Proc. Natl. Acad. Sci. USAVol. 92,, pp. 5022-5026, May 1995Biochemistry

Molecular cloning and characterization of a conserved nuclearserine(threonine) protein kinase

(signal transduction/protein phosphorylation)

THOMAS MILLWARD, PETER CRON, AND BRIAN A. HEMMINGS*Friedrich Miescher-Institut, Postfach 2543, CH-4002 Basel, Switzerland

Communicated by Martha Vaughan, National Institutes of Health, Bethesda, MD, February 27, 1995 (received for review December 22, 1994)

ABSTRACT Human, Drosophila melanogaster, and Cae-norhabditis elegans cDNA clones encoding homologues of aserine(threonine) protein kinase (EC 2.7.1.37) (designatedNdr protein kinase) have been isolated and sequenced. Thehuman and Drosophila cDNAs predict polypeptides of 54 kDaand 52 kDa, respectively, which share 809o amino acidsimilarity. Northern analysis of human tissues revealed aubiquitously expressed 3.9-kb transcript. Recombinant GST-Ndr underwent intramolecular autophosphorylation onserine and threonine residues in vitro but failed to trans-phosphorylate several standard protein kinase substrates.Transfection of the human cDNA into COS-1 cells resulted inthe appearance ofan intense nuclear staining in cells analyzedby indirect immunofluorescence; deletion mutagenesis iden-tified a short basic peptide, KRKAETWKRNRR, responsiblefor the nuclear accumulation ofNdr. Thus, Ndr is a conservedand widely expressed nuclear protein kinase. The closestknown relative of this previously uncharacterized kinase isDbf2, a budding yeast protein kinase required for the com-pletion of nuclear division.

Reversible protein phosphorylation is a major mechanism forthe coordinated control of many fundamental cellular func-tions in eukaryotic organisms, including metabolism, growth,and differentiation (1). The phosphorylation status, and con-sequently the activity, of specific target proteins is regulated bythe opposing actions of protein kinases and protein phospha-tases. Generally, these enzymes are specific either for serine/threonine or for tyrosine phosphoacceptors, although somedual-specificity kinases and phosphatases have also been de-scribed (2). The importance of phosphorylation cascades isreflected by the finding that many kinases, phosphatases, andthe signal transduction pathways in which they participate havebeen highly conserved during the course of evolution (3). Inrecent years, interest has focused on the role of proteinphosphorylation in the control of the cell cycle (4); a numberof cellular protooncogenes encode members of the serine(threonine) kinase family (5-7) and it has become increasinglyclear that certain serine(threonine) kinases function as keycomponents of the cell cycle regulatory network (8). There-fore, the complete delineation of these pathways is an impor-tant aim for the understanding of oncogenesis and tumorprogression.

In this paper we describe the identification of a humanserine(threonine) protein kinase (EC 2.7.1.37) that is thehomologue of a Caenorhabditis elegans expressed sequence tag(EST) called cmllb8 (9) and which is also highly conserved inDrosophila melanogaster; additionally, we report that this ki-nase is a ubiquitously expressed nuclear protein whose closestknown relative is Dbf2, a serine(threonine) protein kinaserequired for normal progression through the cell cycle in the

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budding yeast Saccharomyces cerevisiae (10). This kinase isreferred to as Ndr (nuclear, Dbf2-related) protein kinase.t

MATERIALS AND METHODSPCR and Molecular Cloning. The C. elegans EST clone

cmllb8 (9) was used to screen a Drosophila embryo cDNAlibrary constructed in AZAPII (Stratagene) at low stringency(11), resulting in the isolation of a 2.1-kb clone, SDE-Ndr-12.Degenerate oligonucleotides were then designed correspond-ing to amino acid sequences conserved between C. elegans andDrosophila for amplification of the human homologue. PCRwas performed on reverse-transcribed total RNA isolatedfrom HeLa cells by the guanidium isothiocyanate method (11)using primers 5'-AATCTAGAAARGAIACIGARTAYYT-IMGIYTIAA-3' (sense) and 5'-AAAAGCTTGGIGCDAT-RTARTCIGGIGTICCIAC-3' (antisense). The PCR productwas subcloned and used to screen several human cDNAlibraries in AZAPII (Stratagene) following standard protocols(11). The PCR product and library cloneswere fully sequencedon both strands using Sequenase (United States Biochemical)and custom-synthesized primers.

Plasmids. Amplifications for mutagenesis were achievedusing Pfu polymerase (Stratagene), and amplified regions weresequenced after subcloning to confirm the absence of addi-tional mutations. The following mutagenic primers were used:P1, 5'-AAGGATCCATGGCAATGACAGGCTC-3'; P2, 5'-GAGTCGTTTCTCCTCATC-3'; P3, 5'-TTTCTGCTTTC-CTTTTG-3'; P4, 5'-ACAAGACTTGGATTGGAAG-3'; P5,5'-CATGCCATGGGATTGGAAGATTTTGAG-3'; P6, 5'-TCTAGCTAGCTGGGAATTCATGTTCTG-3'; P7, 5'-GTTTTCCCAGTCACGACGTTGTAAAACG-3'; P8, 5'-GGAGTATTGCCATTGCAT-3'; P9, 5'-ATGCAATG-GCAATACTCC-3'.pBLM-Ndr was generated by ligation of the overlapping

clones BBZ-Ndr-16b and BBZ-Ndr-3a using the commonXmnI site (nt 1045-1054). The assembled cDNA was excised withHindIl andXba I and transferred to HindIII/Xba I-cut pECE(12) to create pECE-Ndr. pECE-Ndr-A65-81 was obtained byamplifying pBLM-Ndr using the primer pairs P1/P2 andP3/P4. The products were blunt-end ligated to each other andused to replace the corresponding Nco I/EcoRI fragment inpECE-Ndr. To generate pECE-Ndr-A1-84, a region ofpBLM-Ndr was amplified using primers P5 and P3. The amplifiedproduct was digested with Nco I and EcoRI and clonedbetween the corresponding sites in pECE-Ndr. pECE-Ndr-A265-276 was made by amplifying pBLM-Ndr using primers P1and P6. The resulting product was cut with Nco I and Nhe I andligated between the corresponding sites in pECE-Ndr. To

Abbreviations: GST, glutathione S-transferase; NLS, nuclear local-ization signal; EST, expressed sequence tag.*To whom reprint requests should be addressed.tThe sequences reported in this paper (human, D. melanogaster, andC. elegans Ndr protein kinase) have been deposited in the GenBankdata base (accession nos. Z35102, Z35103, and Z34989, respectively).

5022

Proc. NatL Acad Sci USA 92 (1995) 5023

make pGEX2T-Ndr, BBZ-Ndr-16b was amplified with primersP1 and P7. A BamHI/Xmn I fragment of the product wasligated together with the 5' Xmn I/EcoRI fragment of BBZ-Ndr-3a into BamHI/EcoRI-cut pGEX2T (13). The resultantplasmid was cut with EcoRI and ligated to the 3' EcoRIfragment of BBZ-Ndr-3a. To change Lys-118 to Ala, a two-stepPCR procedure was used: pBLM-Ndr was amplified usingprimer pairs P1/P8 and P3/P9. The two products were gelpurified, denatured, annealed to each other, and reamplifiedusing primers P1 and P3. The secondary PCR product was cutwith Nco I and EcoRI and cloned between the correspondingsites in pGEX2T-Ndr to generate pGEX2T-Ndr-K118A.

Northern Analysis. The 2.2-kb Nco I-Bgl II fragment ofpBLM-Ndr was 32P-labeled to a specific activity of -2 x 109cpm/,g by random priming and used to probe human tissueRNA blots (Clontech) as described (14).

Bacterial Expression ofHuman Ndr. Mid-logarithmic phaseJM109 cells containing the appropriate plasmid were inducedwith 0.1 mM isopropyl ,B-D-thiogalactopyranoside overnight atroom temperature. Bacteria were lysed by sonication andfusion proteins were purified on glutathione-agarose (Sigma)as described (14). Recombinant proteins were assayed forkinase activity in 10-35 ,ul of 50mM Tris HCl, pH 7.5/10mMMgCl2/1 mM dithiothreitol containing 100 ,uM [y-32P]ATP(0.5-1 gCi/,ul; 1 Ci = 37 GBq). After 30 min at 30°C, reactionswere resolved by 10% SDS/PAGE and autoradiographed. Forquantitation, gels were exposed to a PhosphorImager andscanned using IMAGEQUANT software (Molecular Dynamics).Phosphoamino acid analysis was carried out as described (15).Transient Overexpression in COS-1 Cells. Cells were incu-

bated in Dulbecco's minimal essential medium (DMEM)containing 0.7 ,ug of plasmid DNA per ml and 7 ,lI ofLipofectin per ml (GIBCO/BRL). After 5 hr, an equal volumeof DMEM/20% fetal calf serum was added. The transfectionwas terminated 12 hr later by replacing the medium with freshDMEM/10% fetal calf serum or by passaging the cells ontoglass coverslips (for immunolocalization). Protein expressionwas analyzed 24 hr later.Immunochemistry. A rabbit antiserum (Ab452-465) was

raised against a synthetic peptide, TARGAIPSYMKAAK,that had been conjugated to keyhole limpet hemocyanin (16).Peptide-specific antibodies were purified on protein A-Sepharose (Pharmacia) followed by Affi-Gel 10 (Bio-Rad) towhich the immunogenic peptide had been coupled. Antibodieswere eluted with 50 mM Tris-HCl, pH 7.4/6 M urea and thendialyzed against phosphate-buffered saline (PBS).Immunocytochemical analysis was performed on COS-1

cells that had been seeded onto acid-washed, poly(lysine)-coated glass coverslips 12 hr after transfection. Cells were fixedin 3.7% paraformaldehyde/PBS for 20 min and then perme-abilized with acetone (-20°C for 30 sec). Coverslips wereincubated sequentially with PBS/3% bovine serum albumin,affinity-purified Ab452-465 (1:5 in PBS/0.2% bovine serumalbumin), biotin/goat anti-rabbit IgG (1:100, Amersham), andstreptavidin/Texas red (1:200, Amersham), all at 370C.Stained cells were viewed with a Leica TCS confocal micro-scope equipped with an argon/krypton laser, and projectionswere assembled from 10 scanned sections of 1 gm.

Immunoblotting was carried out as described (12), usingAb452-15 at a dilution of 1:20. The primary antibody wasdetected using an ECL kit (Amersham).

RESULTS AND DISCUSSIONIdentification ofa Conserved Protein Kinase. The C. elegans

ESTs cmllb7 and cmllb8 are overlapping cDNA clones thatwere originally described (9) as worm homologues of humanRAC kinase (12) and the S. cerevisiae cell cycle-regulatedkinase DBF2 (10), respectively. These homologies were as-signed on the basis of single sequencing reads from the 5' end

of each clone. However, complete sequencing in this labora-tory revealed two identical partial-length cDNAs encoding apredicted serine(threonine) kinase that appeared to be distinctfrom RAC and Dbf2. Therefore, cml1b8 was used as a probeto screen a Drosophila embryo cDNA library at low stringency,resulting in the isolation of a 2.1-kb clone containing a

complete open reading frame of 455 amino acids (Fig. 1). Wenext designed degenerate PCR primers corresponding toamino acid sequences conserved between C. elegans andDrosophila (KETEYLRLK and VGTPDYIAP) to amplify a

region of the human homologue. A single -680-bp productwas amplified using cDNA from HeLa, T47-D, and MCF7 cells(data not shown). The HeLa cell PCR product was subcloned,sequenced, and subsequently used to screen human cDNAlibraries derived from fetal brain, fetal retina, placenta, andadult heart. Partial-length clones were found in each of theselibraries.An overlapping pair of clones (BBZ-Ndr-16b and BBZ-Ndr-

3a) encompassing a complete open reading frame was isolatedfrom the fetal brain library (Fig. 1A). The clones cover a totalof 3018 bp, with a single open reading frame extending fromnt 566 to nt 1990. The first methionine in the open readingframe (at nt 596-598) is surrounded by a nucleotide sequence(CAGCCATGG) that contains 6 of 7 nt in agreement with theKozak consensus for translation initiation (18). Translationfrom this methionine would give a 465-amino acid polypeptidewith a pI of 7.2 and a molecular mass of 54.2 kDa. Alignmentof the deduced amino acid sequence with those from Dro-sophila and from the partial-length C. elegans clone cmllb8(Fig. 1B) reveals many highly conserved regions; the overallamino acid identity between human and Drosophila Ndrprotein kinase is 68%.

Relationship of Human Ndr to Previously CharacterizedProtein Kinases. The Ndr amino acid sequence contains all ofthe 12 protein kinase catalytic subdomains defined by Hanksand Quinn (17); the presence of the sequences DIKPDN(subdomain VIb) and GTPDYIAPE (subdomain VIII) indi-cate that Ndr is likely to have serine/threonine specificity (17).No sequences with homology to SH2, SH3, or PH domains areevident. Alignment of Ndr sequences with those of otherknown protein kinases reveals several interesting characteris-tics. (i) Ndr has an unusual catalytic domain structure, in thattwo catalytic subdomains (VII and VIII) that are contiguousin the primary structure of most other protein kinases areseparated by =30 amino acids in Ndr. This feature is shared bythe S. cerevisiae protein kinases Cdc7 (19), Dbf2 (20), and theDbf2 isoform Dbf2O (21). (ii) The closest previously describedrelatives of Ndr are Dbf2 and Dbf2O, two highly related S.cerevisiae serine(threonine) protein kinases that regulate nu-clear division and DNA synthesis (32% overall amino acididentity, 39% identity in the catalytic domain). Temperature-sensitive mutants of DBF2 show delayed initiation of DNAreplication at the restrictive temperature and arrest in latemitosis with a "dumbbell" morphology due to a failure tocomplete nuclear division. In normal cells, Dbf2 transcriptlevels and kinase activity fluctuate during the cell cycle in amanner consistent with these functions (10). However, thesimilarity between human Ndr and Dbf2 is much lower thanthat between human Ndr and Drosophila Ndr (68% identity),suggesting that Ndr and Dbf2 are related, but not specieshomologues. Possibly, Dbf2 and Ndr represent distinct mem-bers of a common subfamily.

Expression of Ndr in Human Tissues. Northern analysisrevealed an -3.9-kb transcript in all tissues analyzed (Fig. 2),with the possible exception of adult brain. The mRNA isparticularly abundant in peripheral blood leukocytes. Thepresence of the Ndr transcript in several cell lines tested byPCR from cDNA, and in all human cDNA libraries screened,is consistent with the idea that Ndr is a widely expressedenzyme. Taken together with its conservation across divergent

Biochemistry: Millward et aL

5024 Biochemistry: Millward et at

2A 3 kb

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----- HeLa PCRHuman BBZ-Ndr-16b

BBZ-Ndr-3a

B h-Ndrd-Ndrc-NdrDbf 2

h-Ndrd-Ndrc-NdrDbf2

h-Ndrd-Ndrc-NdrDbf2

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h-Ndrd-Nddrc-NdrDbf2

h-Ndrd-Ndrc-NdrDOf2

MMSSRTQDADGASIRF --

... DTDVSSPKXLPPXFXYKRA2R40RVESVC(YFLEYhCDK

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c-Ndr --A P 18.Dbf2 EXKE e EQS 7

VIII L IX36n ~~~~~~m -~~~~369

h-Nd3r 2 ,X dd-Ndr DNER=I= -_c-Ndr I 0---DOf2 S ELDGQBDRQ T

d-tr AS -- - ILL

h-Ndr A-LKtVASMBP-d-Ndr AS t; VX I Vc-Ndlr NU g Q $1 Rt- V VDb f2; --RS F NST SI PPFTPQIl

h-Ndr L NH ---3 * < =d-Ndr BI APIPQGcS-----IA---c-Ndr PTLPB-----QPGRRGEGV--DDbf2 -MAKAADV1VRSDKLSAM¶DAnSSstLva S

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FIG. 2. Northern analysis of Ndr expression in human tissues.Lanes contain 2 Mug of poly(A)+ RNA from the indicated tissues. Blotswere washed to a stringency of 1x SSC/0.1% SDS at 60°C. Positionsof molecular size markers, in kb, are indicated.

37PIw

eukaryotes, this further implies that Ndr plays an important1FDY role in an essential cellular function.83 Ndr Enzyme Activity. For the in vitro characterization ofNdr

protein kinase activity, the human cDNA was cloned into them- bacterial expression vector pGEX2T to generate a glutathionetIMK S-transferase (GST)-Ndr fusion protein. As a negative control,

a mutant form of Ndr was generated in which Lys-118 waschanged to Ala. Lys-118 corresponds to the invariant Lys of all

A

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0.5M GST-Ndr (WT)5Ag GST5pg GST-Ndr (Kl 18A)

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0 20 40 60

Time (min)465 Catalytic domain i.detit3y

h-Ndr ARQAXPSYNMAM* 100%d-Ndr LE* 77%c-Ndr QIMRYSDJ&M.... 76%Dhf2 BSDPFSSTF... 39%

FIG. 1. Molecular cloning of Ndr protein kinase from three species.(A) Schematic representation of Ndr mRNA and corresponding cDNAclones from C. elegans, Drosophila embryo, HeLa cells, and human fetalbrain. The predicted coding region is shown as a box, with 5' and 3'untranslated regions indicated by lines; the kinase catalytic domain isshaded. (B) Comparison of the amino acid sequence of human Ndr(h-Ndr) with that of Drosophila Ndr (d-Ndr), the partial sequence fromC. elegans (c-Ndr), and amino acids 66-560 of Dbf2. Amino acids that areidentical between human Ndr and one or more of the other sequences arehighlighted. Numbers at the right correspond to the human Ndr sequence.Stop codons are represented by asterisks. Roman numerals indicate theprotein kinase catalytic subdomains defined by Hanks and Quinn (17);the region between subdomains VII and VIII does not align withpreviously defined catalytic subdomains of protein kinases. "Ndr NLS"marks the position of the minimal Ndr nuclear localization signal definedby deletion analysis (see text).

FIG. 3. Enzyme activity of recombinant human Ndr protein kinase.(A) GST-Ndr (WT) and GST-Ndr (K118A) were subjected to 10%SDS/PAGE and then stained with Coomassie blue. Each lane contains3 jig of protein. Molecular mass markers (in kDa) are indicated. (B)One microgram of GST-Ndr (WT) or GST-Ndr (K118A) was assayedfor autophosphorylation for 30 min; reactions were resolved by 10%SDS/PAGE followed by autoradiography. (C) After autophosphory-lation, 32P-labeled GST-Ndr (WT) was hydrolyzed for 1 hr in 6M HCIat 110°C and then electrophoresed at pH 1.9 (right to left) and at pH3.5 (bottom to top). The plate was exposed to a Phosphorlmager.Positions of ninhydrin-stained standards are shown (pY, phosphoty-rosine; pS, phosphoserine; pT, phosphothreonine). (D) Autophos-phorylation of GST-Ndr (WT) was measured as a function of time (e,enzyme concentration = 0.2 ,ug/,ul) and of concentration (*, reactiontime = 30 min; total protein concentration was kept at 0.2 ,xg/p±l usingfree GST; error bars represent the SD of triplicate determinations).Quantitation was achieved by Phosphorlmager scanning. (E) Combi-nations of GST, GST-Ndr (WT), and GST-Ndr (K118A) as indicatedwere subjected to kinase assay (30 min; reaction volume = 35 ,l) andthen analyzed by SDS/PAGE and autoradiography.

_ _- _- _ __ _ _ _____-_'-_--_w_ Ya A5-Ld

Proc. Natl Acad ScL USA 92 (1995)

1

Proc. Natl. Acad Sci USA 92 (1995) 5025

protein kinases that contacts the a and 13 phosphates of ATPand is essential for catalysis (22). In both cases, purification onglutathione-agarose yielded proteins of the expected size (-82kDa; Fig. 14). About 50% of the purified protein was full-length; the minor species migrating at 28-35 kDa are assumedto be degradation products of the fusion proteins, sincepurification of free GST under identical conditions yieldedvirtually homogenous protein.

Initially, GST-Ndr was tested in kinase assays using variousnonspecific kinase substrates, including histone Hi, myelinbasic protein, casein, and phosvitin; no phosphorylation of anyof these exogenous substrates could be detected (data notshown). However, phosphorylation of an -82-kDa band wasconsistently observed. This was subsequently shown to repre-sent autophosphorylation of GST-Ndr, since a point mutationin the catalytic domain of Ndr abolishes this phosphorylation(Fig. 3B). Phosphoamino acid analysis of in vitro autophos-phorylated Ndr revealed the presence of phosphoserine andphosphothreonine (Fig. 3C). Thus, recombinant Ndr is anactive serine(threonine) protein kinase and can undergo au-tophosphorylation on at least two sites. These two sites arelikely to be located within Ndr itself, since the fusion proteinwas not able to phosphorylate free GST.The inability of GST-Ndr to phosphorylate a number of

protein substrates widely used in kinase assays may be anindication that Ndr has a restricted substrate specificity com-pared to other protein kinases, as was observed in the case ofMAPKAP kinase 2 (23). The same enzymatic characteristics(i.e., autophosphorylation, lack of activity toward standardkinase substrates) were observed using Ndr immunoprecipi-tated from transfected overexpressing COS-1 cells (data not

shown). Alternatively, activity toward exogenous substratesmay be absolutely dependent on a posttranslational modifica-tion, such as phosphorylation/dephosphorylation or associa-tion with an activating subunit. A third possibility is that Ndrrequires an as yet unidentified second messenger or cofactorfor activity toward exogenous substrates.

Autophosphorylation of GST-Ndr was linear for at least 60min (Fig. 3D); moreover, when assayed under linear reactionconditions (30 min) phosphate incorporation was observed tobe independent of enzyme concentration (Fig. 3D), implyingan intramolecular mechanism of autophosphorylation. Thisconclusion was supported by the finding that autophosphory-lation could not be stimulated by the addition of a 10-foldexcess of inactive GST-Ndr (K118A) to the active enzyme (Fig.3E). Given that autophosphorylation of Ndr is intramolecular,linear phosphate incorporation over a period of 60 minsuggests a very slow reaction rate and thus further reinforcesthe notion that Ndr may require some form of activation forits full protein kinase activity.

Expression and Localization ofHuman Ndr in COS-1 Cells.To express Ndr in COS-1 cells, the overlapping fetal braincDNA clones (Fig. iB) were ligated at a common restrictionsite and subcloned into the simian virus 40-based expressionvector pECE (12). The overexpressed protein was then de-tected using an affinity-purified polyclonal antiserum raisedagainst the predicted carboxyl terminus of the protein.

Transfection of the human cDNA into COS-1 cells led to theappearance of an -55-kDa immunoreactive polypeptide onWestern blots of whole-cell lysates (Fig. 4A, lane 2). Thisspecies was normally not observed in lysates from cells trans-fected with the pECE vector alone, although extreme overex-

FIG. 4. Expression and immunolocalization of wild-type and mutant Ndr proteins in COS-1 cells. (A) Western blot of whole-cell lysates (40 i&gper lane) from COS-1 cells transfected with pECE (lane 1) or with derivative Ndr expression plasmids as indicated (lanes 2-5). Positions of molecularsize markers (kDa) are indicated. (B-F) The localization of wild-type and mutant polypeptides was analyzed by indirect immunofluorescence: cellswere transfected with pECE-Ndr (B), pECE only (C), pECE-Ndr-A65-81 (D), pECE-Ndr-A1-84 (E), or pECE-Ndr-A265-276 (F). (Bars = 10 Am.)

Biochemistry: Millward et aL

5026 Biochemistry: Millward et aL

posure of blots revealed a weak -55-kDa band in mock-transfected cells that comigrated with the overexpressed pro-tein from transfected cells. Using the same antibody, cells wereanalyzed by indirect immunofluorescence to assess the sub-cellular localization of Ndr. Cells transfected with the humancDNA showed an intense nuclear staining and a weakercytoplasmic signal (Fig. 4B). The majority of the staining wasspecific under the conditions used, since mock-transfectedcells showed only a very weak background staining (Fig. 4C).

Inspection of the Ndr amino acid sequence suggests severalconserved clusters of basic amino acids that could be potentialNLSs. The amino-terminal domain contains a sequence,RRSAHARKETEFLRLKR (amino acids 65-81), that fits tothe bipartite nuclear targeting motif defined by Dingwall andLaskey (24). Moreover, the amino-terminal domain as a whole(amino acids 1-84) contains a rather high proportion of basicresidues (the pI of this region is - 10). Lastly, the insertsequence between kinase subdomains VII and VIII contains astrongly basic peptide, KRKAETWKRNRR (amino acids265-276). Therefore, plasmids were constructed to expressmutant forms of Ndr lacking these sequences (Fig. 4A).Deletion of amino acids 65-81, containing the consensus

bipartite motif, had no effect on the nuclear accumulation ofNdr (Fig. 4D); similarly, deletion of the entire amino-terminaldomain did not reduce nuclear uptake (Fig. 4E). Therefore,these sequences do not appear to play a role in the nuclearlocalization of Ndr. However, deletion of amino acids 265-276in the catalytic domain insert led to a significant redistributionof the expressed protein (Fig. 4F). Instead of an intensenuclear signal, cells showed a more diffuse pattern of staining.In many cells, the nuclei were visible as darker regions againstthe cytoplasm. The expressed protein was not completelyexcluded from the nuclei (exclusion from the nucleoli is stillvisible), but this maybe explained by an ability to diffuse slowlyinto the nucleus, as the size of the deletion mutant is near thecutoff size (40-60 kDa) for passive nuclear entry (25). Thus,the NLS of Ndr appears to be contained within the peptideKRKAETWKRNRR.The identification of the Ndr NLS raises the question ofwhy

the consensus bipartite sequence is not active, while thenonconsensus NLS is. One explanation might be that thebipartite motif does not lie in an appropriate context. Thesurrounding sequence context is known to influence theactivity of NLSs; for example, the NLS of p53 directs p-ga-lactosidase to the nucleus when fused to its carboxyl terminus,but the same NLS fused to the amino terminus of ,3-galacto-sidase is inactive (26). There is also reason to believe that activeNLSs usually lie in nonhelical regions of proteins, since theyare frequently flanked by helix-destabilizing residues such asPro and Gly (27). Computer prediction of secondary structuresuggests that the bipartite basic sequence of Ndr is likely tooccupy an a-helical region. In agreement with this, the regionamino-terminal to the catalytic domain in cAMP-dependentprotein kinase is an extended a-helix (28), and this structuralfeature is believed to occur frequently in protein kinases ingeneral (29). In contrast, the nonconsensus active NLS of Ndris predicted to reside at a turn and lies near to what has beentermed the "lip" region of protein kinases (30); thus, byanalogy with the structures of cAMP-dependent protein ki-nase and ERK2 (31), the active Ndr NLS may constitute partof an extended L12 loop.

The presence of Ndr protein kinase in the nucleus ofmammalian cells suggests that it may function in the regulationof some nuclear event and that its physiological substrate(s)may also be nuclear. These possibilities are interesting in thelight of its relatedness to Dbf2, a budding yeast protein kinase

necessary for the completion of mitosis. Future work must bedirected toward identification of the substrates and regulatoryproteins that interact with Ndr in vivo. In addition, theavailability of cDNAs from Drosophila and C. elegans wfllgreatly facilitate genetic dissection of the function of Ndr.

We thank Dr. Alan Coulson for C. elegans EST clones, Dr. StanZolnierowicz for providing cDNA from cell lines, Drs. Jorg Hagmannand Kurt Ballmer for extensive help with confocal microscopy, Dr. PatDennis for help with phosphoamino acid analysis, Franz Fischer forpeptide synthesis and purification, and Georg Aeschbacher and PeterMuller for oligonucleotides. We are also indebted to Drs. KurtBallmer, Rick Pearson, and Evan Ingely for comments on the manu-script.

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Proc. NatL Acad ScL USA 92 (1995)