ¢GALys GlU Asp Tyr Nis Lys get Lew Gin Asp Ale LnUgotfer Asp hir Ser Gly Ale Pb.e Ar, Arg 11 Leu Ile 5cr Leu ale lhr Gly lis Arg Glu Glu Gly GlyGM aaC C10 Gac CMc OCA COO SMa SaT cCC CMa01 SC? OCT GAM ATc ITS SA ATA OCA SAC ACA CCT aMT CGA SAC AMA ACT TCC TG GMa ACA COT STC a£1 ACGCGU Asn Len Asp Cln Ale Ar,g GiU Ap Ale Gln ValAle Ale Slu Ile Leu Glu 11 AleaAsp TAr Pro Ser Giy Asp Lys lir Set Leu GiU Thr Arg Ph. NT ThraTC C10 1? acC COG aMC IA? CCC Cac CsC COG ASA TC TIC CMe GMa lTC AIc AMO a£1 acC MAC TAT SaC cGaSAG CAC ACC ATC AAM AaM SAG a1 TCT 00 SaTtie Len Cys lher Arg Set Tyr Pro Nis Leu Arg Arg vTl P1 Gin Sln PMa II Lyes R Thr Asn Tyr Asp VTl Glu His lhr Ile Lys Lys Glu NET S5r Gly AspSC aMeOATe CA ITTm0 OCC ATT GTS CMa aMT OsC AMc aaC AAM CC? CIC TTC TT 0CC Gac AA CIT TAC AMA ICC a£1 aAG SOT OCT GGC ACA SAT SaC AM AC?Tel Ar. AsP Ale PM Tal Ale 1. VTl Gln 5cr VTl Lys Asn Lys Pro Leu Pi. Phe Ala Asp Lys Leu Tyr Lys 5er NET Lys Gly Ale Gly lir Asp Asp Lys lirC10 acC AMG AIC a£1 01£ ICC COC aM GeM AlT Gac C10 CsC AMC AIC COG AM SGM TC AlT G aMa TAT SAC AAM TC? CTC CAC CM OCC ATT GMG 0? SAC ACCLOn lir Ar, 1lb ET Val Ser Ar,gor Gnlu 1. Asp Leu Leu Asn tie Ar, Arg Clu P_a 11. Clu Lys Tyr Asp Lys 5er Lu lis ClGn Ale Ile Glu Sly Asp ThrICC OSA GMCTC C10 AM OCC 110 CTG OC? CIC 101 001 00? GM GM MCcCcMCT10SCSA-AcCITCCgor Gly Asp Pe LeU Lye Ale Lou Lnu Ale Lou Cys Gly Gly Slu Asp

_A?CC10ICCACCICCCTG.-ascsCCCIC10T AAAMAAA

1279

23545

343II

451117

559153

6671i9775225553261

991297

1099333

1207369

1315405

1423441

1531477

1639513

1747549

1655585

1963621

2071657

2196673

2341

2468

Fig. 4. p68 cDNA sequence and the predicted amino acid sequence. Of the pairs of numbers at the right hand side, the upper ones refer tonucleotide positions and the lower ones to amino acid positions. Underlined amino acid sequences correspond to those identified by analysis of thep68 tryptic peptides (see Table I).

This insert, and its restriction fragments generated by diges-tion with the enzymes PvuH, BamHI, AluI and BglI, weresubcloned into M 13 and sequenced on both strands. InsertA2 contained a continuous open reading frame that ter-minated at its 3' end which coincided with an EcoRI site.Re-screening of the J6 library with 32P-labelled A2 failedto identify any cDNA clones containing a sequence whichcrossed this EcoRI site. However, this probe, when usedto screen 3 x 104 colonies of a human liver cDNA library,allowed the isolation, after two rounds of screening, of ashort insert (C2) which encoded such a sequence. A 32p_labelled EcoRI fragment of - 100 bp from C2 was used tore-screen 105 plaques of the J6 library, allowing the isola-tion of four apparently identical clones. One of these (10.6)contained an insert of -0.45 kb which was sequenced onboth strands using the EcoRI sites at the ends and an inter-nal TaqI site. The sequence revealed an open reading frameending in a stop codon. Sequencing of a HaeIII fragmentfrom insert C2 confirmed that the EcoRI site at the 3' endof the open reading frame in A2 was coincident with thatat the 5' end of the open reading frame in 10.6.

cDNA sequenceThe cDNA sequence (Figure 4) contained an open readingframe extending from position 1 to a stop codon beginningwith nucleotide 2120. However, it is predicted that transla-tion of p68 is initiated at the methionine codon beginningwith nucleotide 101. This assignment is made on the basis

that this potential initiation codon is the most 5', and fur-thermore is contained within a nucleotide sequence (ACC-ATGG) which has been shown by Kozak (1986) to beoptimal for initiation by eukaryotic ribosomes. In this case,the predicted open reading frame (Figure 4) encodes a pri-mary translation product of mol. wt 75 874. Although thissize is larger than that predicted from SDS-PAGE analysesof the purified protein, initiation at methionine codons fur-ther 3' is very unlikely because none of these is containedwithin a 'Kozak' sequence. All of the p68 tryptic peptideamino acid sequences that were determined directly (TableI) were contained within the predicted primary sequence(Figure 4). However, the cDNA sequence predicted differentamino acid residues at the fifth positions of peptides 3 and4 (which were sequenced together), than those assigned.These differences can be readily explained if it is assumedthat the respective amino acids were merely assigned to thewrong peptide (Figure 1).

Re-screening of the J6 cDNA library with 32P-labelledA2 as a probe gave a number of shorter cDNA clones whosesequences were contained entirely within that of A2. Unex-pectedly, one of these lacked an 18 base segment encodingthe amino acid sequence VAAEIL (Figure 6), a structurewhich could have been derived by the utilization of an alter-nate splice acceptor site. No full-length open reading frameswith this difference were characterized. As a result, it can-not be concluded that there are two p68 protein species dif-fering by an insert of six amino acid residues. However,

23

M.R.Crompton et al.

if this were the case then it would account for the closely-spaced 'doublet' of p68 bands observed on SDS-PAGEanalysis of the purified protein (Figure 2).The 3' non-coding region comprised 349 nucleotides, with

seven A residues at the 3' end. It is unlikely that theseresidues correspond to the polyadenylation site of the p68mRNA, because there is no AATAAA polyadenylationsignal or any of its functional variants (Birnstiel et al., 1985)located close to the 3' end. Northern blotting analysis of J6RNA using 32P-labelled A2 revealed a single band whosesize was estimated to be - 2.8 kb and a faint smear whichis most likely a result of degradation (Figure 5). Assumingthis size estimation is correct, then the cDNA sequencesanalysed, which totalled 2468 bases, do not account for theentire p68 mRNA.

Fig. 5. Estimation of the size of the p68 mRNA. 32P-Labelled A2cDNA was hybridized to a Northern blot containing - 10 jg of RNAfrom the J6 T-leukaemia cell line. The arrows show the positions ofthe marker ribosomal 28S and 18S structural RNAs. The size of thep68 mRNA was estimated on the basis of its mobility relative to thesebands.

I

p68 amino acid sequence and homology with otherproteinsExamination of the p68 predicted amino acid sequencerevealed that it can be divided after amino acid residue 325to give two segments which show 45% overall homologyto each other. As shown in Figure 6, each of the two halvescan be arranged into four serially repeated segments givingeight segments in all. Each segment has an N-terminal'variable' sequence and a C-terminal consensus sequence of62 amino acid residues. The variable regions are from 10to 46 amino acid residues in length and the fourth and eighthsegments have homologous C-termini of four amino acidresidues. Comparison of the nucleotide sequences encodingthe eight segments revealed that the highest number ofnucleotide matches were obtained by aligning segment onewith five, two with six, three with seven and four with eight.As a result, it appears that p68 has evolved by the duplica-tion of a gene which itself arose by two duplications of aprimordial gene encoding a polypeptide whose amino acidsequence resembled that one of the p68 segments.Comparison of the p68 amino acid sequence with those

of other Ca2+-binding proteins revealed that it possesses ahigh degree of homology with three other proteins, namelyp36, lipocortin I and protein II (Huang et al., 1986; Weberet al., 1987). In particular, its N-terminal half gavehomologies of 46, 43 and 54% respectively with these pro-teins, whereas the C-terminal half showed slightly lowerhomologies of 43, 41 and 51% respectively. Furthermore,p36, lipocortin I and protein II have internally-repetitivestructures which resemble that of p68, each of the formerproteins being divisible into four homologous segments(Figure 6). Alignment of the amino acid sequences of therepetitive segments of all the proteins (i.e. eight from p68,and four each from p36, lipocortin I and protein II) allowedthe definition of a core 62 amino acid consensus sequencefor the protein family. As judged by the physico-chemicalcharacteristics of the amino acid side chains, 20 of the 62amino acid residues of the core sequence were shared inmore than half of the repeats of each protein. In seven of

MAKPAQGAKYRGSIHDFPGFDPNQDAEALYTAMKGFGSDKEAILDIITSRSNRQRQEVCQSYKSLYGKDLIADLKYELTGKFERLIVGLMRPPAYCDAKEIKDAISGIGTDEKCLIEILASRTNEQMHQLVAAYKDAYERDLEADIIGDTSGHFQKMLVVLL

QGTREEDDVVSEDLVQQDVQDLYEAGELKWGTDEAQFIYILGNRSKQHLRLVFDEYLKTTGKPIEASIRGELSGDFEKLMLAVVKCIRSTPEYFAERLFKAMKGLGTRDNTLIRIMVSRSELDMLDIREIFRTKYEKSLYSMIKNDTSGEYKKTLLKLSGGDD

DAAGQFFPEAAQVAYQMWELSAVARVELKGTVRPANDFNPDADAKALRKAMKGLGTDEDTIIDIITHRSNVQRQQIRQTFKSHFGRDLMTDLKSEISGDLARLILGLMMPPAHYDAKQLKKAMEGAGTDEKALIEILATRTNAEIRAINEAYKEDYHKSLEDALSSDTSGHFRRILISLA

TGHREEGGENLDQAREDAQVAAEILEIADTPSGDKTSLETRFMTILCTRSYPHLRRVFQEFIKMTNYDVEHTIKKEMSGDVRDAFVAIVQSVKNKPLFFADKLYKSMKGAGTDEKTLTRIMVSRSEIDLLNIRREFIEKYDKSLHQAIEGDTSGDFLKALLALCGGED

2MSTVHEILCKLSLEGDHSTPPSAYGSVKAYTNFDAERDALNIETAIKTKGVDEVTIVNILTNRSNAQRQDIAFAYQRRTKKELASALKSALSGHLETVILGLLKTPAQYDASELKASMKGLGTDEDSLIEIICSRTNQELQEINRVYKEMYKTDLEKDIISDTSDDFRKLMVALA

KGRRAEDGSVIDYELIDQDARDLVDAGVKRKGTDVPKWISIMTERSVPHLQKVFDRYKSYSPYDMLESIRKEVKGDLENAFLNLVQCIQNKPLYFADRLYUSMKGKGTRDKVLIRIMVSRSEVDMLKIRSEFKRKYGKSLYYYIQQDTKGDYD ALLYLCGGDD

3 MAMVSEFLKQAWFIENEEQEYVQTVKSSKGGPGSAVSPYPTFNPSSDVAALHKAIMVKGVDEATIIDILTKRNNAQRQQIKAAYLQETGKPLDETLKKALTGHLEEVVLALLKTPAQFDADELRAAMKGLGTDEDTLIEILASRTNKEIRDINRVYREELKRDLAKDITSDTSGDFRNALLSLA

KGDRSEDFGVNEDLADSDARALYEAGERRKGTDVNVFNTILTTRSYPQLRRVFQKYTKYSKHDMNKVLDLELKGDIEKCLTAIVKCATSKPAFFAEKLHQAMKGVGTRHKALIRIMVSRSEIDMNDIKAFYQKMYGISLCQAILDETKGDYEKILVALCGGN

4 AAKGGTVKAASGFNAAEDAQTLRKAMKGLGTDEDAIISVLAYRSTAQRQEIRTAYKSTIGRDLLDDLKSELSGNFEQVILGMMTPTVLYDVQELRRAMKGAGTDEGCLIEILASRTPEEIRRINQTYQLQYGRSLEDDIRSDTSFMFQRVLVSLS

AGGRDEGNYLDDALVRQDAQDLYEAGEKKWGTDEVKFLTVLCSRNRNHLLHVFDEYKRISQKDIEQSIKSETSGSFEDALLAIVKCMRNKSAYFAERLYKSMKGLGTDDNTLIRVMVSRAEIDMMDIRANFKRLYGKSLYSFIKGDTSGDYRKVLLILCGGDD

5 * GTD .. *I.**. ....R*.* . .0. L ...***.*

Fig. 6. p68 amino acid sequence aligned to highlight repeating sequences (segments) as well as homologies with other proteins. The sequences ofhuman p68 (1) (this paper), p36 (2) and lipocortin 1 (3) (Huang et al., 1986) and that of porcine protein II (4) (Weber et al., 1987) are aligned tomaximize their homology one to another. The results show that each protein contains multiple copies of a core consensus sequence (5). Underlinedamino acids in p68 are missing in an alternative form (see text). The dots above amino acids in p36 and lipocortin I indicate known in vivophosphorylation sites. In sequence 5, * represents a hydrophobic side chain, 0 an aromatic side chain and - an acidic side chain.24

Structure of Ca2+ -binding protein p68

the 62 positions a consensus amino acid residue was iden-tified. The arginine at position 22 of the core sequence wasconserved throughout all of the repeat segments (Figure 6).The greatest differences between the primary structures ofthe four proteins were located at their N-termini, which ex-tended for varying distances in front of their respective coresequences.A search of the National Biomedical Research Founda-

tion protein sequence database revealed that p68 had nosignificant level of homology with any proteins other thanthose already described. In particular, no homology wasdetected with any other known Ca2+-binding proteins.

DiscussionThe shared structural features of p68 with p36, lipocortinI and protein H presumably reflect shared biological func-tions. Although all of the proteins bind Ca2', (Owens andCrumpton, 1984; Shadle et al., 1985; Glenney, 1986; Glen-ney et al., 1987), examination of their primary structuresfailed to reveal classical 'E - F hand' sequences of the typefound in most other intracellular Ca2'-binding proteins(Tufty and Kretsinger, 1975). In the cases of p36 and lipocor-tin I, the affinity for Ca2+ is increased by the presence ofphospholipid. As phospholipid binding by p36, lipocortinI and protein II is Ca2+-dependent (Glenney, 1986; David-son et al., 1987; Schlaepfer and Haigler, 1987; Shadle andWeber, 1987), these proteins may bind Ca2+ and phospho-lipid at a common binding site. In this case, the structuralbasis of Ca2+-binding is likely to be different from that ofother Ca2+-binding proteins which do not bind phospho-lipids. Equilibrium dialysis using 45Ca2' revealed two highaffinity, phospholipid-dependent Ca2'-binding sites permolecule of p36 (Glenney, 1986), and either two (Glenneyet al., 1987) or four (Schlaepfer and Haigler, 1987) suchsites in lipocortin I. p68 and protein H are apparently dif-ferent in that they both have single high affinity Ca2+-bind-ing sites (Owens and Crumpton, 1984; Shadle et al., 1985).The latter analyses were, however, carried out in the absenceof added phospholipid and further work is required to ascer-tain whether p68 binds phospholipid, whether p68 and pro-tein II co-purify with cellular phospholipid and whetheraddition of phospholipid 'uncovers' otherwise 'hidden'Ca2'-binding sites. It is also not yet apparent whether theCa2+-binding site(s) are generated by a single segment ora combination of segments of the repetitive structure.

In vivo, p36 is a substrate for tyrosine kinase and proteinkinase C. Similarly, lipocortin I is phosphorylated by tyrosinekinase, whereas protein II is phosphorylated in vitro by pro-tein kinase C. As shown in Figure 6, the phosphorylationsites are located close to the proteins' N-termini (Glenneyand Tack, 1985; Gould et al., 1986; Pepinsky and Sinclair,1986; Weber et al., 1987). Although no evidence has beenobtained for the in vivo phosphorylation of either p68 (un-published observations) or protein H, p68 is a good substratein vitro for tyrosine kinases present in the membrane frac-tion of A431 cells (J.M.Hexham and C.J.Pallen, personalcommunication). Also, p68 has serine (13) and tyrosine (10)residues in very similar positions, relative to the first coreconsensus sequence, to those which are phosphorylated inp36, raising the possibility that p68 is also a substrate forprotein kinases in vivo.

Apart from their common characteristic of binding Ca2+with high affinity, the individual members of the family

possess unique features. For example, whereas p36, pro-tein H and p68 occur naturally in association with cellularmembranes, lipocortin I occurs in serum. Also, p36 has beenpurified in association with a polypeptide of - 10 000 mol.wt, as a quaternary complex (p362 pl02), in which the lightchains are bound specifically by the N-terminal, proteolytic-ally-cleavable tails of the p36 polypeptides (Glenney et al.,1986), whereas neither protein H, lipocortin I nor p68 havebeen isolated in association with another peptide. These andother unique features are conceivably mediated by the N-termini (and the 'spacer' sequence connecting the two halvesof p68) of the respective proteins, which contain the mostpronounced differences between the proteins' amino acid se-quences.The relationships between the biochemical properties of

the respective proteins and their physiological functions arenot yet apparent. Lipocortin has been identified as mediatingthe anti-inflammatory effects of glucocorticoids by inhibitingphospholipase A2 (Hirata, 1984; Wallner et al., 1986), andboth lipocortin I and p36 have been purified from humanplacenta on the basis of their capacity to inhibit porcine pan-creas phospholipase A2 in vitro (Huang et al., 1986).However, the mechanism by which this inhibition is achievedappears to be relatively non-specific in that it probably oc-curs by sequestration of the substrate rather than by directinteraction with the enzyme (Davidson et al., 1987). Thelatter assignment is consistent with the observation that phos-pholipases C and D as well as A2 from different sourceswere inhibited (Hirata, 1984). On the other hand, the phos-pholipase inhibition model is supported by the observationthat recombinant lipocortin I mimics the effects of glucocor-ticoids on thromboxane release from perfused lung (Cirinoet al., 1987). Whether such a broad-specificity mechanismis effective in regulating intra- and extracellular phospho-lipases in vivo and whether phosphorylation/dephosphoryla-tion affects the proteins' inhibitory activities (Hirata et al.,1984) remain to be established. Extracellular forms of lipo-cortin I have been well characterized (Pepinisky et al., 1986),although the route by which lipocortin I is externalized isunknown. In contrast, protein H, p36 and p68 are apparent-ly located only intracellularly. As a result, lipocortin I alonehas the potential to regulate extracellular as well as intra-cellular phospholipases.Other investigators have suggested a role for these pro-

teins in the mediation of membrane-associated processes suchas exocytosis (Geisow et al., 1984; Sudhof et al., 1984;Geisow et al., 1986). This suggestion is based upon the bio-chemical and structural similarities of p68, p36, lipocortinI and protein H to calelectrin, a protein from Torpedo mar-morata which self-aggregates and which enhances the ag-gregation of secretory vesicle/granule membranes in aCa2+-dependent manner. Calelectrin is apparently the onlymember of the protein family in the electric fish and it may,therefore, perform a number of functions, which in mam-mals are mediated by a variety of more specialized proteinsthat have evolved via gene duplication.An insight into the functions of the mammalian family

members may be gained from the characterization of theirpatterns of tissue/cell type expression. Western blottinganalyses of human cell lysates have revealed that p68 is wellrepresented in mature T-leukaemia and B-lymphoblastoid celllines, a monoblastic cell line (U937) and human foreskinfibroblasts, whereas it is poorly expressed or absent invarious Burkitt lymphoma cell lines and an epidermally-

25

M.R.Crompton et al.

derived cell line (A431) (unpublished observations). Further-more, immunohistological studies on human lymphoid tissueshave provided evidence that levels of p68 differ in differentlymphocyte sub-populations increasing dramatically duringB-cell but not T-cell maturation, whereas studies of someepithelial tissues have revealed complex patterns withexpression being restricted to particular cell types (unpublish-ed observations). In view of these complex patterns ofexpression, studies based on cDNA/mRNA hybridizationwill need to be carried out on cloned cell lines and on tissuesections in situ in order to yield informative data.p68 is also potentially interesting from an evolutionary

point of view. Because of its 'two-layered' repetitive nature(i.e. its probable evolution by the duplication of a quadrupli-cated structure), analysis of the intron/exon patterns withinthe gene should provide valuable insights into the mechan-isms by which protein diversity is generated via the multi-plication of a primordial gene. If, as we suggest, the p68gene has arisen during evolution by duplication of a primor-dial gene coding for a polypeptide of - 8000 mol. wt, thenit appears likely that this will be reflected at the humangenomic level by the presence of at least eight exons. Pre-liminary experiments show that p68 cDNA sequenceshybridize with at least seven bands in EcoRl digests ofhumangenomic DNA (unpublished observations). Since the com-bined lengths of these genomic fragments total over 40 kb,and the p68 cDNA sequence encodes only one EcoRI site,one interpretation of this data is that p68 is encoded by alarge gene containing at least five introns. Other explana-tions are, however, possible such as the human genome con-tains multiple p68 genes and/or pseudogenes. A clear pictureof the number and structure of the p68 gene(s) will onlyemerge through the isolation and characterization of genomicclones.

Materials and methodsPurification of p68Human tonsils were collected from hospitals in the Greater London areaand were frozen within 24 h. Typically 40 tonsils (160 g wet weight oftissue) were disrupted with an 'Oster osterizer' at full power for 3 min in3 vol of phosphate-buffered saline (PBS), containing 1 mMphenylmethylsulphonyl fluoride (PMSF) and 10 mM iodoacetamide (IAA).Homogenates were centrifuged at 9000 g for 7.5 min and the microsomefraction was recovered from the supematant by centrifuging at 35 000 gfor 30 min. After two washes with the 'disruption buffer' at 4°C, themicrosomes were extracted in 250 ml of 10 mM Tris-HCI buffer, pH 7.4,0.15 M NaCl, 1% (v/v) Nonidet P40 (BDH), 1 mM CaCl2, 1 mM MgCl2,2 mM PMSF, 20 mM IAA, 0.05 mg/ml soybean trypsin inhibitor, 2 mg/mileupeptin, 1 mg/mi bovine serum albumin for 60 min on ice. Insolublematerial was recovered by centrifugation at 150 000 g for 60 min and washedonce with the extraction buffer without Nonidet P40. The detergent-insolubleresidue was resuspended in 100ml of 10 mM Tris-HCI buffer, pH 7.4,0.15 M NaCl, 5 mM ethyleneglycol-bis-(,-aminoethyl ether) N,N,N',N'-tetraacetic acid (EGTA) and stirred for 60 min on ice. Aftercentrifugation at 150 000 g for 60 min, the supernatant was concentratedto -20 ml in dialysis tubing using 'Aquacide II' (Calbiochem), dialysedagainst 20 mM triethanolamine-HCI buffer, pH 7.4, 1 mM EGTA andloaded onto a Pharmacia FPLC Mono Q HR5/5 column in the same buffer.p68 was retained on the column under these conditions and application ofa linear gradient of increasing NaCl concentration resulted in the elutionof about 600 .tg of purified p68 at -0.24 M NaCI. This preparation wassubsequently fractionated by preparative SDS-PAGE under reducing con-ditions (Laemmli, 1970). Protein was visualized with 4 M sodium acetate(Higgins and Dahmus, 1979) and the p68 was electro-eluted from theappropriate gel slice (Kelly et al., 1983), concentrated with Aquacide II,dialysed against distilled water and Iyophilized.

Anti-(p68) serumA rabbit was injected s.c. at multiple sites with - 100 jig of SDS-PAGE-purified p68 in 1 ml of an emulsion of Freund's complete adjuvant withPBS (1:1, v/v), followed by 20 1tg in 1 mi of PBS/Freund's incompleteadjuvant two weeks later and after a further two weeks with 20 yg in 100yl of PBS injected i.v. The rabbit was bled 6 days later. The antiserumwas tested by Western blotting as described by Krissansen et al. (1986),except that biotinylated goat anti-(rabbit IgG) serum (Vector laboratories)was used to visualize bound antibody.

Amino acid sequence analysisThe p68 used for amino acid sequence determination was purified fromthe plasma membrane fraction of human B-lymphoblastoid cells (cell lineBRI 8) as previously described (Owens and Crumpton, 1984). The protein(0.4 mi of 1 mg/mi) was reduced and alkylated by treatment with 10 mMDTT for 2 h at 37°C followed by 20 mM 1AA for 2 h on ice. After dilutingto 1.0 ml with 100 mM ammonium bicarbonate, p68 was digested withtrypsin (Sigma) at a ratio of 10:1 (w/w) for 18 h at 37°C. The digest wasadjusted to pH 8.8 with 1 mM Tris-HCI buffer, pH 8.8 and TFA wasthen added to a final concentration of 0.1%. Peptides were purified byreverse-phase HPLC on a Synchropak RPP C-8 column (250 mm x 5 mm)developed with a 0-40% linear gradient of acetonitrile in 0.1% (v/v) TFA,followed by a Synchropak RPP C18 column (46 mm x 16 mm) eluted witha 0-45% (v/v) acetonitrile gradient in 10 mM ammonium acetate, pH 6.5.Amino acid sequences were determined using a gas phase sequencer

(Hewick et al., 1981) and high sensitivity detection methods for phenyl-thiohydantoin amino acid analysis (Waterfield et al., 1986).

cDNA librariesThe Xgtl 1 expression library used for the initial screening was derived fromcDNA synthesized from purified poly(A)+ RNA isolated from J6 cells (asubclone of the human T-leukaemia cell line, Jurkat); the library was con-structed and donated by Drs H.Kataoka, M.Collins, M.J.Owen and T.Lin-dahl (Krissansen et al., 1986). The human liver cDNA library wasconstructed in a plasmid vector based on pAT 153 and was donated by theMRC Immunochemistry Unit, Oxford (Bentley, 1986).

Screening of cDNA librariesA 1/100 dilution of the rabbit anti-(p68) serum in PBS was pre-cleared withEscherichia coli and Xgtl 1 proteins as described by Huynh et al. (1985),except that a lysogen of non-recombinant Xgtl 1 in Y 1089 was used insteadof BNN97. The pre-cleared antiserum was then used to screen the expres-sion library (Huynh et al., 1985); bound antibody was detected as describ-ed by Krissansen et al. (1986).

Libraries were also screened using cDNA fragments labelled with 32p(Feinberg and Vogelstein, 1983). Lifts of the Xgtl 1 library were probedusing methods recommended for use with 'Hybond-N' membranes (Amer-sham International), whereas the plasmid library was screened using 'Milli-pore-HA' filters (Millipore) and the procedures described by Maniatis etal. (1982).

RNA blottingRNA was purified from J6 cells (Chirgwin et al., 1979). Following electro-phoresis in formaldehyde-containing gels, RNA was transferred to 'PallBiodyne A' membranes (Pall Ultrafine Filtration) by capillary blotting(Maniatis et al., 1982). Hybridization and washing was as described byChurch and Gilbert (1984).

DNA sequencingSequencing of restriction fragments in M13 vectors was by the chain-termination method (Sanger et al., 1977).

Computer analysisNucleotide sequences were aligned by the method of Wilbur and Lipman(1983) using the following parameters; K-tuple length 3, window size 20,gap penalty 7.

AcknowledgementsWe are indebted to Dr M.Owen (ICRF, Dominion House Labs, St Bar-tholomew's Hospital) and the MRC Immunochemistry Unit (Oxford) forthe cDNA libraries. We also thank A.Kelly, Dr J.Young and Prof. R.Craigfor helpful discussions and C.Sinclair and G.Paine for excellent secretarialassistance.

26

Structure of Ca2+ -binding protein p68

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Received on September 23, 1987; revised on October 30, 1987

Note added in proofThese data have been submitted to the EMBL/GenBank data libraries underthe accession number Y00097.

27