Cloning and Characterization of the Murine Nek3 Protein Kinase, a Novel Member of the NIMA Family of Putative Cell Cycle Regulators

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Cloning and Characterization of the Murine Nek3 Protein Kinase, a Novel Member of the NIMA Family of Putative Cell Cycle Regulators

TANAKA, Kayoko, NIGG, Erich A.

Abstract

We have cloned and characterized murine Nek3 (NIMA-related kinase 3), a novel mammalian gene product structurally related to the cell cycle-regulatory kinase NIMA of Aspergillus nidulans. By RNase protection, low levels of Nek3 expression could be detected in all organs examined, regardless of proliferative index. In contrast to Nek1 and Nek2, Nek3 levels were not particularly elevated in either the male or the female germ line. Nek3 levels showed at most marginal variations through the cell cycle, but they were elevated in G0-arrested, quiescent fibroblasts. Furthermore, no cell cycle-dependent changes in Nek3 activity could be detected, and no effects upon cell cycle progression could be observed upon antibody microinjection or overexpression of either wild-type or catalytically inactive Nek3. Finally, Nek3 was found to be a predominantly cytoplasmic enzyme. These data indicate that Nek3 differs from previously characterized Neks with regard to all parameters investigated, including organ specificity of expression, cell cycle dependence of expression and activity, and subcellular localization. Hence, the structural [...]

TANAKA, Kayoko, NIGG, Erich A. Cloning and Characterization of the Murine Nek3 Protein Kinase, a Novel Member of the NIMA Family of Putative Cell Cycle Regulators. Journal of Biological Chemistry , 1999, vol. 274, no. 19, p. 13491-13497

DOI : 10.1074/jbc.274.19.13491

Available at:

http://archive-ouverte.unige.ch/unige:129363

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Cloning and Characterization of the Murine Nek3 Protein Kinase, a Novel Member of the NIMA Family of Putative Cell Cycle Regulators*

(Received for publication, November 30, 1998, and in revised form, January 26, 1999)

Kayoko Tanaka‡ and Erich A. Nigg§

From the Department of Molecular Biology, Sciences II, University of Geneva 30, Quai Ernest-Ansermet, 1211 Geneve 4, Switzerland

We have cloned and characterized murine Nek3 (NIMA-related kinase 3), a novel mammalian gene prod- uct structurally related to the cell cycle-regulatory ki- nase NIMA of Aspergillus nidulans. By RNase protec- tion, low levels of Nek3 expression could be detected in all organs examined, regardless of proliferative index.

In contrast to Nek1 and Nek2, Nek3 levels were not particularly elevated in either the male or the female germ line. Nek3 levels showed at most marginal varia- tions through the cell cycle, but they were elevated in G₀-arrested, quiescent fibroblasts. Furthermore, no cell cycle-dependent changes in Nek3 activity could be de- tected, and no effects upon cell cycle progression could be observed upon antibody microinjection or overex- pression of either wild-type or catalytically inactive Nek3. Finally, Nek3 was found to be a predominantly cytoplasmic enzyme. These data indicate that Nek3 dif- fers from previously characterized Neks with regard to all parameters investigated, including organ specificity of expression, cell cycle dependence of expression and activity, and subcellular localization. Hence, the struc- tural similarity between mammalian Neks may not nec- essarily be indicative of a common function, and it is possible that some members of this kinase family may perform functions that are not directly related to cell cycle control.

Cyclin-dependent kinases (Cdks)¹are well established as key regulators of the eukaryotic cell cycle (1–3). In addition, genetic data demonstrate a cell cycle-regulatory role for several protein kinases that are not regulated by associated cyclin subunits.

One such cyclin-independent kinase, termed NIMA (for never in mitosis, gene A), was identified in the filamentous fungus Aspergillus nidulans, and shown to play a crucial role at the G₂ to M transition (4, 5). InAspergillus, lack of NIMA function results in a cell cycle arrest in G₂(6, 7), while overexpression of thenimA gene causes the premature onset of mitotic events,

such as depolymerization of cytoplasmic microtubules, forma- tion of abnormal mitotic spindles, and chromatin condensation (4, 8). Moreover, the overexpression of NIMA in heterologous organisms and cell types was reported to result in untimely chromatin condensation, reminiscent of the effects produced by NIMA overexpression in Aspergillus (9, 10). In view of the evolutionary conservation of Cdks and other important cell cycle regulators, these data suggested that regulatory cascades involving NIMA-related kinases might be conserved in higher eukaryotes (11).

A search for potential NIMA homologues has led to the description of NIMA-related kinases (Neks) from several spe- cies. These include Kin3p/Npk1p fromSaccharomyces cerevi- siae(12, 13), fin1 fromSchizosaccharomyces pombe (14), and the mammalian kinases Nek1, Nek2, Nek3, and Stk2 (15–17).

However, the only kinase shown to functionally complement a nimA ts mutation inAspergillusis NIM-1, isolated from the related filamentous fungus Neurospora crassa(18). Thus, although all the NIMA-related kinases described above share considerable sequence similarity with NIMA, one critical, un- resolved question concerns the functional relationships between these kinases and NIMA. Another important task will be to determine to what extent the mammalian Neks (Nek1, Nek2, Nek3, and Stk2) share common or overlapping functions.

Mammalian Neks display a high degree of sequence similarity over the N-terminal catalytic domains, but, at the level of primary structure, they show little if any conservation over the C-terminal noncatalytic domains (19). In order to assess whether the multiple mammalian Neks are likely to perform divergent, similar, or redundant functions, it is therefore essential to carry out biochemical studies on the individual family members.

The mammalian kinase most closely related to NIMA is Nek2, and accordingly, Nek2 has received most attention so far. Similar to NIMA, Nek2 is cell cycle-regulated in terms of both abundance and activity (17, 20). However, whereas NIMA has been implicated predominantly in chromatin condensation (11), recent studies indicate that one important function of Nek2 relates to the centrosome cycle (21). Nek2 localizes to the centrosome, and overexpression of active Nek2 profoundly af- fects centrosome structure in cultured cells. Nek2 is highly expressed in testis (22–24), and the same is true for Nek1 (15).

Interestingly, however, Nek1 and Nek2 display different expression patterns during spermatogenesis, suggesting that they may perform distinct functions.

We have previously isolated a partial cDNA for human Nek3 (17), but this cDNA lacked the coding information for the N- terminal end domain, and no biochemical studies on the Nek3 protein have been reported. Here, we describe the molecular cloning and biochemical characterization of a 56-kDa murine protein kinase, termed mNek3, that almost certainly repre-

* This work was supported in part by Swiss National Science Foun- dation Grant 31-50 576.97 and by the Canton of Geneva. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement”

in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EBI Data Bank with accession number(s) AF093416.

‡ Supported during the earliest stages of this project by a postdoctoral fellowship from the Swiss Institute for Experimental Cancer Reseach.

Present address: University of Manchester, Manchester M13 9PT, United Kingdom.

§ To whom correspondence should be addressed. Tel.: 41-22-702- 6127; Fax: 41-22-702-6868; E-mail: erich.nigg@molbio.unige.ch.

1The abbreviations used are: Cdk, cyclin-dependent kinase; BrdUrd, bromodeoxyuridine; PAGE, polyacrylamide gel electrophoresis; FCS, fetal calf serum; Nek, NIMA-related kinase.

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sents the mouse homologue of human Nek3. Using cDNA probes and monospecific anti-Nek3 antibodies, we have studied several basic properties of this kinase. In particular, we have asked whether Nek3 is likely to play a role in cell cycle regulation, and to what extent its properties might resemble those determined recently for Nek2. Our results do not rigorously exclude an involvement of Nek3 in cell cycle control, but they provide no positive evidence to support such a role. Moreover, they clearly show that the properties of Nek3 differ profoundly from those determined previously for Nek2. Thus, despite their structural similarities, mammalian NIMA-related kinases may well perform widely different functions.

MATERIALS AND METHODS

cDNA Cloning and Mutagenesis—A mouse brain cDNA library in lZap II (Stratagene) was screened with a human Nek3 cDNA fragment (XhoI-XbaI, nucleotides 617–1555; Ref. 17), labeled with [a-³²P]dCTP (NEN Life Science Products). Phages were converted into pBlueScript (SK2) byin vivoexcision, and inserts were sequenced. One plasmid, termed pBS-mNek3, was found to contain the entire coding sequence of mouse Nek3. APvuII-PvuII fragment excised from pBS-mNek3 was inserted into thePstI site of a pBluescript-myc vector carrying the myc epitope tag (25), to yield pBS-myc-mNek3. Myc-tagged mNek3 was then excised as aHindIII-XbaI fragment and inserted into the eukaryotic expression vector pRcCMV (Invitrogen Corp.), to generate pCMV-myc- mNek3. A catalytically inactive mNek3 mutant (mNek3-D143A) was prepared by changing Asp-143 to Ala, using the Transformer site- directed mutagenesis kit (CLONTECH).

Tissue Protein Extractions and RNA Techniques—For preparation of protein extracts, testis tissue from 5-week-old mice was homogenized in lysis buffer (50 mMHepes/KOH pH 7.4, 5 mMMnCl2, 10 mMMgCl2, 2 mMEDTA, 100 mMNaCl, 5 mMKCl, 0.1% Nonidet P-40, 30 mg/ml DNase I, 1 mMphenylmethylsulfonyl fluoride, 10mg/ml leupeptin, 10 mg/ml pepstatin A, 1% aprotinin, 20 m^Mb-glycerophosphate, 20 m^M sodium fluoride, 0.3 mMsodium orthovanadate, and 2mg/ml heparin).

Protein extracts from mouse intestine were prepared in the same way, except that, prior to homogenization in lysis buffer, intestines were thoroughly rinsed with ice-cold phosphate-buffered saline containing protease inhibitors (1 mMphenylmethylsulfonyl fluoride, 10mg/ml leupeptin, 10mg/ml pepstatin A, 1% aprotinin). Following the determina- tion of protein concentration, each sample was mixed with an equal volume of gel sample buffer and boiled for 10 min. RNA purification and RNase protection assays were performed as described (23). An anti- sense RNA probe was prepared by transcribing a cDNA fragment from pBS-mNek3 (nucleotides 738 –1145; GenBank™ AF093416).

Immunochemical Techniques—The affinity-purified anti-Nek2 anti- body PepN2 (23), the anti-small nuclear ribonucleoprotein monoclonal antibody D5 (26), and the affinity-purified anti-Cdc2 antibody AR8 (27) were described previously, and the anti-a-tubulin monoclonal antibody was obtained commercially (Amersham Pharmacia Biotech). A rabbit antibody (PepN3) was raised against a synthetic 29-amino acid peptide (pepN3), corresponding to residues 424 – 452 of human Nek3 (17). This peptide was coupled to keyhole limpet hemocyanin and injected into rabbits (Zymed Laboratories Inc.). Antibodies were affinity-purified, using the pepN3 peptide coupled to an insoluble support (Zymed Lab- oratories Inc.). A mouse monoclonal antibody, 5F2G6, was prepared using the same pepN3 peptide for immunization (Zymed Laboratories Inc.). Peptide competition experiments were carried out as described (23), using the antigenic peptide pepN3 or a structurally unrelated peptide, pepN2 (Zymed Laboratories Inc.), for control. Immunoblotting was performed using primary antibodies at 1mg/ml (23), and chemilu- minescence (ECL; Amersham Pharmacia Biotech) for detection.

Cell Culture, Cell Synchronization, and Transfections—Swiss 3T3 mouse fibroblasts and U2OS human osteosarcoma cells were grown in Dulbecco’s modified Eagle’s medium containing 10% fetal calf serum (FCS) and penicillin-streptomycin (100 IU/ml and 100mg/ml, respectively). For G0arrest, cells were serum-starved for 48 h in Dulbecco’s modified Eagle’s medium without FCS. Cells were synchronized at G1/S by a 24 h addition of medium containing 20% FCS and aphidicolin (5 mg/ml) to G0-arrested cells, and at S phase by a 4-h release of aphidicolin-arrested cells into drug-free medium. M phase-arrested cells were collected by gentle pipetting after a 7-h incubation in nocodazole (0.4 mg/ml, Sigma), and G1cells were obtained by a 5-h release from the nocodazole block. Cell cycle profiles were determined by flow cytometric analysis of DNA content (20), using a Becton Dickinson FACScan and Lysis II software. Transient transfections were performed on U2OS

cells seeded onto HCl-treated glass coverslips at a density of 1310⁵ cells per 35-mm dish with 5mg of plasmid DNA, using calcium phosphate precipitates as described (28).

Preparation of Cell Extracts and Subcellular Fractionation—Swiss 3T3 cells were lysed on ice for 10 min in NEB buffer (50 mMHepes/

KOH, pH 7.4, 5 mMMnCl2, 10 mMMgCl2, 2 mMEDTA, 100 mMNaCl, 5 mMKCl, 30mg/ml DNase I, 0.1% Nonidet P-40, 0.1% deoxycholic acid, 20 mMb-glycerophosphate, 20 mM sodium fluoride, 0.3 mM sodium orthovanadate, 2mg/ml heparin, 1 m^Mphenylmethylsulfonyl fluoride, 10mg/ml leupeptin, 10mg/ml pepstatin A, 1% aprotinin), and lysates were clarified by centrifugation at 12,000 3 g for 10 min at 4 °C.

Nuclear and cytoplasmic fractions from G0-arrested Swiss 3T3 cells were prepared as described previously (29).

Immunofluorescence Microscopy—Immunofluorescence microscopy was performed as described (21), except that cells were fixed with 3%

formaldehyde in phosphate-buffered saline for 10 min at room temper- ature, followed by permeabilization with acetone for 30 s at220 °C. The anti-myc monoclonal antibody 9E10 was used as undiluted hybridoma supernatant, whereas affinity-purified PepN3 antibodies were used at 10mg/ml.

Immunoprecipitations and in Vitro Kinase Assays—In vitrotranslated mNek3 or whole cell lysates were diluted 10 times with NEB buffer, and subjected to immunoprecipitation using anti-Nek3 antibody (5F2G6) or nonspecific mouse IgG (Sigma), both at 20 mg/ml, and immune complexes were isolated using protein G-Sepharose beads (Amersham Pharmacia Biotech). Immunoprecipitates were washed three times with 0.53NEB buffer and twice with 50 mMHepes/KOH (pH 7.4), 5 mMMnCl2, and incubated for 30 min at 30 °C in a total volume of 30ml of 50 m^MHepes/KOH (pH 7.4), 5 mMMnCl2, 5 mM

b-glycerophosphate, 5 mMNaF, 7.5mg/ml heparin, 1 mMdithiothreitol, 0.5m^MATP, 5mCi of [g-³²P]ATP (3000 Ci/mmol, Amersham Pharmacia Biotech), in the presence of casein as a substrate (1.4 mg/ml). Kinase reactions were stopped by addition of 0.5 volumes of gel sample buffer.

Samples were boiled for 5 min, and proteins separated by SDS-PAGE.

Following Coomassie Blue staining, gels were dried and exposed to RX film (Fuji Photo Film).

RESULTS

Cloning and Sequencing of a Murine Nek3 cDNA—To isolate the mouse homologue of human Nek3, a mouse brain cDNA library was screened with a human Nek3 cDNA fragment. Of two independent phages isolated from 10⁶plaques, one carried a cDNA insert that encompassed the other. Sequencing of the longer cDNA revealed a single open reading frame, coding for a 511-amino acid protein (GenBank™ accession number AF093416). As described below, this protein almost certainly represents the murine homologue of human Nek3, and, therefore, we refer to it as murine Nek3 (mNek3). Although the presumptive 59-untranslated region of this cDNA lacks an in- frame stop codon, we believe that the first methionine represents the initiating methionine of mNek3, and that the isolated cDNA codes for the entire mNek3 protein. First, the nucleotide sequence surrounding this methionine conforms to the Kozak consensus sequence for initiation methionines (30). Second, and more importantly, thein vitrotranslated mNek3 protein was found to comigrate exactly with the endogenous mNek3 protein detectable by anti-Nek3 antibodies in Swiss 3T3 whole cell lysates (see Fig. 3A).

The mNek3 protein displays a typical serine/threonine protein kinase domain within its N-terminal half. Data base searches revealed that this domain shares greatest similarity with NIMA-related kinases, as expected. Within the C-terminal non-catalytic region, however, there were no obvious similarities to any known proteins, except for human Nek3. As shown in Fig. 1, mNek3 shows 74% overall identity to human Nek3. The published human Nek3 sequence lacks the first two kinase subdomains, but over the remainder of the catalytic domain, human and murine Nek3 show 90% identity. In comparison, the catalytic domain of mNek3 shows significantly lower degrees of identity to murine Nek1 (55%), Nek2 (43%), or Stk2 (44%), and it shows 42% identity withA. nidulansNIMA.

Concerning the non-catalytic C-terminal end domains of

Cloning and Characterization of Murine Nek3 Protein Kinase 13492

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NIMA-related kinases, it is striking that both murine and human Nek3 lack predicted coiled-coil regions. This contrasts with the evolutionary conservation of coiled-coil regions in other NIMA-related kinases (19), includingAspergillusNIMA itself (4), mammalian Nek1 and Nek2 (15, 17), budding yeast Kin3p/Npk1p (12, 13), and fission yeast fin1 (14).

Expression of mNek3 in Adult Mouse Organs—To study the organ specificity of the expression of mNek3 mRNA, RNase protection assays were performed on total RNA prepared from various mouse organs, i.e.liver, lung, kidney, spleen, brain, heart, small intestine, large intestine, ovary, testis, skin, and thymus. As an internal control, the expression of the transcription factor NF-Ya was analyzed in parallel. This factor had previously been shown to be expressed in a fairly uniform manner among different types of cells (31). When DNA-equivalent amounts of RNA from the different organs were analyzed (Fig. 2A), the greatest abundance of Nek3 mRNA was detected in small intestine (IS). Much weaker signals were obtained for most other organs, including testis. It seems, therefore, that no correlation exists between Nek3 expression levels and the proliferative index of a particular tissue. Overall, Nek3 transcript levels appeared to be roughly comparable to those of NF-Ya, which has previously been estimated to be present at approximately 10 transcripts per cell (31). This suggests that Nek3 mRNA is expressed at relatively low levels in all organs analyzed here.

It is important to bear in mind that the amount of total RNA per DNA equivalent differs widely among different cell types (31). When the amounts of Nek3 transcripts were standardized to equal amounts of RNA (Fig. 2B), rather than to DNA equivalents of RNA (Fig. 2A), qualitatively similar results were obtained, although the differences between organs were quan-

titatively less pronounced (compare Fig. 2Bwith 2A); again, highest levels of Nek3 mRNA were seen in intestine, while testis and ovaries showed no particularly high levels of Nek3 expression. This latter result is in striking contrast to data reported previously for murine Nek1 and Nek2. Both of these kinases are in fact highly expressed in testis, although their precise expression patterns during spermatogenesis differ (15, 22–24).

To extend the above expression studies to the protein level, an antibody (PepN3) was raised against a peptide that is highly conserved between mouse and human Nek3 (see Fig. 1). When tested by immunoblotting on total extracts from G₀-arrested Swiss 3T3 cells, the PepN3 antibody recognized a single protein migrating at approximately 56 kDa (Fig. 3A,lane 3). This band comigrated exactly with in vitro translated mNek3 (Fig. 3A, lanes 1and2), confirming that the coding sequence reported here almost certainly represents the complete mNek3 protein.

The PepN3 immune reaction was completely abolished by pre- incubation of PepN3 with the immunogenic peptide, pepN3, but not by incubation with a control peptide, pepN2 (Fig. 3A, lanes 4and5, respectively). Finally, a single band migrating at the exact same position was also revealed when immunoblots were performed on Swiss 3T3 total cell lysates with the monoclonal anti-Nek3 antibody 5F2G6 (Fig. 3A,lane 6).

Fig. 3B shows the abundance of Nek3 protein in murine intestine and testis, respectively (central panel). For comparison, the left-hand panel shows the protein profiles of these samples, as revealed by Coomassie Blue staining, confirming equal loading. Theright-hand panelshows the results of prob- ing the same samples with antibodies against Nek2, one of the Neks shown previously to be expressed predominantly in testis (22–24). From these data it is clear that Nek3 is expressed at comparable levels in both testis and intestine, in striking contrast to Nek2. It is also noteworthy that the Nek3 expression seen at the protein level does not strictly parallel that seen at the mRNA level (compare Figs. 3Band 2A). This suggests that posttranscriptional mechanisms may contribute to control the FIG. 1.Sequence comparison between mouse and human Nek3

proteins.The alignment was generated using the BESTFIT program from the University of Wisconsin GCG sequence analysis software package. Identical residues are indicated byvertical lines, similar residues by either one or two dots (Similarity according to BESTFIT default settings). Residues that are highly conserved in the catalytic domains of serine/threonine protein kinases are indicated byasterisks (42).

FIG. 2.Nek3 mRNA expression in mouse organs.A, RNase protection analysis of Nek3 mRNA expression in mouse organs. The amounts of RNA corresponding to 10-mg DNA equivalents of each tissue were calculated as described previously (23). Each RNA sample was then supplemented to 100mg with yeast RNA and hybridized with 10 fmol of both Nek3 and NF-Ya probes. The control contained 100mg of yeast RNA and 10 fmol of each probe. All organs except thymus were isolated from 4-month-old adult mice. Thymus was isolated from 3-week-old mice. Abbreviations: Li, liver; Lu, lung; Ki, kidney; Sp, spleen;Br, brain;He, heart;IS, small intestine;IL, large intestine;Ov, ovary;Te, testis;Sk, skin;Th, thymus.B, histogram showing quantifi- cation of data in relation to RNA (rather than DNA equivalents).

Signals obtained by RNase protection (seepanel A) were scanned and normalized to the amounts of RNA loaded in each lane.

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abundance of Nek3 protein in particular cell types.

Nek3 Protein Expression during the Cell Cycle—As NIMA- related kinases might be expected to play a role in cell cycle regulation, we examined the protein level of mNek3 throughout the cell cycle. To this end, extracts were prepared from synchronized Swiss 3T3 fibroblasts and analyzed by immunoblotting with the PepN3 antibody. Cells were synchronized in G₀by serum starvation, at the G₁/S phase boundary by aphidicolin treatment, in S phase by release from the aphidicolin block, in M phase by nocodazole treatment, and in early G₁ phase by release from the nocodazole block. Exponentially growing cells were analyzed in parallel. Cell extracts were normalized for protein content, before the amounts of mNek3 were determined by immunoblotting (Fig. 4A,upper panel). For control, aliquots of each sample were also probed with antibodies against the Cdc2 protein kinase (Fig. 4A,lower panel), and subjected to analysis by flow cytometry (Fig. 4B). As shown in Fig. 4A, mNek3 protein levels displayed at most minor variations throughout the cell cycle, and no evidence could be obtained for mobility shifts that might be indicative of posttranslational modifications. As expected, levels of Cdc2 protein were also constant throughout the cell cycle (27). However, whereas Cdc2 protein was virtually absent from G₀-arrested, quiescent cells (Fig. 4A,lane 6, lower panel), we have consistently ob- served that Nek3 was more highly expressed in G₀-arrested

cells than in proliferating cells (Fig. 4A,upper panel; compare lane 6withlanes 1–5). Thus, in striking contrast to Cdc2, the expression of mNek3 is not limited to proliferating cells, and, in fact, is enhanced in quiescent cells.

mNek3 Kinase Activity through the Cell Cycle—Next, we wished to determine the activity of mNek3 through the cell cycle. To optimize the conditions for measuring kinase activity intrinsic to mNek3,in vitrotranslated mNek3 was used. Wild- type mNek3 and a mutant form of mNek3, carrying a point mutation in the putative ATP binding site (mNek3-D143A), were produced in a reticulocyte lysate, and samples were diluted into NEB buffer containing 0.1% deoxycholic acid. (For presently unknown reasons, the presence of this detergent was found to be critical for our ability to measure mNek3 kinase activityin vitro.) Following immunoprecipitation of the trans- lation products, kinase assays were performed in the presence ofb-casein, a protein previously shown to be a good substrate for several NIMA-related kinases (15, 20, 32, 33). Fig. 5 shows that both wild-type mNek3 and the catalytically inactive mNek3-D143A mutant were produced at similar levels (panel A), yet only wild-type mNek3 showed strong kinase activity towardb-casein, while mNek3-D143A was inactive (panel B).

Likewise, only wild-type mNek3 displayed apparent autophosphorylation activity (panel B,lane 1). These data show that the above assay conditions allow a reliable measurement of mNek3 activity.

To determine the activity of endogenous mNek3 through the cell cycle, mNek3 activity was measured in immunoprecipitates prepared from Swiss 3T3 fibroblasts synchronized at different stages of the cell cycle (Fig. 5D,laneslabeleda). For each sample, the recovery of mNek3 was monitored by immunoblotting (Fig. 5C, lanes labeled a). As a control for specificity, parallel analyses were performed on immunoprecipitates prepared from each sample using control mouse IgG (Fig. 5,Cand D, lanes labeled c). As shown in Fig. 5C, roughly similar FIG. 3.Detection of Nek3 by anti-Nek3 antibodies.A,in vitro

translated Nek3 (IVT,lane 1), total protein from G0-arrested Swiss 3T3 fibroblasts (lanes 3– 6), and a mixture of the two samples (lane 2) were separated by SDS-PAGE and transferred to a nitrocellulose membrane.

Western blot analysis was performed with polyclonal (PepN3;lanes 1–5) or monoclonal (5F2G6;lane 6) anti-Nek3 antibodies, raised against a C-terminal peptide. Lane 2 shows that in vitro translated Nek3 comigrated exactly with the endogenous Nek3.Lanes 4and5illustrate the results of pre-incubating the PepN3 anti-Nek3 antibody with the pepN3 peptide (lane 4) or the unrelated pepN2 peptide (lane 5). The arrowheadpoints to the 56-kDa Nek3 protein.B, Western blot analysis of total protein extracts from mouse intestine and testis. Extracts (20mg of protein) were resolved by SDS-PAGE and subjected to Coomassie Blue staining (left panel) and immunoblotting (centralandright panel).

Immunoblotting was performed with anti-Nek3 and anti-Nek2 antibodies, as indicated. The positions of Nek3 (56 kDa) and Nek2 (51 kDa) are indicated byarrowheads.

FIG. 4.Expression of Nek3 protein during the cell cycle in Swiss 3T3 fibroblasts. A, immunoblotting analyses. Total cell ex- tracts were prepared from an asynchronously growing population of Swiss 3T3 fibroblasts (lane 1) or from cells treated in the following ways: blocked with aphidicolin (lane 2), released from the aphidicolin block for 4 h (lane 3), blocked with nocodazole (lane 4), released from the nocodazole block for 5 h (lane 5), or arrested in G0by serum starvation (lane 6). Equal amounts of protein were resolved by SDS-PAGE, transferred to nitrocellulose, and probed with antibodies against Nek3 (upper panel) and Cdc2 (lower panel). Note that a slower migrating form of Cdc2 (known to result from tyrosine phosphorylation) could be observed in the G1/S and S phase samples.B, flow cytometric analysis of cell cycle progression. Aliquots of the samples analyzed inpanel Awere used for staining of DNA with propidium iodine, and subjected to fluorescence- activated cell sorting analyses. Thenumbersin each panel correspond to thelane numbersinpanel A, and the positions of cells with 2N and 4N DNA contents are indicated byarrowheads.

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amounts of mNek3 were recovered from exponentially growing cells (Expo) and from cells synchronized at particular cell cycle stages (as indicated), although a slightly higher amount of mNek3 was precipitated from G₀-arrested cells, consistent with the data shown in Fig. 4A. As is evident from Fig. 5D, mNek3 activity roughly paralleled the amount of mNek3 protein present in each sample, and thus showed little variation through the cell cycle. These data also show clearly that mNek3 is active

in lysates prepared from G₀-arrested cells. Attesting to the specificity of these immunoprecipitations and activity meas- urements, no mNek3 protein and virtually nob-casein kinase activity were detectable in the control samples (Fig. 5,CandD, laneslabeledc). Taken together, these data strongly indicate that mNek3 activity is determined largely, if not exclusively, at the level of protein expression, and they provide no evidence for posttranslational regulation of mNek3 kinase activity during the cell cycle. Most importantly, both mNek3 protein and activity levels were largely constant throughout the cell cycle, but increased in quiescent cells.

Subcellular Localization of mNek3—We have attempted to localize endogenous mNek3 protein by indirect immunofluorescence microscopy, both in cultured cells and on cryostat sec- tions prepared from mouse intestine. However, although a variety of standard fixation procedures were used, we have so far been unable to detect a signal that could reliably be attrib- uted to mNek3. It is possible that this reflects the low abundance (Fig. 2) and rather uniform distribution (see below) of mNek3. Alternatively, we cannot exclude the possibility that the epitopes recognized by presently available antibodies may be maskedin vivo. To overcome this difficulty and obtain some information about the subcellular localization of mNek3, we have taken two approaches. First, indirect immunofluorescence microscopy was used to study the localization of myc- epitope tagged mNek3 after ectopic expression; and second, the partitioning of endogenous mNek3 was determined after biochemical fractionation of cultured cells.

In the first type of experiment, cDNAs encoding myc epitope- tagged wild-type or catalytically inactive mNek3 were intro- duced by transient transfection into human U2OS osteosarcoma cells, and the corresponding proteins detected with a monoclonal antibody against the myc epitope. Both myc- mNek3 (Fig. 6A) and myc-mNek3-D143A (data not shown) were found to be diffusely distributed throughout the cytoplasm. As the related kinase Nek2 has recently been shown to localize to centrosomes (21), we have carefully examined FIG. 5.Analyses of Nek3 kinase activity.AandB, establishment

of a specific kinase activity assay, usingin vitrotranslated mNek3.A, wild-type Nek3 (lane 1) and catalytically inactive Nek3 (D143A;lane 2) were translatedin vitroand immunoprecipitated with the anti-Nek3 monoclonal antibody 5F2G6. The position of Nek3, as detected by immunoblotting, is indicated by the arrowhead; theasteriskmarks the position of the immunoglobulin heavy chain. Note equal recovery of wild-type and mutant Nek3 protein.B, immunoprecipitated wild-type Nek3 (lane 1) and D143A mutant Nek3 (lane 2) were subjected to anin vitro kinase assay, in the presence of [g-³²P]ATP and b-casein as a substrate. Phosphate incorporation was determined by autoradiogra- phy. Note that only wild-type Nek3 was able to phosphorylateb-casein and to undergo apparent autophosphorylation.CandD, measurement of Nek3 kinase activity during the cell cycle. Extracts were prepared from Swiss 3T3 fibroblasts arrested at different stages of the cell cycle, as described in the legend to Fig. 4. These were then subjected to immunoprecipitation using either the anti-Nek3 antibody 5F2G6 (lanes labeleda) or mouse IgG (laneslabeledc) for control. To monitor the recovery of endogenous Nek3, aliquots of each sample were analyzed by immunoblotting with the anti-Nek3 antibody PepN3 (C), whereas the bulk of each sample was used to measure Nek3-specific kinase activity, usingb-casein as a substrate (D). The positions of Nek3 andb-casein are indicated byarrowheads; theasteriskinDmarks the position of the immunoglobulin heavy chain. Densitometric scanning was used to determine the Nek3 activity relative to the amount of Nek3 protein recovered from each sample. These values are indicated underneath panel D.

FIG. 6.Subcellular localization of Nek3.A, Myc epitope-tagged Nek3 localizes predominantly to the cytoplasm. U2OS cells were tran- siently transfected with CMV-Myc-mNek3. After 24 h, transfected cells were stained with anti-Myc antibody (left panel) and DNA was visual- ized by Hoechst dye 33258 (right panel).Bar525mm.B, subcellular distribution of endogenous Nek3 in Swiss 3T3 fibroblasts. Cytoplasmic (C) and nuclear (N) extracts were prepared as described under “Mate- rials and Methods,” and subjected to immunblotting with antibodies against Nek3, a small nuclear ribonucleoprotein, anda-tubulin.

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whether a similar localization might be discernible for mNek3.

However, co-staining with centrosomal markers provided no evidence for an association of mNek3 with centrosomes (data not shown).

To corroborate these findings and extend them to endogenous mNek3, subcellular fractionation experiments were performed. When Swiss 3T3 fibroblasts were subjected to a fractionation procedure that allows the separation of nuclear and cytoplasmic proteins (29), mNek3 was found predominately in the cytoplasmic fraction, although minor amounts could also be seen in the nuclear fraction (Fig. 6B,top panel). Reprobing of the same fractions with an antibody against an small nuclear ribonucleoprotein(26) revealed a predominantly nuclear localization, as expected, whilea-tubulin could be detected in both fractions (Fig. 6B,middle and bottom panels). These results confirm that mNek3 is a predominantly cytoplasmic protein, although a quantitatively minor population may also be present in the nucleus. Furthermore, our data do not exclude the possibility that a fraction of mNek3 might associate with cel- lular membranes.

Antibody Injection and Kinase Overexpression Experi- ments—To directly address a possible function of mNek3 in cell cycle regulation, two types of experiments were performed.

First, we have microinjected polyclonal anti-Nek3 antibodies into Swiss 3T3 cells, and scored the injected cells for their ability to undergo one or two successive cell divisions. Anti- Nek3 antibody-injected cells were found to divide as efficiently and with the same kinetics as cells injected with control rabbit IgG (data not shown). Thus, this assay provided no evidence to suggest a requirement for Nek3 during cell cycle progression.

We cannot exclude that the antibodies used in these experiments were unable to neutralize Nek3 function, but we note that a very similar approach has readily allowed us to demonstrate a requirement for another kinase, Polo-like kinase 1, during cell division (34). In a second series of experiments, we have overexpressed myc epitope-tagged versions of either wild- type or catalytically inactive mutant Nek3 in U2OS cells. After 22 h, the ability of transfected cells to undergo DNA replication was determined by a 1-h pulse labeling with bromodeoxyuridine (BrdUrd). As a positive control, the Cdk inhibitor p27^Kip1 was overexpressed in parallel. When monitoring BrdUrd-incorporation in cells overexpressing wild-type Nek3 or catalytically inactive Nek3, the percentage of BrdUrd-positive cells was found to be very similar to that in surrounding cells (data not shown). In contrast, BrdUrd incorporation was almost completely suppressed in cells in which a G₁ block had been im- posed by overexpression of p27^Kip1, as expected (data not shown). These results do not rigorously exclude a cell cycle function for Nek3, but they certainly lend no support for such a function.

DISCUSSION

InA. nidulans, NIMA is an essential gene, and the NIMA kinase appears to be a key regulator of the G₂to M transition (reviewed in Refs. 11, 35, and 36). The precise molecular function of NIMA remains uncertain, but the kinase has been implicated in mediating chromatin condensation (37), DNA structure checkpoint signaling (38), and, most recently, the nuclear localization of the Cdc2/cyclin B complex (39). To what extent these proposed roles reflect a single underlying function of NIMA remains to be determined. With Nim-1, a functional homologue of NIMA has been described inN. crassa(18). Ki- nases structurally related to NIMA have also been identified in budding yeast and fission yeast, but these kinases, termed Kin3p/Npk1p (12, 13) and fin1 (14), respectively, are non-essential. The function of Kin3p/Npk1p is largely unknown, but fin1 has been proposed to play a role in chromatin condensation

(14), in line with early studies on NIMA.

Whetherbona fidefunctional homologues of NIMA exist in mammals remains unclear, but several NIMA-related kinases (Neks) have been identified. Sequencing of cDNA fragments indicates that the mammalian genome harbors at least six distinct Neks (35). It is an important task, therefore, to determine whether these kinases perform unique, related, or redundant functions. On the one hand, it is possible that different mammalian Neks may all perform NIMA-related functions, but that, during evolution, these kinases may have assumed spe- cialized tasks during the cell cycle, or be expressed specifically in particular organs or at particular developmental stages.

Alternatively, it would be premature to exclude the possibility that Neks might perform fundamentally distinct functions.

Some Neks might functionally resemble NIMA, but others might regulate entirely different processes, not necessarily related to cell cycle progression altogether. Precedents for both scenarios exist in the Cdk family. In line with the first viewpoint, a single major Cdk regulates cell cycle progression in yeast, whereas multiple Cdks cooperate in mammals. In line with the second viewpoint, most Cdks control cell cycle progression, but some appear to function primarily in transcription (reviewed in Refs. 3, 40, and 41).

In this study, we have characterized mNek3, a novel member of the mammalian Nek family. mNek3 resembles the other known mammalian Neks in that it carries the catalytic domain at the N terminus. In the C-terminal non-catalytic domain, however, mNek3 differs from the other Neks (as well as from NIMA and the yeast NIMA family members Kin3p/Npk1p and fin1) in that it lacks predicted coiled-coil regions. In fact, the C-terminal end domain of mNek3 shows no detectable similarity to any other NIMA family member, except for the putative human Nek3 homologue (17).

RNase protection experiments suggest that mNek3 is expressed at rather low levels (in the order of 10 copies of mRNA/cell) in all organs analyzed. When data were calibrated on a per cell basis (i.e.standardized for DNA equivalents; Ref.

31), highest expression of mNek3 was seen in the small intestine; when calibrated for equivalent amounts of RNA, relatively high expression was seen in small intestine, thymus, lung, and spleen. Regardless of the precise mode of data analysis, however, no particularly high expression of mNek3 could be seen in either the male or the female germ line. This result was confirmed also by comparing the amounts of mNek3 protein present in intestine and testis. It is clear, therefore, that the organ specificity of mNek3 expression differs very mark- edly from that of Nek1 and Nek2, both of which were recently shown to be expressed to far higher levels in the germ lines than in any other mammalian tissue (15, 22–24).

Considering the role of NIMA in cell cycle regulation, it was of considerable interest to determine the expression and activity of mNek3 at various stages of the cell cycle. Immu- noblotting experiments, performed on lysates prepared from synchronized Swiss 3T3 fibroblasts, revealed that mNek3 levels vary at most marginally during the cell cycle, and no evidence could be obtained for posttranslational modifications that would result in altered gel electrophoretic mobility.

Interestingly, however, we found that mNek3 protein levels were 2–3-fold higher in G₀-arrested, quiescent cells than in proliferating cells. This somewhat surprising result contrasts with data reported for other NIMA family members. In particular, both NIMA and Nek2 levels vary drastically through the cell cycle (20, 32).

Using b-casein as an exogenous substrate, we have also measured mNek3 activity during the cell cycle in Swiss 3T3 fibroblasts. We found that mNek3 activity essentially paral-

Cloning and Characterization of Murine Nek3 Protein Kinase 13496

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leled the amounts of mNek3 protein, suggesting that mNek3 may be regulated primarily, and perhaps exclusively, at the level of protein expression. It remains possible, of course, that accessory proteins and/or posttranslational modifications may modulate mNek3 activityin vivo, but that such hypothetical regulators or modifications may have been lost during cell solubilization and/or immunoprecipitation.

Finally, we have examined the subcellular localization of mNek3. While we have been unable to reliably detect endogenous mNek3 by immunofluorescence microscopy, a myc epitope-tagged mNek3 was found to localize predominantly to the cytoplasm. Furthermore, a predominantly cytoplasmic localization was observed for endogenous mNek3, when subcellular fractionation was combined with immunoblotting. Our data do not exclude the possibility that mNek3 may also associate with membranes, or that minor amounts of this kinase may be present in the nucleus. They clearly indicate, however, that mNek3 is not associated with centrosomes. This is inter- esting in view of our recent finding that mammalian Nek2 localizes to centrosomes and functions in relation to the centrosome cycle (21).

In conclusion, we have shown that murine Nek3 differs in virtually all aspects examined from both NIMA and Nek2, the mammalian kinase most closely related to NIMA. In particular, Nek3 displays no major changes in either abundance or activity during the cell cycle, and antibody microinjection as well as overexpression experiments provide no evidence for a cell cycle-related function. While the precise molecular function has not yet been determined for any of the NIMA family members, our present study, in conjunction with previous work on Nek1 and Nek2 (15, 17, 20, 21), clearly indicates that the mammalian Nek family members display widely different properties. Taken at face value, the available evidence is diffi- cult to reconcile with the idea that all mammalian Neks functionally resemble each other and/orAspergillusNIMA. Instead, the emerging picture suggests that kinases structurally related to NIMA may have assumed rather disparate functions during evolution. To positively identify these functions remains a major challenge for the future.

Acknowledgments—We thank E. E. Schmidt, T. Kondo, and J.

Zakany for their generous help, and D. Duboule for the use of animal facilities. We also thank D. Wohlwend for the fluorescence-activated cell sorting analyses, and G. van Houwe for technical assistance.

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