From the
The human Nek2 protein kinase is the closest known mammalian
relative of the mitotic regulator NIMA of Aspergillus
nidulans. The two kinases share 47% sequence identity over their
catalytic domains and display a similar cell cycle-dependent expression
peaking at the G
In the filamentous fungus, Aspergillus nidulans, entry
into mitosis requires the activation of a serine/threonine protein
kinase termed NIMA
(1, 2) (reviewed in Ref. 3). Cells
carrying a temperature-sensitive copy of the nimA gene arrest
specifically in G
The striking evolutionary conservation of cell
cycle control mechanisms (8) suggests that protein kinases related to
fungal NIMA may also exist in higher eukaryotes. In support of this
possibility, three NIMA-related protein kinases, termed
Nek1,
Among all vertebrate kinases known to date, Nek2
is most closely related to fungal NIMA; furthermore, the two kinases
display similar cell cycle-dependent expression patterns, being low in
G
To overcome this major difficulty and make Nek2 amenable to
biochemical analyses, we have now generated recombinant baculoviruses
and expressed wild-type human Nek2, as well as a catalytically inactive
mutant, in insect cells. We show here that recombinant human Nek2 is
indeed active as a serine/threonine-specific protein kinase.
Furthermore, we report that the biochemical properties and substrate
specificity of human Nek2 are remarkably similar to those of fungal
NIMA; in contrast, Nek2 can readily be distinguished from Nek1, as well
as from casein kinase II, a ubiquitously expressed enzyme which also
prefers casein as its in vitro substrate. Finally, having
optimized the conditions for assaying Nek2 activity, we have been able
to return to cultured HeLa cells and demonstrate endogenous Nek2 kinase
activity. We found that Nek2 activity fluctuates during the cell cycle,
strengthening the hypothesis that this NIMA-related human protein
kinase may play a role in cell cycle regulation.
A catalytically inactive mutant
of Nek2 (Nek2-K37R, carrying a replacement of Lys-37 by Arg) was
created by site-directed mutagenesis of pGEM-Nek2, using the Clontech
mutagenesis kit, and the mutation was confirmed by double-stranded
plasmid sequencing. The mutagenic oligonucleotide used was
AGATATTAGTTTGGAGAGAACTTGACTATGGC, with the underlined codon
corresponding to residue 37 in Nek2; as a selection primer, an
oligonucleotide eliminating the PstI site in the pGEM
multicloning site was used. The full-length mutant cDNA was then
excised from pGEM-Nek2-K37R as a NaeI-XbaI fragment
and ligated into the PstI (blunted)-XbaI site of the
vector pVL1392 (Pharmingen Corp.) to create pVL1392-Nek2-K37R.
Recombinant Nek2-K37R baculovirus was generated by cotransfection of
pVL1392-Nek2-K37R and BaculoGold DNA (Pharmingen Corp.) and subsequent
amplification of viral DNA.
We found that Nek2 kinase was inhibited by several
detergents, notably those commonly used in cell extraction buffers.
Nonidet P-40 (1%), Triton X-100 (1%), or SDS (0.1%) all strongly
inhibited the activity of immunoprecipitated Nek2 (data not shown).
However, provided that extracts were prepared under appropriate
conditions (see above) and stored at -80 °C, Nek2 kinase
activity in whole cell extracts or immunoprecipitates was stable for
several months.
Prior to immunoprecipitation, extracts were
equalized for protein content where appropriate, and precleared with
protein A-Sepharose beads for 30 min. Extracts were then incubated for
60 min on ice with 0.01 volume of R31 anti-Nek2 polyclonal rabbit
antiserum
(12) , followed by protein A-Sepharose for a further 45
min. Immune complexes were collected by centrifugation and washed at
least five times with Sf9 lysis buffer before they were used for in
vitro kinase assays. Alternatively, they were stored at -80
°C or mixed with an equal volume of 3
Following
optimization of assay conditions, the activity of endogenous, human
Nek2 kinase was detected as follows. HeLa cells were lysed in 50
mM Hepes, pH 7.5, 5 mM MnCl
Peptide phosphorylation was assayed by both phosphocellulose P81
paper binding
(15) and by thin layer chromatography in buffer C
(isobutyric acid:pyridine:acetic acid:butanol:water =
65:5:3:2:29), essentially as described previously
(16) ; 500
µM peptide was used in each assay.
Phosphoamino acid analyses and tryptic
phosphopeptide mapping experiments were carried out as described
elsewhere
(16) , except that the electrophoresis of tryptic
peptides was carried out in pH 1.9 buffer (glacial acetic acid:formic
acid (100%):deionized water, 156:44:1800) and EDTA was omitted from the
pH 3.5 electrophoresis buffer used for the second dimension of
phosphoamino acid separation. For phosphoamino acid analysis of the
peptide PLM(64-72), the radiolabeled peptide was scraped from a
TLC plate
(17) .
To detect
Nek2-associated kinase activity, in vitro kinase assays were
carried out on either whole insect cell lysates
(Fig. 1A) or Nek2 immunoprecipitates
(Fig. 1B). Where indicated, dephosphorylated casein was
added as an exogenous substrate, since
The results of in vitro kinase assays using a
series of synthetic peptides are also summarized in .
Quantitative analyses of peptide phosphorylation were performed using a
phosphocellulose P81 filter binding assay (Fig. 5A), or,
where appropriate, TLC followed by autoradiography
(Fig. 5B). The peptides chosen for analysis
(Fig. 5C) represent preferred substrates for several
important families of protein kinases, and most of them have been
tested previously for their ability to be phosphorylated by the fungal
NIMA protein kinase
(21, 25) . For control, all peptide
phosphorylation experiments were carried out in parallel with both
active Nek2 and the inactive Nek2-K37R mutant; none of the peptides
studied here was phosphorylated by the catalytically inactive Nek2
kinase (Fig. 5B, and results not shown), ruling out the
possibility that contamination of immunoprecipitates by insect kinases
might complicate the interpretation of results. Of seven peptides
tested, three were found to be excellent substrates for human Nek2
kinase (Fig. 5, A and B). Two of these were
phospholemma-derived peptides, PLM(42-72) and PLM(64-72);
the third was a known preferred substrate of kinases acting on the
ribosomal protein S6. Since the short PLM(64-72) peptide contains
only one serine and one threonine residue (and no tyrosines), the
phosphorylated peptide was recovered from a TLC plate and subjected to
phosphoamino acid analysis. Only phosphoserine could be detected (data
not shown), demonstrating that the serine in the sequence IRRL-S-TRRR
constitutes one phosphoacceptor site for the human Nek2 protein kinase.
Indeed, a serine in the context RRL-S-(S/T)XR is present in
all three Nek2 substrate peptides identified here.
In this study, we have been able to demonstrate that
baculovirus-encoded recombinant Nek2 is active as a
serine/threonine-specific protein kinase, and we have carried out an
extensive biochemical characterization of this novel enzyme. Nek2
kinase activity can readily be monitored in vitro, using
Both Nek2 and NIMA phosphorylate
exclusively serine or threonine residues. Thus, among the NIMA-related
kinases studied so far, Nek1 appears to be unique in its ability to
phosphorylate tyrosine in addition to serine/threonine, and one may
question whether it is appropriate to consider Nek1 and NIMA/Nek2 as
members of the same family. We believe, however, that the difference in
amino acid specificity between Nek1 and NIMA/Nek2 may be more apparent
than real. Although Nek1 clearly displays dual specificity under
appropriate experimental conditions
(9, 10) , it is by no
means established that Nek1 will act as a tyrosine kinase under
physiological conditions.
So far, we have been unable to purify
sufficient quantities of recombinant Nek2 in an active state to warrant
an exhaustive analysis of substrate specificity, and the use of
immunoprecipitated Nek2 as a source of active kinase has precluded
precise determinations of kinetic parameters. Nevertheless, our survey
of a number of potential target proteins and peptides provides a first
glimpse of the requirements for substrate recognition by Nek2. Whereas
the precise sites phosphorylated by Nek2 have not been mapped for most
of the proteins studied here, we found that the peptide IRRLSTRRR was
phosphorylated exclusively on serine, suggesting that basic residues
may contribute to substrate recognition by Nek2. In support of this
view, the Nek2 protein contains acidic residues within the kinase
domain which, based on homology to protein kinase
A
(22, 28, 29) , would suggest a requirement for
arginine residues at positions -2 and -3 in the target
sequence. However, since Kemptide also contains N-terminal arginines,
yet is not a substrate of Nek2, it is likely that basic residues
C-terminal to the phosphorylation site may also be important for
substrate recognition by Nek2. More systematic studies on synthetic
peptides with permutations at various positions would be necessary to
more definitively assess the relevance of individual residues within
the above sequence.
Interestingly, recent studies on NIMA have
suggested a requirement for a phenylalanine N-terminal to the
phosphorylated serine or threonine; hence, the motif FRXS/T
was proposed to constitute a consensus for a NIMA phosphorylation
site
(25) . The results reported here indicate that this
purported phenylalanine requirement cannot be extended to Nek2 (see
Fig. 5
and ). However, we note that the proposed NIMA
consensus sequence is clearly not absolute, since several good
substrates of NIMA, including
Finally, and perhaps most importantly, the
present study has allowed us to identify conditions under which the
kinase activity of endogenous Nek2 can now be assayed in
immunoprecipitates prepared from human cells. This in turn made it
possible to carry out a first analysis of the regulation of Nek2
activity during the cell cycle. Using drug arrest-release protocols, we
have shown that Nek2 kinase activity is low in M and early G
Biochemical properties associated with human
Nek2 as determined in this study are compared with those of A.
nidulans NIMA, murine Nek1, and mammalian casein kinase II.
The relative levels
of phosphorylation of a series of protein and peptide substrates by
human Nek2 kinase are indicated (+++, excellent;
++, good; +, poor; -, not at all). Results are
compared to the in vitro substrate specificity reported for
A. nidulans NIMA (21,25) and murine Nek1 (10).
We thank N. Lydon (Hoffmann-LaRoche, Basel) for a kind
gift of insect cell lysates, G. Thomas (FMI, Basel) and A. Means (Duke
University, Durham, NC) for synthetic peptides; M. Allegrini (ISREC,
Epalinges) for photography; C. Knabenhans for help with FACS analysis;
and S. Osmani (Weis Center for Research, Danville, PA) for
communicating results prior to publication.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
to M phase transition. Hence, it is
attractive to speculate that human Nek2 and fungal NIMA may carry out
similar functions at the onset of mitosis. To study the biochemical
properties and substrate specificity of human Nek2 and compare them to
those reported previously for other NIMA-related protein kinases, we
have expressed Nek2 in insect cells. We show that recombinant Nek2 is
active as a serine/threonine-specific protein kinase and may undergo
autophosphorylation. Both human Nek2 and fungal NIMA phosphorylate a
similar, albeit not identical, set of proteins and synthetic peptides,
and
-casein was found to be a suitable substrate for assaying Nek2
in vitro. By exploiting these findings, we have studied the
cell cycle regulation of Nek2 activity in HeLa cells. We show that Nek2
activity parallels its abundance, being low during M and G
but high during S and G
phase. Taken together, our
results suggest that human Nek2 resembles fungal NIMA in its primary
structure, cell cycle regulation of expression, and substrate
specificity, but that Nek2 may function earlier in the cell cycle than
NIMA.
when shifted to the restrictive
temperature (hence nim for never in
mitosis), and they enter mitosis synchronously when released
from the block
(4, 5) . Conversely, strong overexpression
of a wild-type nimA allele drives cells into a premature
mitotic state from any point in the cell cycle, the phenotype of such
cells being characterized by the formation of mitotic spindles and the
maintenance of chromosomes in a condensed
state
(1, 6, 7) . Expression of catalytically
inactive NIMA or the carboxyl-terminal noncatalytic domain of NIMA also
causes a G
arrest, even in cells expressing wild-type NIMA,
indicating that these NIMA mutants act in a dominant-negative
fashion
(7) . How exactly NIMA interacts or cooperates with the
p34
/cyclin B protein kinase to trigger entry into
mitosis in A. nidulans is an interesting but still unresolved
question
(2) .
(
)
Nek2, and Nek3 (for
NIMA-related kinase), have recently been
cloned from mammalian species. Nek1 was identified by screening of a
mouse cDNA expression library with anti-phosphotyrosine antibodies, and
this kinase was shown to display dual specificity for tyrosine and
serine/threonine residues
(9, 10) . Nek1 may play a role
specifically during meiosis, since high levels of nek1 mRNA
were expressed in male and female germ cells
(10) .
Independently, three cDNAs coding for human NIMA-related kinases were
isolated by using a polymerase chain reaction approach (11). One cDNA
was closely related to murine nek1, but the two others
represented novel NIMA-related kinases and hence were named Nek2 and
Nek3
(12) .
and increasing through S and G
to reach a
plateau in late G
/M, suggesting that they may both play a
role at the onset of mitosis
(12, 13) . Unfortunately,
further progress toward understanding Nek2 function had been hampered
by the lack of biochemical information on Nek2. In particular, it had
previously not been possible to measure specific Nek2-associated kinase
activity in Nek2 immunoprecipitates prepared from mammalian cells, and
it could not be determined whether these negative results were due to
the low abundance of Nek2 in exponentially growing cells, a narrow
window of activity during the cell cycle, the presence of inhibitory
factors in whole cell lysates, the choice of inappropriate substrates
or assay conditions, or a combination of these parameters
(12) .
Construction of Recombinant Baculoviruses
To
create a baculovirus encoding wild-type human Nek2 (pBlueBac-Nek2), a
full-length human Nek2 cDNA was excised as a
NaeI-PstI fragment from pGEM-Nek2
(12) and
subcloned into the BamHI (blunted)-PstI site of the
baculovirus transfer vector pBlueBac (Invitrogen Corp.). In this
construct the Nek2 cDNA is placed behind the viral polyhedrin promoter,
but the authentic ATG is used for translational initiation. Nek2
recombinant baculovirus was then generated by in vivo homologous recombination following cotransfection of pBlueBac-Nek2
and wild-type baculoviral DNA into Sf9 cells. The transfection
supernatant was used to infect Sf9 cells in a standard plaque assay,
recombinant virus was purified by three further rounds of plaque
assays, and purity was confirmed by the absence of polyhedrin occlusion
bodies. All procedures relating to Sf9 insect cell growth,
transfections, infections, plaque assays, and viral amplification were
as previously described
(14) .
Protein Expression, Preparation of Cell Extracts, and
Immunoprecipitation
Sf9 cells were infected with recombinant
baculoviruses in 10-cm culture plates at a multiplicity of infection of
10. Cell lysates were prepared at 48 h post-infection (unless indicated
otherwise) after harvesting both floating and adherent cells. Cells
were washed once in ice-cold PBS, 1 mM phenylmethylsulfonyl
fluoride, and resuspended at 10 cells/ml in Sf9 lysis
buffer (50 mM Hepes/KOH, pH 7.5, 100 mM NaCl, 5
mM KCl, 10 mM MgCl
, 2 mM EDTA, 5
mM EGTA, 0.2% Nonidet P-40, 1 mM phenylmethylsulfonyl
fluoride, 10 µg/ml leupeptin, 10 µg/ml pepstatin A, 10
µg/ml aprotinin, 0.3 mM sodium vanadate, 20 mM
-glycerophosphate, 20 mM sodium fluoride). After 30 min
on ice, cells were lysed by Dounce homogenization, and lysis was
checked by phase contrast microscopy. Lysates were spun at full speed
in a microcentrifuge, and the supernatants are referred to as whole
cell extracts.
gel sample buffer and
analyzed by SDS-PAGE.
In Vitro Kinase Assays
During the initial studies,
immunoprecipitated recombinant proteins were washed three times in Nek2
kinase buffer (50 mM Tris-HCl, pH 7.5, 10 mM
MgCl). Then, kinase reactions were carried out for 20 min
at 30 °C in Nek2 kinase buffer supplemented with 4 µM ATP, 1
mM dithiothreitol, and 10 µCi of
[
-
P]ATP (Amersham Corp.), in a total volume
of 50 µl. Dephosphorylated casein (Sigma, C-4032) was included at
0.5 mg/ml as an exogenous substrate, unless indicated otherwise.
Reactions were stopped by the addition of 50 µl of 3
gel
sample buffer and heating to 95 °C. Reactions products were
visualized by SDS-PAGE and autoradiography. Casein kinase II (Promega
Corp.; 2.5 units/assay) was assayed under the same conditions except
that 200 mM NaCl was added to the kinase buffer.
, 10
mM MgCl
, 5 mM EGTA, 2 mM EDTA,
100 mM NaCl, 5 mM KCl, 0.1% Nonidet P-40, 30
µg/ml of DNase I, 30 µg/ml of RNase A, 1 mM
phenylmethylsulfonyl fluoride, 1 µg/ml each of leupeptin and
pepstatin A, 0.1% aprotinin, 1 µM okadaic acid, and 1 µg/ml
heparin. Then, Nek2 was immunoprecipitated as described above.
Immunoprecipitates were given three final washes in lysis buffer, and
kinase assays were carried out as described above, except for using the
following, modified assay buffer: 50 mM Hepes, pH 7.5, 5
mM MnCl
, 5 mM NaF, 5 mM
-glycerophosphate, 1 µM okadaic acid, and 1 µg/ml heparin.
Metabolic Labeling, Phosphoamino Acid Analysis, and
Phosphopeptide Mapping
Metabolic labeling of
baculovirus-infected Sf9 cells was carried out as follows. 24-h
post-infection cells were washed twice in either methionine- or
phosphate-free medium, and then replated, respectively, in
methionine-free medium containing 50 µCi/ml
[S]methionine or phosphate-free medium
containing 500 µCi/ml [
P]ortho-phosphate;
both labeling media also contained 10% dialyzed serum and 10% normal,
fully supplemented Sf9 medium. Labeling was allowed to proceed for 16 h
before preparation of cell extracts and immunoprecipitation of
recombinant proteins.
Production of Nek2-specific Monoclonal
Antibodies
A histidine-tagged fusion protein encoded by a
partial nek2 cDNA (HsPK 21)
(11) was expressed in
Escherichia coli and purified by nickel column chromatography,
as previously described
(12) . This fusion protein was then
injected subcutaneously into Balb/c mice (about 50 µg/injection),
and hybridoma cell lines were generated as described previously, using
a dot blot assay for screening of supernatants
(18) . The
monoclonal antibody used in this study, termed NK1, is an
IgG.
Immunoblotting
Proteins were resolved on 12%
SDS-polyacrylamide gels and transferred to nitrocellulose by semi-dry
blotting. Detection of Nek2 protein was usually performed using
affinity-purified R31 anti-Nek2 antibodies (aR31)
(12) at 0.72
µg/ml, followed by an alkaline phosphatase-conjugated anti-rabbit
secondary antibody. However, for immunoblotting of Nek2
immunoprecipitates, undiluted supernatant of the Nek2-specific
monoclonal antibody NK1 was used, followed by an alkaline
phosphatase-conjugated anti-mouse secondary antibody. Detection of
phosphotyrosine was performed using the affinity-purified
anti-phosphotyrosine monoclonal antibody 4C10 (Upstate Biotechnology,
Inc.) at 1 µg/ml, followed by alkaline-phosphatase conjugated
anti-mouse secondary antibody. All blotting methods were as described
by Harlow and Lane
(19) .
Cell Cycle Synchronization
HeLa cells were
synchronized at the G/S boundary by the addition of 2
µg/ml aphidicolin (Sigma) for 16 h. For synchronization at
prometaphase, cells were incubated for 16 h with 500 ng/ml nocodazole
(Janssen), and mitotically arrested cells were collected by gentle
pipetteting. Cells were released from these blocks by three washes in
PBS and replating into fresh medium. The cell cycle distribution of the
various HeLa cell populations was monitored by flow cytometric (FACS)
analysis. For this purpose, 10
cells were washed twice in
ice-cold PBS and then suspended in 0.3 ml of PBS. Then, samples were
fixed by slow addition of 0.9 ml of 95% ethanol (-20 °C)
while vortexing. At this stage, cells were stored for up to 24 h.
Immediately prior to FACS analysis, fixed cells were gently washed
three times in ice-cold PBS (centrifugation at 2000
g,
5 min, 4 °C), and finally resuspended in 0.5 ml of PBS. Then, 20
µl of RNase A (Sigma, 10 mg/ml, pre-boiled) and 25 µl of
propidium iodide (200 µg/ml in 50 mM sodium citrate, pH
7.6) were added. Samples were incubated on ice for 30 min before the
DNA contents were determined by measuring fluorescence intensities on a
FACS II (Becton Dickinson) instrument.
Expression of Active Human Nek2 Protein Kinase in
Insect Cells
The predicted amino acid sequence of Nek2 displays
all the hallmarks of a protein kinase
(20) . Yet, no direct
biochemical evidence for Nek2 kinase activity had previously been
obtained. To be able to characterize this novel protein kinase, we
expressed it from a recombinant baculovirus in insect Sf9 cells. Sf9
cells were infected with either a wild-type baculovirus or a
recombinant Nek2 virus, and whole cell lysates were prepared at
increasing times after infection. As determined by immunoblotting,
expression of the 46-kDa Nek2 protein could first be detected at
18-24 h post-infection and reached maximal levels by 36-48
h (data not shown). At these latter times, a second, slower migrating
form of Nek2 became visible, suggesting that Nek2 may be subject to
post-translational modification in insect cells.
-casein had previously been
reported to be a preferred substrate of A. nidulans NIMA
(13, 21) . Whereas no major specific substrates
of Nek2 could be detected among total insect cell proteins
(Fig. 1A, compare lanes 1 and 2),
-casein was strongly phosphorylated in lysates from Nek2 virus
infected insect cells (Fig. 1A, lane 4), but
not in lysates from uninfected cells (Fig. 1A, lane
3). Similarly, strong
-casein phosphorylation was observed in
Nek2 immunoprecipitates prepared from Nek2 virus infected cells
(Fig. 1B, lane 8), but not in
immunoprecipitates prepared from noninfected cells
(Fig. 1B, lane 6) or in control
immunoprecipitates prepared using preimmune serum
(Fig. 1B, lanes 5 and 7). In Nek2
immunoprecipitates from Nek2 virus infected cells, a second protein
migrating at approximately 46 kDa was strongly phosphorylated
(Fig. 1B, lane 8). As indicated by comparison
with immunoprecipitates prepared in parallel from
[
S]methionine labeled cell lysates
(Fig. 1B, lanes 1-4), this protein
comigrated exactly with the recombinant human Nek2
(Fig. 1B, lane 4), suggesting that Nek2 protein
kinase might undergo autophosphorylation (see also
Fig. 1A).
Figure 1:
Casein kinase activity of
baculovirus-expressed Nek2 protein. Insect Sf9 cells were infected with
the Nek2-recombinant baculovirus (Nek2), and whole cell
extracts were prepared 36 h post-infection; for control (C),
uninfected cells were analyzed in parallel. A, crude extracts
were incubated with [P]ATP in the absence
(lanes 1 and 2) or presence (lanes 3 and
4) of casein; then, phosphoproteins were separated by SDS-PAGE
and detected by autoradiography. Note that viral infection caused an
overall decrease in protein expression. Molecular masses (kDa) are
shown on the left, and the positions of the Nek2 protein and
-casein are indicated on the right. B, Nek2 was
immunoprecipitated from insect cell extracts using either the
Nek2-specific R31 polyclonal immune serum (I) or the preimmune
serum (P). Immunoprecipitates (lanes 5-8) were
incubated in the presence of
[
P]ATP and casein before SDS-PAGE
and autoradiography. To confirm the specificity of the antibody and the
size of Nek2, immunoprecipitates were prepared in parallel from in
vivo [
S]methionine-labeled Sf9 cells
(lanes 1-4) and also analyzed by SDS-PAGE and
autoradiography. Molecular masses (kDa) are shown on the left,
and the positions of the Nek2 protein and
-casein are indicated on
the right.
The above results strongly suggested that
the recombinant Nek2 protein displayed intrinsic kinase activity, but
it remained formally possible that insect protein kinases might
contaminate Nek2 immunoprecipitates. Thus, we constructed a recombinant
baculovirus expressing a mutant Nek2 protein, termed Nek2-K37R, in
which a critical lysine of the Nek2 catalytic domain (residue 37) was
changed to arginine; by analogy to corresponding mutations made
previously in other protein kinases including NIMA
(21) , this
mutation was expected to render Nek2 catalytically
inactive
(22) . As shown in Fig. 2A, the Nek2-K37R
mutant was expressed to a similar level as the wild-type Nek2, and it
was also immunoprecipitated with comparable efficiency (not shown).
Interestingly, however, the Nek2-K37R protein did not display the
retarded electrophoretic mobility that was characteristically observed
with wild-type Nek2, indicating that the inactive kinase was not
post-translationally modified to the same extent
(Fig. 2A, compare lanes 1 and 2).
Also, whereas the extent of -casein phosphorylation catalyzed by
wild-type Nek2 increased linearly with reaction time, the Nek2-K37R
mutant was completely inactive in an in vitro kinase assay
(Fig. 2, B and C).
Figure 2:
Kinase activity of wild-type but not K37R
mutant Nek2. A, detection of Nek2-K37R (lane 1) or
wild-type Nek2 (lane 2) protein in whole cell extracts
prepared from insect Sf9 cells infected with the corresponding
recombinant baculoviruses. Immunoblots were carried out with the R31
anti-Nek2 antibody. Molecular masses (kDa) are shown on the
left. Immunoreactive bands migrating below 46 kDa represent
degradation products arising during late viral infection. B and C, casein kinase activity associated with wild-type
Nek2 (upper panel in B; dots in C)
and Nek2-K37R mutant protein (lower panel in B;
triangles in C). Kinase assays were carried out with
immunoprecipitates prepared from baculovirus infected Sf9 cells. These
were incubated at 30 °C, and reactions were stopped at the times
indicated (minutes). Samples were separated on SDS-PAGE, then
the gels were dried and autoradiographed. The positions of - and
-casein are indicated. For Panel C, the
-casein band
was cut out of the dried gels, and the radioactivity (cpm)
associated with each band was determined by scintillation
counting.
Biochemical Characterization of Recombinant Nek2 Protein
Kinase
Prompted by the description of Nek1 as a dual-specificity
kinase
(10) , we next asked whether human Nek2 might display any
tyrosine kinase activity. To this end, Sf9 cells were infected with
viruses encoding either wild-type Nek2 or the Nek2-K37R mutant, and
extracts were probed by immunoblotting with an anti-phosphotyrosine
antibody (Fig. 3, lanes 2 and 3). To provide
negative and positive controls, lysates from uninfected Sf9 cells and
from cells infected with a virus encoding the tyrosine kinase
p60 were analyzed in parallel (Fig. 3,
lanes 1 and 4). Only lysates from cells expressing
p60
showed an increase in the overall level of
phosphotyrosine, with p60
itself being the most
prominent tyrosine-phosphorylated protein visible (Fig. 3,
arrow). In contrast, no specific signals were produced by
anti-phosphotyrosine antibodies on immunoblots of either lysates of
Nek2 virus infected cells (Fig. 3, compare lane 2 with
lanes 1 and 3) or Nek2 immunoprecipitates (data not
shown). These data argue that Nek2 is likely to be a
serine/threonine-specific kinase, like Aspergillus NIMA but
unlike the murine dual-specificity kinase Nek1. This conclusion is
supported also by the results of phosphoamino acid analyses performed
on several
P-labeled substrates of Nek2 (see below).
Figure 3:
Expression of Nek2 in Sf9 cells does not
cause an increase in tyrosine phosphorylation levels. Sf9 insect cell
lysates were prepared 48 h post-infection with no virus (lane
1, C), wild-type Nek2 virus (lane 2,
wt), mutant Nek2-K37R virus (lane 3, K37R),
or p60-encoding virus (lane 4, src). Equal amounts of
protein from each lysate were then processed by SDS-PAGE and probed by
immunoblotting with the anti-phosphotyrosine antibody, 4C10. Molecular
masses are indicated on the left, and the position of the
tyrosine phosphorylated pp60 protein is marked on the
right.
As
summarized in , the casein kinase activity of human Nek2
displays several characteristics that are reminiscent of the
casein-kinase activity described for A. nidulans NIMA, but can
readily be distinguished from that of another major casein kinase,
casein kinase II (for review see Ref. 23). First, like NIMA but unlike
casein kinase II, Nek2 kinase was not able to utilize
[P]GTP as a phosphate donor, and
phosphotransfer from [
P]ATP could not be
inhibited by the addition of unlabeled GTP up to concentrations of 500
µM. Second, Nek2 casein kinase activity was unaffected by heparin
(up to 5 µg/ml), whereas substantial inhibition of casein kinase II
occurred at the same concentrations, as expected
(23) . In
further studies, we found that the
-casein kinase activity of Nek2
was sensitive to increases in ionic strength, with 200 mM NaCl
leading to an approximate 50% (reversible) reduction in activity.
Importantly, Nek2 activity was stimulated about 5-fold by 10
mM Mn
, unaffected by 10 mM
Ca
, and completely inhibited by 10 mM
Zn
. Variation of the Mn
concentration (from 0 to 50 mM), in the presence or
absence of 10 mM Mg
, revealed that maximal
stimulation of Nek2 activity occurred at Mn
concentrations of 2-10 mM; also, at these
concentrations of Mn
, Nek2 activity was independent
of the presence of Mg
. In the absence of divalent
cations, Nek2 kinase was inactive. Finally, the presence of the
phosphatase inhibitor okadaic acid (1 µM) in extraction and kinase
assay buffers stimulated Nek2 activity, suggesting that Nek2 may need
to be phosphorylated for maximal activity.
Evidence for Autophosphorylation of Nek2 Protein
Kinase
The observed shift in the electrophoretic mobility of
Nek2 suggested that human Nek2 was being post-translationally modified
in Sf9 cells. In particular, it appeared possible that the retarded
electrophoretic mobility of wild-type Nek2 might be the result, at
least in part, of autophosphorylation. In support of this view, no
molecular weight shift was observed with the catalytically inactive
Nek2-K37R mutant (Fig. 2A). To determine whether Nek2
protein was indeed phosphorylated in Sf9 cells in vivo,
baculovirus-infected cells were labeled with
[P]orthophosphate and Nek2 as well as Nek2-K37R
proteins were immunoprecipitated. As determined by immunoblotting, both
proteins were recovered in comparable amounts (Fig. 4A,
lower panel); however, only the wild-type Nek2 protein had
incorporated detectable amounts of
P
(Fig. 4A, upper panel), indicating that active
Nek2 kinase was required for phosphorylation of Nek2 in insect cells.
Phosphoamino acid analysis of the in vivo labeled Nek2 protein
showed predominantly phosphoserine with a minor contribution of
phosphothreonine, but no phosphotyrosine (Fig. 4B).
Tryptic phosphopeptide mapping revealed at least eight distinct spots,
indicating that the phosphorylation of Nek2 kinase in Sf9 cells is
complex (data not shown).
Figure 4:
In vivo phosphorylation of Nek2
but not Nek2-K37R protein in insect Sf9 cells. A, insect Sf9
cells were infected with either wild-type Nek2 (lane 2) or
Nek2-K37R (lane 1) recombinant baculoviruses and grown in the
presence of [P]orthophosphate. Nek2
immunoprecipitates were then prepared from cell extracts using the R31
Nek2-specific antiserum and separated by SDS-PAGE before
autoradiography (top panel,
P) or immunoblotting
with the Nek2-specific monoclonal antibody, NK1 (bottom panel,
IB). B, the phosphorylated Nek2 protein was cut out
of the dried polyacrylamide gel and subjected to phosphoamino acid
analysis. The positions of phosphoserine (S), phosphothreonine
(T,) and phosphotyrosine (Y) standards were
determined by ninhydrin staining of the TLC plate prior to
autoradiography.
Substrate Specificity of Nek2 Protein Kinase
To
compare the substrate specificity of human Nek2 with that of
Aspergillus NIMA and murine Nek1, a range of protein and
peptide substrates were assayed for their ability to be phosphorylated
in vitro by immunoprecipitated Nek2 (for a summary of results,
see ). In addition to -casein, Nek2 strongly
phosphorylated myelin basic protein and microtubule-associated protein
2 (MAP2); phosvitin, and histone H1 were phosphorylated moderately,
whereas enolase, a good in vitro substrate of several tyrosine
kinases
(24) , was not phosphorylated at all. Also, no
phosphorylation occurred on bovine serum albumin or immunoglobulin G,
and none of the Nek2 substrates was phosphorylated by the Nek2-K37R
mutant. Phosphoamino acid analyses were performed on
-casein,
myelin basic protein, and MAP2, as well as on in vitro phosphorylated Nek2, and all substrates were found to be
phosphorylated exclusively on serine and threonine residues; no
phosphotyrosine could be detected even after prolonged exposures (data
not shown).
Figure 5:
Phosphorylation of synthetic peptides by
human Nek2 kinase. In vitro kinase assays were performed on a
series of peptides, using Nek2 or Nek2-K37R immunoprecipitates.
Phosphorylation was then analyzed either by P81 phosphocellulose paper
assays (squares, PLM(42-72); diamonds,
PLM(64-72); circles, S6 peptide; triangles,
Kemptide, cdc2 peptide CK II peptide and poly(EY)) (A) or by
thin-layer chromatography (B), where phosphorylated peptides
are indicated with arrowheads as detailed in the text. The
amino acid sequence of the synthetic peptides tested is indicated in
single-letter code (C).
In contrast,
three synthetic peptides previously shown to be preferred substrates of
cAMP-dependent protein kinase (Leu-Arg-Arg-Ala-Ser-Leu-Gly;
Kemptide)
(26) , casein kinase II
(27) , and
p34 /cyclin B
(16) , respectively, were not
detectably phosphorylated by Nek2 (Fig. 5, A and
B). Likewise, as determined by SDS-PAGE on a 15% gel (data not
shown), Nek2 did not detectably phosphorylate poly(Glu,Tyr), although
this peptide represents a good substrate for many tyrosine kinases and
dual-specificity kinases, including murine Nek1
(10) . A
comparison of the available data on the substrate specificity of the
three Nek family members studied so far shows that there is a
remarkable similarity in the substrate specificity of fungal NIMA and
human Nek2, although the correspondence between the two kinases is not
perfect (see and ``Discussion'').
Cell Cycle Regulation of Endogenous Nek2 Activity in HeLa
Cells
The availability of recombinant Nek2 made it possible to
optimize extraction and assay conditions for detecting kinase activity
associated with this enzyme. These results set the stage for making
another attempt at detecting kinase activity associated with endogenous
Nek2 present in cultured human cells. As shown in
Fig. 6A, we are now able, for the first time, to detect
-casein kinase activity associated specifically with Nek2
immunoprecipitates (lane 2) but not with control
immunoprecipitates (lane 1). Attesting to the specificity of
this phosphorylation, it displayed the same characteristics (e.g. insensitivity to heparin; stimulation by Mn
ions) that had been determined for recombinant Nek2 (data not
shown). Phosphorylation of Nek2 itself was also evident in Nek2
immunoprecipitates (Fig. 6A), consistent with the
possibility that Nek2 may undergo autophosphorylation.
Figure 6:
Nek2 activity fluctuates during the cell
cycle in HeLa cells. A, immunoprecipitates were prepared from
asynchronous HeLa cells, using either preimmune (lane 1,
P) or anti-Nek2-specific immune serum (lane 2,
I). Kinase assays were then carried out in the presence of
casein, as indicated under ``Experimental Procedures''
(optimized conditions). Proteins were subjected to SDS-PAGE and
autoradiography. Molecular masses are indicated on the left,
and the positions of Nek2 and -casein are marked on the right.
B, HeLa cell extracts were prepared from an exponentially growing
population (lane 1), from cells blocked with nocodazole
(lane 2) or aphidicolin (lane 4), and from cells that
had been released for 6 h from the nocodazole block (lane 3)
or the aphidicolin block (lane 5). Samples were equalized for
protein content and subjected to SDS-PAGE and immunoblotting
(IB) (upper panel) or immunoprecipitation, using the
anti-Nek2 antibody. Immunoprecipitates were then used for kinase assays
carried out in the presence of [
P]ATP and
exogenous
-casein as a substrate. Reaction products were analyzed
by SDS-PAGE and autoradiography (lower panel;
P). The positions of Nek2 and
-casein are
indicated. C, a portion of each cell population used for
obtaining the data in Panel B was processed for the
determination of DNA content by FACS analysis, as described under
``Experimental Procedures.'' For each sample, the positions
of the G
phase peak and the G
/M phase peak are
marked by arrowheads.
To determine
whether Nek2 kinase activity might fluctuate during the cell cycle,
HeLa cells were synchronized as described under ``Experimental
Procedures.'' The abundance of Nek2 protein in each sample was
determined by immunoblotting (Fig. 6B, upper
panel), and Nek2 kinase activity was measured in Nek2
immunoprecipitates, using -casein as a substrate
(Fig. 6B, lower panel). In parallel, the DNA
content of each sample was analyzed by flow cytometry
(Fig. 6C). In comparison to an exponentially growing,
asychronous population of cells (Fig. 6, B, lane
1, and C, Panel 1), Nek2 activity was low in
mitotically arrested (Fig. 6, B, lane 2, and
C, Panel 2) and in early G
phase cells
(Fig. 6, B, lane 3, and C, Panel
3), but high in G
/S (Fig. 6, B,
lane 4; C, Panel 4) and G
/M
phase cells (Fig. 6B, lane 5, and C,
Panel 5). In all cases, Nek2 activity levels roughly
paralleled the abundance of Nek2 protein (Fig. 6B).
These results are consistent with the notion that Nek2 protein kinase
may play a role in cell cycle events prior to the onset of mitosis.
-casein as an exogenous substrate; alternatively, advantage may be
taken of an apparent autophosphorylation activity associated with Nek2.
Based on a comparison of various biochemical parameters, as well as a
survey of several protein and peptide substrates, we conclude that the
human Nek2 protein kinase is remarkably similar to its potential fungal
homolog NIMA. In particular, casein was found to be a suitable
exogenous substrate for assaying either Nek2 or NIMA. Importantly,
however, the casein-kinase activities of Nek2 and NIMA can readily be
distinguished from that of the ubiquitously expressed casein kinase II:
Nek2 and NIMA specifically target the
- rather than the
-isoform of casein, utilize only ATP but not GTP as a phosphate
donor, are unaffected by heparin, and are inhibited rather than
stimulated by 200 mM NaCl (see ). One striking
difference between Nek2 and NIMA concerns their response to
Mn
; whereas Nek2 is stimulated by this divalent
cation, NIMA is inhibited (21). Interestingly, in this respect Nek2
resembles Nek1 which was also reported to be stimulated by
Mn
(10) .
-casein and NIMA itself, do not
contain this sequence.
phase cells, but high throughout S and G
phase of the
cell cycle. We cannot rigorously exclude that these results might be
influenced by the use of drugs for cell cycle synchronization, but note
that our previous studies performed with size-fractionated HeLa cells
yielded very similar data on the expression of Nek2
protein
(12) . Interestingly, Nek2 activity was found to
fluctuate in parallel with the amounts of Nek2 protein, suggesting that
cell cycle regulation of this kinase may occur primarily, although
perhaps not exclusively, at the level of expression. These initial
studies on the regulation of Nek2 activity are consistent with the
hypothesis that human Nek2 may play a role in cell cycle events leading
to the onset of mitosis. A similar role has also been proposed for
fungal NIMA
(2) , but in contrast to Nek2 (this study), NIMA has
been reported to be maximally active in mitotically arrested
cells
(13) . This difference does not exclude that NIMA and Nek2
might carry out related biological functions, since the temporal
organization of certain cell cycle events (e.g. spindle
formation) is known to differ between fungi and mammals, but it raises
the possibility that vertebrate Nek2 may be required already at an
earlier cell cycle stage than fungal NIMA. Physiological substrates
remain to be identified for both NIMA and Nek2, and definitive judgment
on the potential functional homology between these kinases must await
discovery of such substrates.
Table:
Biochemical properties of NIMA-related kinases
and casein kinase II
Table:
Comparison of the substrate specificity of
human Nek2 with A. nidulans NIMA and murine Nek1
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.