From the Department of Molecular Biology, Sciences II, University of Geneva 30, Quai Ernest-Ansermet, 1211 Geneve 4, Switzerland
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ABSTRACT |
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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 similarity between mammalian Neks
may not necessarily 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)1 are 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). In
Aspergillus, lack of NIMA function results in a cell cycle
arrest in G2 (6, 7), while overexpression of the
nimA gene causes the premature onset of mitotic events, such as depolymerization of cytoplasmic microtubules, formation 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 species. These include
Kin3p/Npk1p from Saccharomyces cerevisiae (12, 13), fin1
from Schizosaccharomyces 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 in
Aspergillus is 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, unresolved 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 affects
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 represents 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.
cDNA Cloning and Mutagenesis--
A mouse brain cDNA
library in Tissue Protein Extractions and RNA Techniques--
For
preparation of protein extracts, testis tissue from 5-week-old mice was
homogenized in lysis buffer (50 mM Hepes/KOH pH 7.4, 5 mM MnCl2, 10 mM MgCl2,
2 mM EDTA, 100 mM NaCl, 5 mM KCl, 0.1% Nonidet P-40, 30 µg/ml DNase I, 1 mM
phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 10 µg/ml
pepstatin A, 1% aprotinin, 20 mM Immunochemical Techniques--
The affinity-purified anti-Nek2
antibody 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- 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 100 µg/ml,
respectively). For G0 arrest, 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 µg/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 µg/ml, Sigma), and G1 cells 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 1 × 105 cells per 35-mm dish with 5 µg 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 mM Hepes/KOH, pH 7.4, 5 mM
MnCl2, 10 mM MgCl2, 2 mM EDTA, 100 mM NaCl, 5 mM KCl, 30 µg/ml DNase I, 0.1% Nonidet P-40, 0.1% deoxycholic acid, 20 mM 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
temperature, followed by permeabilization with acetone for 30 s at
Immunoprecipitations and in Vitro Kinase Assays--
In
vitro translated 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 µg/ml,
and immune complexes were isolated using protein G-Sepharose beads
(Amersham Pharmacia Biotech). Immunoprecipitates were washed three
times with 0.5× NEB buffer and twice with 50 mM Hepes/KOH
(pH 7.4), 5 mM MnCl2, and incubated for 30 min
at 30 °C in a total volume of 30 µl of 50 mM Hepes/KOH
(pH 7.4), 5 mM MnCl2, 5 mM
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 106 plaques, 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 (GenBankTM 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 5'-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, the
in vitro translated 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 with A. nidulans NIMA. Concerning the non-catalytic C-terminal end domains of 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), including Aspergillus NIMA 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 quantitatively less pronounced
(compare Fig. 2B with 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 1 and 2), confirming that the
coding sequence reported here almost certainly represents the complete
mNek3 protein. The PepN3 immune reaction was completely abolished by
preincubation of PepN3 with the immunogenic peptide, pepN3, but not by
incubation with a control peptide, pepN2 (Fig. 3A,
lanes 4 and 5, 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. The
right-hand panel shows the results of probing 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.
3B and 2A). This suggests that
posttranscriptional mechanisms may contribute to control the 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 G0 by 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 observed that Nek3 was more
highly expressed in G0-arrested cells than in proliferating cells (Fig. 4A, upper panel; compare
lane 6 with lanes 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 vitro translated 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 activity
in vitro.) Following immunoprecipitation of the translation
products, kinase assays were performed in the presence of
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, lanes labeled a). 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, C
and D, lanes labeled c). As shown in
Fig. 5C, roughly similar 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 measurements, no mNek3 protein and
virtually no Subcellular Localization of mNek3--
We have attempted to
localize endogenous mNek3 protein by indirect immunofluorescence
microscopy, both in cultured cells and on cryostat sections 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 attributed 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
masked in 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 introduced 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 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, while Antibody Injection and Kinase Overexpression Experiments--
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
p27Kip1 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 imposed by
overexpression of p27Kip1, 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.
In A. nidulans, NIMA is an essential gene, and the NIMA
kinase appears to be a key regulator of the G2 to 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 in N. crassa (18). Kinases 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.
Whether bona fide functional 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
specialized 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 markedly 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. Immunoblotting 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 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 interesting 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 difficult to reconcile with the idea
that all mammalian Neks functionally resemble each other and/or
Aspergillus NIMA. 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.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
Zap II (Stratagene) was screened with a human Nek3
cDNA fragment (XhoI-XbaI, nucleotides
617-1555; Ref. 17), labeled with [
-32P]dCTP (NEN Life
Science Products). Phages were converted into pBlueScript (SK
) by
in vivo excision, and inserts were sequenced. One plasmid,
termed pBS-mNek3, was found to contain the entire coding sequence of
mouse Nek3. A PvuII-PvuII fragment excised from
pBS-mNek3 was inserted into the PstI site of a
pBluescript-myc vector carrying the myc epitope tag (25), to yield
pBS-myc-mNek3. Myc-tagged mNek3 was then excised as a
HindIII-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).
-glycerophosphate, 20 mM sodium fluoride, 0.3 mM sodium orthovanadate, and 2 µg/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 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 10 µg/ml pepstatin A, 1% aprotinin). Following the determination 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 antisense RNA
probe was prepared by transcribing a cDNA fragment from pBS-mNek3 (nucleotides 738-1145; GenBankTM AF093416).
-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 Laboratories 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 1 µg/ml
(23), and chemiluminescence (ECL; Amersham Pharmacia Biotech) for detection.
-glycerophosphate, 20 mM sodium fluoride,
0.3 mM sodium orthovanadate, 2 µg/ml heparin, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 10 µg/ml pepstatin A, 1% aprotinin), and lysates were clarified by
centrifugation at 12,000 × g for 10 min at 4 °C.
Nuclear and cytoplasmic fractions from G0-arrested Swiss
3T3 cells were prepared as described previously (29).
20 °C. The anti-myc monoclonal antibody 9E10 was used as undiluted
hybridoma supernatant, whereas affinity-purified PepN3 antibodies were
used at 10 µg/ml.
-glycerophosphate, 5 mM NaF, 7.5 µg/ml heparin, 1 mM dithiothreitol, 0.5 µM ATP, 5 µCi of
[
-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
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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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 by vertical
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 by
asterisks (42).
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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-µg
DNA equivalents of each tissue were calculated as described previously
(23). Each RNA sample was then supplemented to 100 µg with yeast RNA
and hybridized with 10 fmol of both Nek3 and NF-Ya probes. The control
contained 100 µg 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 quantification of data in relation to
RNA (rather than DNA equivalents). Signals obtained by RNase protection
(see panel A) were scanned and normalized to the
amounts of RNA loaded in each lane.
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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 4 and 5 illustrate the results of pre-incubating the PepN3
anti-Nek3 antibody with the pepN3 peptide (lane 4) or the
unrelated pepN2 peptide (lane 5). The arrowhead
points to the 56-kDa Nek3 protein. B, Western blot analysis
of total protein extracts from mouse intestine and testis. Extracts (20 µg of protein) were resolved by SDS-PAGE and subjected to Coomassie
Blue staining (left panel) and immunoblotting
(central and right 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 by arrowheads.
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Fig. 4.
Expression of Nek3 protein during the cell
cycle in Swiss 3T3 fibroblasts. A, immunoblotting
analyses. Total cell extracts 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 G0
by 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 in panel A were
used for staining of DNA with propidium iodine, and subjected to
fluorescence-activated cell sorting analyses. The numbers in
each panel correspond to the lane numbers in
panel A, and the positions of cells with 2N and
4N DNA contents are indicated by arrowheads.
-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 toward
-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.
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Fig. 5.
Analyses of Nek3 kinase activity.
A and B, establishment of a specific kinase
activity assay, using in vitro translated mNek3.
A, wild-type Nek3 (lane 1) and
catalytically inactive Nek3 (D143A; lane 2) were
translated in vitro and immunoprecipitated with the
anti-Nek3 monoclonal antibody 5F2G6. The position of Nek3, as detected
by immunoblotting, is indicated by the arrowhead; the
asterisk marks 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 an in vitro kinase assay, in the presence of
[ -32P]ATP and
-casein as a substrate. Phosphate
incorporation was determined by autoradiography. Note that only
wild-type Nek3 was able to phosphorylate
-casein and to undergo
apparent autophosphorylation. C and D,
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 labeled a) or mouse IgG
(lanes labeled c) 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, using
-casein as a substrate (D). The
positions of Nek3 and
-casein are indicated by
arrowheads; the asterisk in D marks
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.
-casein kinase activity were detectable in the control
samples (Fig. 5, C and D, lanes
labeled c). 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.
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Fig. 6.
Subcellular localization of Nek3.
A, Myc epitope-tagged Nek3 localizes predominantly to the
cytoplasm. U2OS cells were transiently transfected with CMV-Myc-mNek3.
After 24 h, transfected cells were stained with anti-Myc antibody
(left panel) and DNA was visualized by Hoechst
dye 33258 (right panel). Bar = 25 µm. B, subcellular distribution of endogenous Nek3 in
Swiss 3T3 fibroblasts. Cytoplasmic (C) and nuclear
(N) extracts were prepared as described under "Materials
and Methods," and subjected to immunblotting with antibodies against
Nek3, a small nuclear ribonucleoprotein, and -tubulin.
-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 cellular membranes.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-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 paralleled 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 activity in vivo, but that
such hypothetical regulators or modifications may have been lost during cell solubilization and/or immunoprecipitation.
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ACKNOWLEDGEMENTS |
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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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FOOTNOTES |
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* This work was supported in part by Swiss National Science Foundation 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. The 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 GenBankTM/EMBL 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{at}molbio.unige.ch.
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ABBREVIATIONS |
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The 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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