©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Isolation of a Functional Homolog of the Cell Cycle-specific NIMA Protein Kinase of Aspergillus nidulans and Functional Analysis of Conserved Residues (*)

(Received for publication, March 3, 1995; and in revised form, May 22, 1995)

Robert T. Pu Gang Xu Liping Wu John Vierula (1) Kerry O'Donnell (2) Xiang S. Ye Stephen A. Osmani (§)

From the  (1)Weis Center For Research, Geisinger Clinic, Danville, Pennsylvania 17822-2617, the Department of Biology, Carleton University, 1125 Colonel By Drive, Ottawa, Ontario, Canada K1S 5B6, and (2)National Center for Agricultural Utilization Research, United States Department of Agriculture, Peoria, Illinois 61604

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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 -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.


INTRODUCTION

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/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.

Temperature-sensitive mutations of nimA reversibly arrest cells in late G (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).

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, 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).

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.


EXPERIMENTAL PROCEDURES

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.

Protein kinase assays and Western blot analysis were carried out as described previously (Ye et al., 1995).

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.


RESULTS

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.


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.

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.

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.

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).



At 32 °C on alcA-inducing media (ethanol) several transformants were unable to form colonies (Table 1). For strains transformed with pAL5B1 and pAL5B1 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).



Cells containing nimA1, nimA5, or nimA7 all arrest in G 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.


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 -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.



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 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.


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 -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.



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 -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).

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) 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.


DISCUSSION

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 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.

Comparison of published protein kinase sequences similar to NIMA and NIM-1 identified a conserved potential phosphorylation site (Thr 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.

If NIMA is normally activated by phosphorylation of Thr, 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).

Several questions remain. Are any of the other NIMA-related protein kinases functional homologs of A. nidulans nimA? In addition, if Thr is phosphorylated in vivo, does the NIMA family of kinases require autophosphorylation of Thr for function, or is there a NIMA-activating kinase?


FOOTNOTES

*
This work was supported by the Geisinger Foundation and by National Institutes of Health Grant GM42564 (to S. A. O.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank®/EMBL Data Bank with accession number(s) L42573[GenBank Link].

§
To whom correspondence should be addressed: Weis Center for Research, Geisinger Clinic, 100 N. Academy Ave., Danville, PA 17822-2617. Tel.: 717-271-6677; Fax: 717-271-6701; SAO{at}SMTP.GEISINGER.EDU

The abbreviations used are: kb, kilobase(s); PAGE, polyacrylamide gel electrophoresis.

X. S. Ye and S. A. Osmani, manuscript in preparation.


ACKNOWLEDGEMENTS

We are most thankful to Liz Cigelnik for excellent technical assistance.


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