(Received for publication, March 3, 1995; and in revised form, May 22, 1995)
From the
To investigate the degree of conservation of the cell
cycle-specific NIMA protein kinase of Aspergillus nidulans,
and to help direct its functional analysis, we cloned a homolog
(designated nim-1) from Neurospora crassa. Over the
catalytic domain NIM-1 is 75% identical to NIMA, but overall the
identity drops to 52%. nim-1 was able to functionally
complement nimA5 in A. nidulans. Mutational analysis
of potential activating phosphorylation sites found in NIMA, NIM-1, and
related protein kinases was performed on NIMA. Mutation of threonine
199 (conserved in all NIMA-related kinases) inhibited NIMA Initiation and exit from mitosis in Aspergillus nidulans requires the activation and inactivation of two cell
cycle-regulated protein kinases, both of which have maximal kinase
activity during mitotic initiation and also require mitotic transition
to down-regulate their elevated mitotic activities (Osmani et
al., 1988b, 1991a, 1991b, 1994; Ye et al., 1995; Pu and
Osmani, 1995). One, the p34 Temperature-sensitive mutations of nimA reversibly arrest cells in late G Several genes that encode protein
kinases with significant similarity (>40% identity over the
catalytic domain) to A. nidulans NIMA have been isolated from
higher eukaryotes. These putative kinases have been termed nek for nimA related kinases or nrk, also for nimA related kinase, due to their protein sequence similarity
to A. nidulans NIMA (Letwin et al., 1992; Schultz and
Nigg, 1993; Levedakou et al., 1994; Schultz et al.,
1994). A nimA-like gene has also been isolated from yeast
(Jones and Rosamond, 1990; Barton et al., 1992; Schweitzer and
Philippsen, 1992), and two related genes have been isolated from Trypanosome (Gale and Parsons, 1993). The role of the
NIMA-related kinases in cell cycle regulation remains to be
established, but mouse nek1 is highly expressed in germ line
cells, suggesting a role in meiosis (Letwin et al., 1992). In
addition, the level of human Nek2 protein is regulated through the cell
cycle, reaching a maximum during G To help identify important functional features of NIMA and to
determine the existence of functional homologs, a nimA-like
gene from Neurospora crassa was isolated. We have carried out
functional analysis of this gene and also defined important amino acid
residues required for NIMA function by mutagenesis and sequencing of
previously isolated temperature-sensitive alleles of nimA.
Protein kinase assays and Western
blot analysis were carried out as described previously (Ye et
al., 1995).
Figure 1:
Functional complementation of A.
nidulans nimA5 by nim-1. a, the region from
which nim-1 sequence was obtained on both strands is indicated
by the thickline. Sequence 5` to the internal EcoRI site was derived from genomic clones. Sequence 3` to the EcoRI site was derived from both genomic and cDNA clones.
Restriction sites used for subcloning are indicated. The location of
the long open reading frame encoding NIM-1 (ORF) is indicated,
ignoring the position of two proposed introns. Panelsb and c, complementation of nimA5 by subclones of N. crassa nim-1 constructs co-transformed with pyrG.
The plates in the upperpanel were incubated
at 32 °C (permissive temperature) for 3 days after transformation.
In the lowerpanel, an identical set of plates was
incubated at 42 °C (restrictive temperature). Only pUCX7-17
plus pyrG co-transformants showed growth at 42
°C.
Hypothetical translation identified a long
open reading frame (Fig. 2) encoding a protein with high
similarity to A. nidulans NIMA (Osmani et al., 1988b) (Fig. 3). The open reading frame was initiated from a methionine
codon in a good context to initiate translation and was preceded
closely by stop codons in each reading frame (underlined in Fig. 2). To maintain the open reading frame we propose the
presence of two introns as indicated in Fig. 2, the first being
107 and the second 52 bases in length. Both putative introns contained
consensus splice sites, and one is positioned in exactly the same
location as one of the two introns present in A. nidulans nimA (see below). We propose to call this locus nim-1 and
designate the translation product NIM-1. By Northern blot analysis nim-1 mRNA was estimated to be 3.0 kb in length (data not
shown).
Figure 2:
nim-1 DNA sequence and predicted
translation product. The nucleotide and amino acid (boldface)
numbers are given to the left. Several stop codons upstream of
the putative translational start codon are underlined. The
positions of two predicted introns are also
indicated.
Figure 3:
Kinase domain alignment of NIMA protein
kinase family. The kinase domains of NIMA, NIM-1, Kin3, Nek1, Nek2,
Stk2, and NrkA were aligned using GCG software. Positions with more
than half-identical residues are shaded. The 11-kinase
subdomains are marked with Romannumeralsbelow the sequences. Kinase signature residues are indicated above the aligned sequences as are the amino acid substitutions of the
three nimA mutant alleles, which are marked in
lowercase.
NIM-1 contains 779 amino acids with a molecular weight of
86,056 and a pI of 10.8, similar to the values for NIMA (Osmani et
al., 1988b). Alignment of NIM-1 to NIMA shows both to contain the
conserved features of protein kinases in their N termini (Hanks et
al., 1988). Over the catalytic domain of 295 amino acids they have
75% identical residues, and over the first 500 amino acids 62% are
identical and 90% similar. Overall the sequence identity of NIM-1 to
NIMA is 52%, and overall similarity 64.8%. Like NIMA, the NIM-1 kinase
domain contains a cysteine residue (amino acid 34) two positions
upstream from the putative ATP interactive lysine, in contrast to other
protein kinases, which generally have an alanine, and occasionally a
valine or an isoleucine, at this position (Fig. 3). The
NIMA-related kinases all have an N-terminal kinase domain and a
C-terminal extension. However, unlike the majority of other kinases,
NIMA has an insert of 22 amino acids in kinase domain VI, as does Kin3
and NIM-1. However, Kin3 also contains a second insert of 38 amino
acids in this region that is absent in NIMA and NIM-1 and most other
protein kinases. NIM-1 is thus overall most similar to NIMA (Fig. 3). One feature found both in NIM-1 (starting at 306)
and Nek2 (starting at 306) is a leucine zipper-like motif. This motif
has been implicated in DNA binding and protein-protein interactions
(Landschulz et al., 1988). The significance of this motif in
NIM-1 and Nek2 is at present unclear.
To address the issue of whether nim-1 could functionally complement nimA5 as a single
copy gene, rather than as a high copy number suppressor, Southern blot
analysis was carried out on DNA isolated from a wild type strain and
five transformants co-transformed with pyrG and the
full-length nim-1. Duplicate blots were hybridized with DNA
probes labeled to an equal specific activity. With the A. nidulans
nimA-specific probe all strains generated an equivalent signal,
indicating equal loading and transfer to the blot (Fig. 4a). With the N. crassa nim-1 probe wild
type DNA showed no hybridization under the high stringency conditions
used. Based on the relative intensities of the signal produced,
transformants 1, 2, 3, and 5 had a single copy of the N. crassa
nim-1 integrated into their genome (Fig. 4a).
Figure 4:
Complementation of nimA5 by
single copy nim-1. a, Southern blot analysis. Aspergillus genomic DNAs from wild type (lane0) and five strains (lanes1-5)
transformed with nim-1 were hybridized with a nim-1-specific or nimA-specific probe as indicated.
Some bands resulting from partial digestion are apparent. b,
growth of nim-1-complemented nimA5 strain. R153 is
the wild type, SO6+pyrG is a nimA5 mutant strain
transformed with pyrG, and
SO6+pyrG+nim-1 is the same strain
co-transformed with pyrG and Neurospora
nim-1.
To establish how well a single copy of N. crassa nim-1 could complement the nimA5 mutation in A. nidulans we grew strains at 32 °C (permissive temperature), at 37
°C (semi-restrictive temperature), and at 42 °C (restrictive
temperature). Colony diameters were measured for 5 days, and the final
colonies were photographed (Fig. 4b). At 32 °C no
difference in growth rate could be detected among the control strain,
the nimA5-containing strain, and a strain containing nimA5 and a single copy of nim-1. However at 37 °C growth
of the nimA5 strain was slightly inhibited, and the control
strain and nimA5 + nim-1 strain grew at
essentially the same rate. At 42 °C the nimA5 strain was
unable to form a colony, but the wild type and nimA5 strain
containing nim-1 could both form a conidiating colony. These
data indicate that a single copy of N. crassa nim-1 can
complement the nimA5 mutation of A. nidulans for
vegetative growth and conidia formation.
At 32 °C on alcA-inducing media (ethanol) several transformants were
unable to form colonies (Table 1). For strains transformed with
pAL5B1 and pAL5B1
Cells containing nimA1, nimA5, or nimA7 all arrest in G
Figure 5:
Effects of nimA1 block release on
NIMA kinase activity and protein levels. A nimA1-containing
strain (SO23) was grown to early log phase (Ex) and then shifted to 42
°C for 3 h before downshifting to 32 °C. Samples were taken at
the times indicated for protein isolation and determination of
chromosome mitotic index. The level of NIMA protein was determined by
immunoprecipition followed by Western blotting using NIMA-specific
antisera visualized by ECL. The immunoprecipitates were also assayed
for NIMA
One hour after the shift to the restrictive
temperature the nimA1 strain had decreased NIMA kinase
activity. The activity then recovered by 3 h to a level slightly higher
than the preshift levels. Upon return to permissive temperature NIMA
kinase activity increased, and cells entered mitosis synchronously. The
kinase activity then decreased as cells completed mitosis (Fig. 5). The level of NIMA protein also showed dramatic changes
in abundance and phosphorylation state during the transition through
G
Figure 6:
Mutational analysis of potential
phosphorylation sites of NIMA. a, location of sites mutated in
NIMA and alignment of these sites with analogous phosphorylation sites
(*) in mitogen-activated protein kinase (MAPK), cyclic AMP
dependent protein kinase (cAPK), and human cdc2 kinase (CDC2Hs). The sites conserved between all
NIMA-related protein kinases within this region of their catalytic
domain are shaded. The numbers refer to NIMA sites. b, the indicated mutant versions of NIMA were expressed in E. coli. Protein extracts from cells expressing equal amounts
of soluble NIMA were assayed for
The T199A mutant form of NIMA
was not completely inactive as a protein kinase, since it was able to
undergo autophosphorylation, as could be observed in the kinase
reactions to which Since the T199A
form of NIMA was not completely inactivated as a kinase we tested its
ability to function in A. nidulans. An alcA promoter-driven form of the T199A mutant in the expression vector
pAL5 (pAL5B1 We have isolated a functional homolog of the A. nidulans
nimA cell cycle regulator from N. crassa called nim-1, the first such homolog isolated. Over the catalytic
domain the two kinases are 75% identical, but their C-terminal
extensions are much less similar. Because of the divergence between the nimA homologs from A. nidulans and N.
crassa, which are both filamentous members of the Ascomycetes fungi, it is unlikely that higher eukaryotic functional homologs
will have highly conserved C-terminal noncatalytic domains. However,
there are important functional domains in the C terminus of NIMA, which
is essential for function in A. nidulans (Pu and Osmani, 1995)
and contains three putative bipartite nuclear localization motifs. This
motif consists of two basic amino acids followed by a spacer region,
which can vary from 10 to 37 amino acids in length, and ends with a
cluster of five amino acids containing at least three basic residues
(Dingwall and Laskey, 1991). The first such motif in NIMA starts at
amino acid 299. A similar motif also occurs at a comparable position in
NIM-1, but the second basic clusters do not align, since the NIM-1
spacer is composed of 19 amino acids and the NIMA spacer is composed of
11 amino acids. Nor do the other two nuclear localization motifs of
NIMA align with comparable motifs in NIM-1. However, NIM-1 does contain
another nuclear localization motif starting at amino acid 633 in a
region that is very poorly conserved between the two kinases.
Inspection of the other NIMA-related kinases identified at least one
putative bipartite nuclear localization motif in Nek1, Nek2, and Stk2
but none in Kin3 or NrkA. This motif is thus common but not universal
in the NIMA-related kinases. At present little data is available
regarding the subcellular localization of any of these kinases, but
given their overall basic nature and the existence of multiple
bipartite nuclear localization signals in several of these kinases, a
nuclear localization appears likely for at least NIMA, NIM-1, Nek2,
Nek1, and Stk2. Indeed, it has recently been shown that NIMA expressed
in HeLa cells does localize to the nucleus (Lu and Hunter, 1995). Another intriguing motif found in the C terminus of NIMA are PEST
sequences, which have been implicated in mediating rapid protein
turnover, and such motifs are typically found in proteins that have
very short half-lives (Rogers et al., 1986). Two regions of
NIMA gave a score of 12.6 (amino acids 406-437) and 16.4 (amino
acids 502-524), and over comparable regions NIM-1 also scored
high for PEST motifs, 5.3 (amino acids 393-444) and 11.0 (amino
acids 522-543). This indicates that NIMA and NIM-1 may have short
half-lives, and we know that removal of the PEST sequences from NIMA
generates a very stable kinase that arrests cells in mitosis (Pu and
Osmani, 1995) and that normally NIMA protein is proteolytically
destroyed during mitotic transition (Ye et al.(1995) and Fig. 5). In light of the mitotic instability of NIMA it is
intriguing that it contains a motif, RXXLXXXXN, in
protein kinase subdomain III, which is similar to a mitotic cyclin
destruction box (Glotzer et al., 1991). None of the other
NIMA-related kinases match at all three conserved positions in this
region, but human Nek2 does contain this motif starting at position
amino acid 361 and is also degraded during the cell cycle (Schultz et al., 1994). Sequence analysis of three different mutant
alleles of nimA identified three amino acid substitutions that
cause temperature sensitivity to NIMA, and each residue changed is in
or near the protein kinase domain. nimA7 has glutamic acid 41
changed to a glycine, and the NIMA-related kinases NIM-1, Nek1, and
Nek2 all contain a glutamic acid at the equivalent position. Lysine 40,
which is adjacent to glutamic acid 41, has been implicated in ATP
binding in protein kinases. This mutation may therefore interfere with
the ability of NIMA to bind ATP correctly at the restrictive
temperature. nimA5 changes tyrosine 91 to an asparagine, and
NIM-1, Kin3, Nek1, and Nek2 also have a tyrosine at this position. This
region is clearly conserved among the NIMA-like protein kinases, and it
has been suggested that a conserved motif, based on this region of
homology, may be a characteristic of NIMA-like protein kinases (Schultz et al., 1994). The nimA1 mutation is located at the
border of the kinase domain and changes leucine 304 to a proline. This
mutation is the weakest of the three, and cells arrested at restrictive
temperature containing this allele still retain some protein kinase
activity. The three temperature-sensitive alleles of nimA arrested cells in G Comparison of published protein kinase sequences similar to NIMA and
NIM-1 identified a conserved potential phosphorylation site
(Thr If NIMA is normally activated by phosphorylation
of Thr Several questions remain. Are any of the other
NIMA-related protein kinases functional homologs of A. nidulans
nimA? In addition, if Thr The nucleotide
sequence(s) reported in this paper has been submitted to the
GenBank®/EMBL Data Bank with accession number(s)
L42573[GenBank Link].
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-casein
kinase activity and abolished its in vivo function. This site
conforms to a minimal consensus phosphorylation site for NIMA
(FXXT) and is analogous to the autophosphorylation site of
cyclic-AMP-dependent protein kinases. However, mutation of a unique
cysteine residue found only in the catalytic site of NIMA and NIM-1 had
no effect on NIMA kinase activity or function. Three
temperature-sensitive alleles of nimA that cause arrest in
G
were sequenced and shown to generate three different
amino acid substitutions. None of the mutations prevented accumulation
of NIMA protein during G
arrest, but all prevented the
p34
/cyclin B-dependent phosphorylation of NIMA
normally seen during mitotic initiation even though
p34
/cyclin B H1 kinase activity was fully
activated.
/cyclin B histone H1
kinase, is a universal regulator of mitosis in organisms ranging from
fungi to humans, and much is known about its regulation through the
cell cycle (Draetta, 1990; Nurse, 1990; Norbury and Nurse, 1992). The
second kinase, called NIMA, has at present only been identified as a
key component of mitotic regulation in A. nidulans (Doonan,
1992). However, expression of A. nidulans nimA in fission
yeast, Xenopus, and human cell lines (O'Connell et al.,
1994; Lu and Hunter, 1995) can induce mitotic events in these systems,
and expression of a dominant negative version of nimA leads to
an arrest of human cells in G
(Lu and Hunter, 1995). These
data strongly indicate that NIMA is a universal component of cell
cycle regulation, but until the current work no functional homolog had
been identified.
(Oakley and Morris,
1983). At the G
arrest point of nimA5 cells
contain a stable interphase microtubule array and show no signs of
chromatin condensation or spindle formation, as determined using either
indirect immunofluorescence or electron microscopy (Morris, 1976;
Oakley and Morris, 1983; Bergen et al., 1984; Osmani et
al., 1987). Surprisingly, however, such arrested cells have
activated p34
H1 kinase activity even though
they are clearly arrested in G
(Osmani et al.,
1991a). This suggests that NIMA could function either downstream or in
parallel to p34
in order to promote mitosis.
Evidence has recently accumulated suggesting that NIMA is activated and
phosphorylated by p34
/cyclin B during mitotic
initiation (Ye et al., 1995). The phosphorylation of NIMA by
p34
/cyclin B is not required for a basal level
of NIMA kinase activity but is required for the final mitotic
activation of NIMA kinase. This interaction may play a role in
coordinating the mitotic promoting activities of these two protein
kinases (Ye et al., 1995). NIMA protein also accumulates
during G
, as does its kinase activity, but it is then
inactivated during mitosis by proteolysis (Ye et al., 1995).
The degradation of mitotic NIMA is required for cells to exit from
mitosis (Pu and Osmani, 1995).
, which indicates that
Nek2 may play a cell cycle-specific role in humans (Schultz et
al., 1994). The cell cycle variation in Nek2 protein level is
similar to the fluctuations seen for NIMA protein during the cell cycle
of A. nidulans (Ye et al., 1995; Pu and Osmani,
1995).
Strains, Media, and General
Techniques
Aspergillus strains used were R153 (wA3; pyroA4), SO6 (nimA5; wA2; pyrG89; cnxE16; choA1; yA2; chaA1), and SO23 (nimA1; wA2; nicB8
or A2; choA1). Growth conditions, media, and transformation
were as described previously (Morris, 1976; Osmani et al.,
1987, 1988a). N. crassa genomic and cDNA libraries (Orbach et al., 1986, 1990) were obtained from the Fungal Genetic
Stock Center (University of Kansas Medical Center). Restriction enzymes
were obtained from Promega (Madison, WI), and T4 DNA ligase was
purchased from New England Biolabs (Beverly, MA). Universal primers for
M13 mp18 and M13 mp19 and specific primers for N. crassa nim-1 were used for sequencing (Sanger et al., 1977) using
Sequenase obtained from U.S. Biochemical Corp. To generate overlapping
clones for sequencing, nested deletion was performed using Cyclone I
Biosystem from IBI (New Haven, CT). For nimA mutant allele
sequencing, two overlapping polymerase chain reaction fragments were
amplified using genomic DNA as template isolated from wild type and the
three nimA mutant strains. A 5` polymerase chain reaction
primer pair (5`-ACAATGGCAATCGCACTGGCG-3` forward primer and
5`-GGCTACACGATCCTGAACCTCGCA-3` reverse primer) and a 3` polymerase
chain reaction primer pair (5`-GGTAAAGGCAAGATTAGAGATCGAT-3` forward
primer and 5`-AAAGATGCAGCAGCGCAAGAAATG-3` reverse primer) were used to
amplify the overlapping fragments (5` fragment = 1269 base
pairs, 3` fragment = 1297 base pairs). After 35-40 cycles
of denaturing (94 °C, 35 s), annealing (65 °C, 55 s), and
extension (72 °C, 2 min), the double stranded DNA templates were
purified with GeneClean II (Bio 101, La Jolla, CA) and sequenced
completely on both stands with the Applied Biosystems (Foster City, CA) Taq Dyedeoxy terminator cycle sequencing kit, using a series
of oligonucleotide primers. Sequencing reactions were purified by G50
Sephadex gel filtration equilibrated in deionized HO and
run on an Applied Biosystems 373A sequencer. For Southern blot analysis
genomic DNAs were isolated and restricted by KpnI followed by
electrophoresis and standard Southern blot procedures. Blots were
probed with a 2.0-kb
(
)PstI nimA fragment or a 2.4-kb nim-1 fragment labeled to equal
specific activity. DNA sequence analysis and alignment were performed
using GCG software (University of Wisconsin) and PC-Gene software
(Intelligenetics Inc). In vitro mutagenesis was performed on
plasmid pET21a-NIMA (Ye et al., 1995) using a site-directed
mutagenesis kit from Clontech (Palo Alto, CA). After mutagenesis a BamHI fragment containing the mutation was used to replace the
corresponding BamHI fragment of plasmid pAL5B (Pu and Osmani,
1995). Primers used were as follows: S198A,
5`-CACGACTTTGCGGCCACCTATGT-3`; T199A,
5`-ACTTTGCGTCCGCCTATGTCGGAACAC-3`; Y200A,
5`-GCGTCCACCGCTGTCGGAACACC-3`; and Y203A,
5`-CCACCTATGTCGGAGCACCATTCTACA-3`. For each mutagenesis the selection
primer was 5`-AACAATTCCCCTCGAGAAATAATTTTG-3`, which destroys an XbaI site in pET21a-NIMA.
Plasmid Constructs for Complementation of
nimA5
pUCX7 contains a 3.2-kb XbaI genomic fragment of nim-1 cloned into the XbaI site of pUC18. To generate
pUCX7-4, a 0.6-kb HindIII fragment from pUCX7-17
was removed before religation. Plasmid pUCX7-27 was generated by
inserting the 2.4-kb HindIII genomic fragment from
pUC7-17 into pUCX7-4 in the reverse orientation.
Molecular Cloning of a NIMA-like Kinase from Neurospora
crassa
An N. crassa J1 genomic DNA library
(Orbach et al., 1986) was probed with nimA cDNA under
low stringency conditions. DNA was isolated from purified clones, and
the hybridizing restriction fragments were cloned into both M13 and
pUC18. We attempted to isolate a full-length cDNA from two different
cDNA libraries (Orbach et al., 1990) but were unable to
isolate a full-length cDNA. The longest cDNA isolated and the genomic
clones were sequenced on both strands as depicted in Fig. 1a.
nim-1 Encodes the First Isolated Functional Homolog of A.
nidulans nimA
To test the ability of nim-1 to
functionally complement the nimA5 mutation of A. nidulans we transformed it into A. nidulans. Plasmid
pUCX7-17 contains nim-1 as a 5.0-kb HindIII-XbaI fragment (Fig. 1b) in
pUC18. This fragment contains the open reading frame encoding NIM-1
flanked 5` and 3` by about 1.6 and 1.0 kb, respectively. Two control
plasmids were also constructed in the same vector (Fig. 1b). Strain SO6 (nimA5 and pyrG89) was transformed with a plasmid containing pyrG (Oakley et al., 1987) alone to complement the pyrG89 mutation or in combination with one of the plasmids described
above. After limited growth at the permissive temperature on selective
media one-half of the plates were shifted to the restrictive
temperature of 42 °C. After 2 days of growth (Fig. 1c) only transformants receiving both pyrG and pUCX7-17 were able to form colonies at 42 °C. The
control plasmids or pyrG alone allowed colony formation at 32
°C but not at 42 °C. Since one of the control plasmids contains
the complete insert, with part being reversed, it indicates that the
complementing activity resides in NIM-1 and not another protein encoded
by the insert DNA of pUCX7-17.
The Conserved Cysteine Residue -2 from the
ATP-interactive Lysine in the Catalytic Site of NIMA and NIM-1 Is Not
Essential for Function
Unlike all other kinases that we are
aware of, both NIMA and NIM-1 have a cysteine -2 from the
conserved lysine residue in the ATP binding pocket. This unusual
conservation led us to ask if this specific residue (cysteine 38) is
important to the function of NIMA in A. nidulans. Three forms
of nimA were cloned into the A. nidulans expression
vector pAL5 to put their expression under control of the alcA promoter (Waring et al., 1989; Doonan et al.,
1991; Pu and Osmani, 1995). These constructs were introduced into a nimA5-containing strain and tested for their ability to
replace nimA5 function at the restrictive temperature (Table 1). Both the wild type and C38S versions of nimA were able to complement the nimA5 mutation at 42 °C
only when the alcA promoter was active due to growth on either
glycerol (alcA noninduced nonrepressed) or ethanol (alcA induced). The kinase-negative version of nimA was unable
to complement under identical conditions. These data indicate that
cysteine 38 is not essential for NIMA function under these test
conditions. A similar result was obtained when cysteine 38 was changed
to an alanine, the most commonly found amino acid at this location in
other protein kinases (data not shown).
this is most likely due to the
presence of multiple copies of the plasmids, since we have previously
shown that high expression levels of active NIMA causes premature
mitotic induction (Osmani et al., 1988b), which is lethal. For
the kinase-negative construct the inhibition of growth is caused by
dominant negative effects as these cells arrest with nuclei in an
interphase configuration (data not shown), a result also observed by Lu
and Means(1994).
Sequence Determination of Three Temperature-sensitive
Alleles of nimA and Activation/Inactivation of NIMA during
Mitosis
To define the nimA1, nimA5, and nimA7 mutations at the DNA level we sequenced DNA amplified by
polymerase chain reaction from the mutants and a wild type strain. This
analysis confirmed the previously published cDNA sequence of nimA (Osmani et al., 1988b) and also identified two introns
after positions 491 (64 base pairs) and 544 (53 base pairs). The first
is in the same position as the first putative intron of nim-1.
For each mutant allele we identified three different point mutations
that generated a single amino acid substitution (Table 2).
when grown or shifted to the
restrictive temperature. Since the level of NIMA protein accumulates
during G
and is destroyed upon mitotic progression (Ye et al., 1995; Pu and Osmani, 1995) we considered the
possibility that the temperature-sensitive alleles of nimA could cause thermal instability. To test this hypothesis, we
arrested cells in G
by shifting them to the restrictive
temperature (42 °C). Cells were then released into mitosis by
shifting to the permissive temperature. The mitotic index of the
cultures and the level of NIMA protein and NIMA kinase activity were
determined. Representative data for the nimA1 allele are shown
in Fig. 5.
-casein kinase activity using
[
-
P]ATP followed by SDS-PAGE and
autoradiography. p34 was immunoprecipitated, and its H1 kinase activity
was assayed using [
-
P]ATP followed by
SDS-PAGE and autoradiography. The chromosome mitotic index was
determined after fixation and staining of DNA with
4',6-diamidino-2-phenylindole.
and mitosis. At the G
arrest point of nimA1 NIMA protein accumulated to high levels, but it was only
partially active. Upon the return to permissive temperature NIMA
protein was hyperphosphorylated, and its mobility decreased during
SDS-PAGE (Fig. 5). As cells progressed through mitosis NIMA
protein was proteolytically destroyed. Similar results were obtained
using the nimA5 and nimA7 alleles (data not
shown),
(
)but these two mutations caused arrest
in G
with much reduced NIMA kinase activity, although all
three mutations arrested in G
with fully activated
p34
/cyclin B H1 kinase activity ( Fig. 5and data not shown). These data demonstrate that the
temperature-sensitive alleles of nimA do not prevent
accumulation of NIMA protein during G
. However, these
alleles do prevent the hyperphosphorylation and full activation of NIMA
that normally occurs during mitotic initiation.
Mutation of a Conserved Potential Autophosphorylation
Site in NIMA Inhibits Its Normal Kinase Activity
Several classes
of serine/threonine-specific protein kinases, including cyclic
AMP-dependent protein kinase (Steinberg et al., 1993), the
cyclin-dependent protein kinases (Booher and Beach, 1986), and
mitogen-activated protein kinases (Payne et al., 1991), all
require phosphorylation in a specific region of their catalytic domains
to activate their protein kinase potential. This region, loop
L, which connects protein kinase domains VII and VIII
(Hanks et al., 1988), contains several potential
phosphorylation sites in NIMA, which are in positions analogous to the
phosphorylation sites required for cyclic AMP-dependent protein kinase,
cyclin-dependent protein kinases, and mitogen-activated protein kinase
activation (Fig. 6a). Interestingly, one particular
site (Thr
of NIMA) is conserved among all of the
NIMA-related kinases, and phosphorylation of the analogous site in
cyclic AMP-dependent protein kinase is required for normal cyclic
AMP-dependent protein kinase activity. We therefore generated four
mutant versions of NIMA in which one of the four potential
phosphorylation sites was changed to an alanine (S198A, T199A, Y200A,
and T203A). These constructs were then expressed in Escherichia
coli, and equal amounts of NIMA protein were tested for
-casein kinase activity. Only the T199A mutant form of NIMA had
greatly reduced
-casein kinase activity compared with either the
wild type or the other mutant forms of NIMA (Fig. 6b).
Thus threonine 199 of NIMA is a candidate phosphorylation site required
for NIMA
-casein kinase activity.
-casein kinase activity using
[
-
P]ATP followed by SDS-PAGE and
autoradiography. An overexposed film is shown to demonstrate the low
level of T199A kinase activity.
-casein was added (Fig. 6b). In
addition, if a peptide substrate was used (Lu et al., 1994),
then significant kinase activity (30% of wild type) could be measured
for the T199A form of NIMA. However, the activity of T199A NIMA was not
improved by increasing the concentration of either
-casein or ATP.
The K40M kinase-negative version of NIMA was still inactive as a kinase
using the peptide as substrate, but the S198A and Y200A versions had
similar activity to the wild type (data not shown).
) was introduced into a nimA5-containing strain and tested for its ability to replace nimA5 function at restrictive temperature. On alcA-inducing media at restrictive temperature no
complementation of nimA5 was observed using the T199A
construct although wild type alcA driven NIMA was able to
complement (Table 1). Thus, Thr
is not only
important for NIMA
-casein kinase activity when expressed in E. coli, it is also essential for NIMA function in Aspergillus.
with elevated NIMA protein levels.
These mutations do not therefore affect the expression of the mutant
proteins but instead render them functionally inactive at restrictive
temperature. The accumulated proteins were not hyperphosphorylated in
G
but were rapidly phosphorylated upon release into
mitosis. This hyperphosphorylation is normally seen after activation of
p34
during mitotic initiation (Ye et
al., 1995). Since each of the nimA alleles causes G
arrest with fully activated p34
/cyclin B
H1 kinase activity it is unclear why NIMA is not hyperphosphorylated by
p34
/cyclin B at the G
arrest point.
One possibility is that at the G
arrest points of nimA1, nimA5, and nimA7 the NIMA protein has
not assumed a conformation that is a suitable substrate for
p34
/cyclin B. Upon release to permissive
temperature, NIMA would be able to assume an active configuration and
then become a substrate for p34
/cyclin B.
Alternatively, if NIMA and p34
are in different
subcellular compartments prior to initiation of mitosis, activation of
NIMA may allow the active p34
/cyclin B kinase
access to NIMA within the cell, which it could then phosphorylate.
in NIMA) analogous to the activating site
autophosphorylated in cyclic AMP-dependent protein kinase. Mutation of
Thr
specifically affected NIMA kinase activity when
expressed in E. coli, although other mutations in the same
region had no effect. Thus, like cyclic AMP-dependent protein kinase,
the NIMA family of protein kinases may require (auto?)phosphorylation
at Thr
for normal protein kinase activity. Although
severely compromised as a
-casein kinase the T199A mutant still
retained the ability to undergo autophosphorylation and partially
retained the ability to phosphorylate a NIMA-specific peptide
substrate. The equivalent mutant form of cyclic AMP-dependent protein
kinase (in which Thr
of cyclic AMP-dependent protein
kinase is also changed to an alanine) is also not completely inactive
as a protein kinase but has greatly increased K
values for both the ATP and protein substrates (Steinberg et al., 1993). Thus mutation of this site in cyclic
AMP-dependent protein kinase and NIMA has similar effects on their
kinase activities, since both kinases are compromised by this mutation
but neither is completely inactivated by it. However, the T199A mutant
form of NIMA, although still partially active as a protein kinase, was
found to be functionally inactive, demonstrating the importance of this
particular residue.
, then NIMA is able to autophosphorylate at this
position when expressed in E. coli. Thr
, and its
equivalent in NIM-1, Kin3, and Nek2, is found within a region that
corresponds to the NIMA phosphorylation consensus sequence
FXXT (Lu et al., 1994), further supporting the notion
that NIMA autophosphorylates at this site. NIMA is known to undergo
autophosphorylation on multiple sites when expressed in E. coli (Lu et al., 1993), and mapping studies will have to be
carried out to determine if NIMA does in fact phosphorylate Thr
when expressed in E. coli. We know that NIMA is
phosphorylated during G
to yield an active protein kinase
in A. nidulans and that it is then further phosphorylated
during mitotic initiation after activation of the
p34
/cyclin B protein kinase (Ye et
al., 1995). As the nimA1, nimA5, and nimA7 alleles all arrest in G
with elevated levels of NIMA
protein, which is largely inactive, these mutations may prevent
autophosphorylation of NIMA at threonine 199. Testing this possibility
will require mapping of the phosphorylation sites of NIMA during cell
cycle progression. This task is likely to be difficult since, based on
molecular weight shifts seen during SDS-PAGE, the NIMA protein is
phosphorylated at multiple sites during the cell cycle (Ye et
al., 1995).
is phosphorylated in
vivo, does the NIMA family of kinases require autophosphorylation
of Thr
for function, or is there a NIMA-activating
kinase?
We are most thankful to Liz Cigelnik for excellent
technical assistance.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.