Mitotic Histone H3 Phosphorylation by the NIMA Kinase in Aspergillus nidulans

Cell, Vol. 102, 293–302, August 4, 2000, Copyright 2000 by Cell Press

Mitotic Histone H3 Phosphorylation by the NIMA Kinase in Aspergillus nidulans Colin P. C. De Souza, Aysha H. Osmani, Li-Ping Wu,† Jeffrey L. Spotts, and Stephen A. Osmani* Henry Hood Research Program Weis Center for Research Geisinger Clinic Danville, Pennsylvania 17822

Summary Phosphorylation of histone H3 serine 10 correlates with chromosome condensation and is required for normal chromosome segregation in Tetrahymena. This phosphorylation is dependent upon activation of the NIMA kinase in Aspergillus nidulans. NIMA expression also induces Ser-10 phosphorylation inappropriately in S phase–arrested cells and in the absence of NIMXcdc2 activity. At mitosis, NIMA becomes enriched on chromatin and subsequently localizes to the mitotic spindle and spindle pole bodies. The chromatin-like localization of NIMA early in mitosis is tightly correlated with histone H3 phosphorylation. Finally, NIMA can phosphorylate histone H3 Ser-10 in vitro, suggesting that NIMA is a mitotic histone H3 kinase, perhaps helping to explain how NIMA promotes chromatin condensation in A. nidulans and when expressed in other eukaryotes. Introduction Key to cell cycle progression are the structural changes in DNA necessary for replication, transcription, repair, and segregation. Mitotic segregation of DNA requires a dramatic compacting of interphase chromatin into highly condensed chromosomes. While this is one of the most fundamental of cellular processes, it is still relatively poorly understood on a molecular level. Recent studies have begun to define the role of the SMC (structural maintenance of chromosomes) proteins in mitotic chromatin condensation (reviewed in Murray, 1998; Hirano, 1999). Mutations in SMC proteins lead to defects in chromosome condensation in fission yeast (Saka et al., 1994; Sutani et al., 1999). In Xenopus and fission yeast, the 13S condensin complex consists of two SMC proteins and three non-SMC subunits (Kimura and Hirano, 1997; Sutani et al., 1999). Xenopus 13S condensin activity promotes chromatin condensation in the presence of topoisomerase II by introducing a globalpositive writhe into interphase DNA in an ATP-dependent manner (Kimura et al., 1999). In Xenopus, 13S condensin is nuclear throughout the cell cycle (Hirano and Mitchison, 1994), and its supercoiling activity is regulated by mitosis-specific phosphorylation that appears to be carried out by the cdc2 kinase (Kimura et al., * To whom correspondence should be addressed (e-mail: sosmani@ geisinger.edu). † Present address: Millennium Pharmaceuticals Inc., Cambridge, Massachusetts 02139.

1998). Phosphorylation by cdc2 is also important for the nuclear localization of 13S condensin complex in S. pombe, which differs from Xenopus in that it is only nuclear during mitosis (Sutani et al., 1999). Chromatin consists of DNA associated with histone proteins in highly conserved structures called nucleosomes. Each nucleosome is composed of two units each of the four highly conserved core histones, H2A, H2B, H3, and H4 and a single linker histone H1 (Kornberg, 1977). Posttranscriptional modifications including acetylation, methylation, and phosphorylation of histones play central roles in transcriptional regulation and chromatin condensation, and in recent years, the functions of these modifications have begun to be understood (reviewed in Roth and Allis, 1996; Spencer and Davie, 1999; Strahl and Allis, 2000). Upon entry into mitosis, histone H1 is phosphorylated on multiple sites, and this was long assumed necessary for chromatin condensation. However, several studies have now demonstrated that histone H1 phosphorylation is not a prerequisite for chromatin condensation (Ohsumi et al., 1993; Dasso et al., 1994) but that the less dramatic phosphorylation of histone H3 may play a role in this event (Guo et al., 1995; Ajiro et al., 1996; Hendzel et al., 1997; van Hooser et al., 1998; Wei et al., 1998, 1999; Sauve´ et al., 1999). Mitotic phosphorylation of histone H3 occurs on serine 10 and serine 28 residues (Gurley et al., 1978; Paulson and Taylor, 1982; Wei et al., 1998; Goto et al., 1999), the former having recently been demonstrated to be necessary for chromosome condensation and segregation in Tetrahymena (Wei et al., 1999). Ser-10 phosphorylation of histone H3 appears to be involved in the initiation but not the maintenance of chromosome condensation (van Hooser et al., 1998). Recent studies have demonstrated that the N-terminal tails of the core histones are able to inhibit chromatin condensation in an in vitro system by sequestering a factor(s) involved in this process (de La Barre et al., 2000). Interestingly, these authors found that a H3 kinase activity could be sequestered in vitro by nucleosome complexes. This supports an earlier study that demonstrated that microinjection of the Ser-10 cognate peptide, but not the phosphorylated peptide, was able to inhibit histone H3 Ser-10 phosphorylation, presumably by competing for the H3 kinase(s) (van Hooser et al., 1998). While the kinase(s) responsible for mitotic phosphorylation of histone H3 Ser-10 have not been identified, this phosphorylation is not carried out by cdc2, and thus, another kinase(s) is responsible for this early mitotic event. In the filamentous fungus Aspergillus nidulans, entry into mitosis requires the activities of the NIMA and NIMXcdc2 kinases (Osmani et al., 1991b; Ye et al., 1995). Temperature-sensitive mutations in nimA or overexpression of dominant-negative forms of NIMA causes cells to arrest in G2 with uncondensed DNA and interphase microtubules (Osmani et al., 1987; Lu and Means, 1994). NIMA mRNA and protein levels peak at G2/M, and its mitotic kinase activity is further activated by multiple phosphorylations to give maximal activity upon entry into mitosis (Osmani et al., 1987; Osmani et al., 1991b; Ye et al., 1995). Additionally, although cdc2/ cyclin B is active as a kinase at the nimA5 G2 arrest

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point (Osmani et al., 1991b), NIMA activity is required for its translocation to the nucleus to allow mitotic entry (Wu et al., 1998). One of the other mitotic functions of NIMA may be to regulate chromatin condensation, as ectopic expression of NIMA can induce chromatin condensation in either asynchronous cells or in cells arrested in S phase with hydroxyurea (Osmani et al., 1988). This appears to be independent of cdc2 kinase activity, as overexpression of NIMA still induces chromatin condensation in the absence of cdc2 activity (Ye et al., 1995). Interestingly, ectopic expression of NIMA in fission yeast, Xenopus, and human cells also leads to chromatin condensation, while expression of dominant-negative forms of NIMA causes a G2 arrest in human cells (O’Connell et al., 1994; Lu and Hunter, 1995). In addition, the fission yeast NIMAlike kinase fin1 is able to promote chromatin condensation when expressed at any point in the cell cycle (Krien et al., 1998). These studies suggest that NIMA-related kinases may regulate chromatin condensation in yeast and higher eukaryotes. To date, the only functional NIMA homolog identified is nim1 from Neurospora crassa (Pu et al., 1995), although there is a growing family of NIMAlike kinases identified from fission yeast (Krien et al., 1998), Tetrahymena (Wang et al., 1998), Xenopus (Uto et al., 1999), and mammalian cells (Letwin et al., 1992; Schultz and Nigg 1993; Chen et al., 1999; Hayashi et al., 1999; Tanaka and Nigg, 1999). Of these, human Nek2 (NIMA-related kinase 2) is maximally active during S and G2 phases and has a role in the regulation of the centrosome (Schultz et al., 1994; Fry et al., 1998). Interestingly, murine Nek2 has been demonstrated to associate with chromosomes in the meiotic cell cycle (Rhee and Wolgemuth, 1997). Phosphorylation of histone H3 on Ser-10 also occurs as part of the immediate-early response of mammalian cells to mitogens (Mahadevan et al., 1991; Chadee et al., 1999; Sassone-Corsi et al., 1999). The precise role of this phosphorylation is unknown, although it may be involved in increased transcription in response to mitogenic stimuli (Chadee et al., 1999). Mitogen-induced phosphorylation of histone H3 occurs in localized areas of the nucleus and appears to be distinct from mitotic phosphorylation and is likely to involve different kinases, including Rsk-2 and/or MSK1 (Sassone-Corsi et al., 1999; Thomson et al., 1999). Here we characterize cell cycle–dependent histone H3 Ser-10 phosphorylation in A. nidulans. Histone H3 Ser-10 phosphorylation initiates prior to spindle formation and is maximal during mitosis-paralleling chromosome condensation. Mitotic phosphorylation of histone H3 Ser-10 occurs downstream of both NIMA and NIMXcdc2 activation and can be induced by NIMA expression in asynchronous cells even in the absence of NIMXcdc2 activity. Moreover, we show that histone H3 Ser-10 phosphorylation is inappropriately induced by NIMA overexpression in S phase–arrested cells. We also characterize the localization of NIMA through mitosis, and at the onset of histone H3 phosphorylation, NIMA displays a chromatin-like staining pattern. NIMA then transiently localizes to the spindle microtubules and spindle pole body following metaphase. Finally, NIMA can phosphorylate histone H3 at Ser-10 in vitro. This establishes that NIMA is both required and sufficient to trigger H3 phosphorylation and that it is present at the correct time and location to perhaps help promote chromosome condensation through histone H3 phosphorylation.

Results Phosphorylation of Ser-10 on Histone H3 during Mitosis in A. nidulans The phosphorylation of the highly conserved Ser-10 residue of histone H3 is tightly correlated with mitotic chromosome condensation (van Hooser et al., 1998; Wei et al., 1998; Sauve´ et al., 1999). In order to study this event in A. nidulans, we used a strain carrying the nimT23cdc25 temperature-sensitive mutation that allows cells to be reversibly arrested in G2. Germlings at the nimT23cdc25 arrest point have low NIMXcdc2 kinase activity because the NIMXcdc2-activating phosphatase NIMTcdc25 is inactive at the restrictive temperature (O’Connell et al., 1992; Ye et al., 1995). Phosphorylation of histone H3 Ser-10 was monitored by indirect immunofluorescence using an antibody that specifically recognizes this epitope (Hendzel et al., 1997) and the percentage of cells that displayed positive staining with this antibody determined. Staining for microtubules was also carried out to determine the spindle mitotic index (SMI). At the nimT23cdc25 arrest point, cells displayed uncondensed DNA and no staining for the phosphorylated histone H3 epitope (Figures 1A and 1B). When cells were rapidly shifted to the permissive temperature to release cells into mitosis, phosphorylation of histone H3 Ser-10 occurred simultaneously with the onset of DNA condensation (Figures 1C and 1D). This indicates that histone H3 Ser-10 phosphorylation occurs downstream of nimXcdc2 activation. Interestingly, the onset of Ser-10 phosphorylation of histone H3 occurred prior to the formation of the mitotic spindle within the nucleus (Figures 1A, 1C, and 2B). Levels of histone H3 phosphorylation remained high from prophase to telophase (Figures 1C–1E) but decreased as cells exited mitosis and DNA became decondensed. During exit from mitosis into G1, slight staining of the nucleolus with the Ser-10 Phos-H3 antiserum was detectable (C. P. C. D. and S. A. O., unpublished data). This results in the separation of the SMI and H3P graphs (Figure 1A) seen during mitotic exit. Phosphorylation of Histone H3 Is Dependent on NIMA Kinase Activity As the NIMA kinase is essential for entry into mitosis in A. nidulans (Osmani et al., 1987), we examined the dependency of Ser-10 phosphorylation of histone H3 on NIMA using a strain that carried the nimA5 temperaturesensitive mutation. Cells were grown to log phase in liquid culture prior to rapid temperature shift to 42⬚C for 3 hr to inactivate NIMA kinase activity and arrest cells in G2. A wild-type strain was used as a control. Cells were then released to the permissive temperature in the presence of nocodazole to trap them in mitosis. Immunoblot analysis indicated that a low level of Ser10 phosphorylated histone H3 was detected in the log phase cultures of both the nimA5 and wild-type strains (Figure 2A). At the nimA5 arrest point, Ser-10 phosphorylation of histone H3 was not observed; however, upon return to the permissive temperature and entry into mitosis, high levels of Ser-10-phosphorylated histone H3 were detected (Figure 2A). The low-level phosphorylation detected in the wild-type strain following temperature shift to 32⬚C is due to the small increase in the mitotic index expected when cells are incubated in nocodazole for 10 min. The results were confirmed by indirect immunofluorescent staining of germlings before

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Figure 1. Cdc2-Dependent Phosphorylation of Histone H3 Ser-10 during Mitosis Cells were released from the G2 nimT23cdc25 arrest point, fixed, and stained with anti-tubulin and anti-phosphorylated Ser-10 histone H3 antibodies and DAPI to visualize DNA. (A) The percentage of cells displaying histone H3 Ser-10 phosphorylation (H3P) and the percentage of cells displaying mitotic spindles (SMI, spindle mitotic index) as determined by immunofluorescent cell staining. (B–E) Typical histone H3–phosphorylated Ser-10 staining at the indicated stages of mitosis as determined by tubulin and DAPI staining. Exposure times were similar respectively for each antibody and DAPI. Bar, 5 ␮m.

and after release from the nimA5 arrest point (Figure 2B). In this experiment, phosphorylation of histone H3 was evident in cells that had not yet begun spindle formation (Figure 2B). Collectively, these data indicate that phosphorylation of the Ser-10 residue of histone H3 occurs downstream of both NIMXcdc2 and NIMA activation. Ectopic Expression of NIMA Promotes Histone H3 Ser-10 Phosphorylation We have previously shown that overexpression of NIMA in A. nidulans leads to the induction of mitosis and maintains chromatin in a condensed state (Osmani et al., 1988). To determine if phosphorylation of Ser-10 on histone H3 was associated with this phenotype, we examined levels of phosphorylated histone H3 in a strain

containing two extra copies of nimA under the control of the inducible alcA promoter. Little Ser-10-phosphorylated histone H3 was detected in asynchronously growing cultures prior to NIMA expression (Figure 3A). However, high levels of this phosphorylated epitope were detected upon the addition of threonine to induce expression of NIMA (Figure 3A), indicating that histone H3 phosphorylation is associated with chromatin condensation induced by overexpression of NIMA. Phosphorylation of Histone H3 Ser-10 in S Phase–Arrested Cells upon Overexpression of NIMA While the above data are supportive of NIMA playing a role in histone H3 Ser-10 phosphorylation, we wished to see if NIMA induction in cell cycle–arrested cells

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Figure 3. Induction of Histone H3 Ser-10 Phosphorylation upon Ectopic Expression of NIMA

Figure 2. NIMA-Dependent Phosphorylation of Histone H3 Ser-10 Cells, either wild type or a nimA5 strain, were grown to early log phase (log) at the permissive temperature prior to temperature shift to 42⬚C for 3 hr to arrest at the nimA5 arrest point (42⬚C). Cells were then synchronously released into mitosis by rapid downshift to 32⬚C in the presence of nocodazole and samples taken after 10 min (32⬚C). (A) Immunoblotting for Ser-10-phosphorylated histone H3 or tubulin as a loading control. (B) The spindle mitotic index (SMI) and the percentage of cells staining positive for Ser-10-phosphorylated histone H3 (H3P) determined by immunofluorescent staining as in Figure 1A following release from the nimA5 arrest point.

would have a similar effect. To address this, we assessed whether NIMA overexpression could promote histone H3 Ser-10 phosphorylation in cells arrested in S phase with hydroxyurea (HU). Cells containing extra copies of alcA::nimA and control cells were grown to log phase and then arrested in S phase by the addition of 50 mM HU for 2.5 hr. Induction of nimA was then allowed by medium exchange still in the presence of HU. As expected, no histone H3 Ser-10 phosphorylation was observed in S phase–arrested cells prior to induction of nimA (Figure 3B). However, high levels of Ser10 phosphorylation were observed in cells by 45 min following induction of nimA (Figure 3B). These data indicate that overexpression of NIMA can inappropriately promote histone H3 phosphorylation in S phase–arrested cells. NIMA Can Promote Histone H3 Phosphorylation in the Absence of nimXcdc2 Kinase Activity To address whether NIMA may be promoting histone H3 phosphorylation indirectly through a kinase pathway

(A) A wild-type strain or a strain containing two extra copies of alcA::nimA (5C) were grown to log phase in minimal medium yeast lactose medium (noninducing). Cultures were then grown for a further 2 hr in the presence of 40 mM threonine (inducing) and samples analyzed by immunoblotting for Ser-10-phosphorylated histone H3 or tubulin as a loading control. (B) A control strain not containing extra copies of nimA (SO65) or a strain containing extra copies of alcA::nimA (RP2) were grown to log phase in minimal medium acetate at 32⬚C (log; repressing). Hydroxyurea was then added to 50 mM and cells grown for another 2.5 hr to arrest cells in S phase (HU). Cultures were then placed in ethanol-inducing medium by media exchange still in the presence of 50 mM HU and grown for a further 45 min (HU ⫹ EtOH) and samples analyzed by immunoblotting.

involving nimXcdc2, we used a strain containing the temperature-sensitive nimX3cdc2 mutation and extra copies of alcA::nimA. NIMXcdc2 was inactivated in log phase cultures by rapid temperature shift to 42⬚C for 3 hr prior to nimA induction by medium exchange while maintaining cultures at 42⬚C (Figure 4). Under these conditions, histone H3 Ser-10 phosphorylation was observed 30 min after nimA induction, while no phosphorylation was detectable in the control nimX3cdc2 strain (Figure 4), indicating that NIMXcdc2 kinase activity is not required for NIMA-induced histone H3 phosphorylation. At Mitosis NIMA Is Transiently Localized to Nuclear Chromatin, Spindle Microtubules, and Spindle Pole Bodies The above data point to NIMA playing a role in regulating chromatin condensation perhaps by promoting histone H3 phosphorylation at mitosis. If this were the case, NIMA would likely be localized to the nucleus at the time of chromatin condensation. Previous attempts to localize NIMA have failed due to the low abundance of the protein and its instability. In order to improve our ability to detect NIMA protein in situ, we constructed versions of nimA which were C-terminally tagged with

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Figure 4. Histone H3 Ser-10 Phosphorylation Occurs in the Absence of NIMXcdc2 Activity upon NIMA Induction A control strain carrying the nimX3cdc2 temperature-sensitive mutation but no extra copies of nimA (SO65) and a strain carrying nimX3cdc2 in combination with extra copies of alcA::nimA (RP2) were grown to log phase in minimal medium acetate at 32⬚C (log; repressing). Cultures were rapidly shifted to 42⬚C for 3 hr to eliminate NIMXcdc2 activity (42⬚C). Ectopic expression of NIMA was then induced by rapid medium exchange into minimal medium ethanol, maintaining the culture temperature at 42⬚C (42⬚C ⫹ EtOH) and samples analyzed by immunoblotting.

four copies of the HA epitope and transformed these constructs into various strains of A. nidulans. Strains containing one extra copy of nimA-HA were selected for further study. We followed the localization of NIMA during the G2/M transition and mitosis by indirect immunofluorescent staining for the HA epitope in a strain containing an extra copy of nimA-HA in a nimT23cdc25 background. At the nimT23cdc25 arrest point, no distinct locale for NIMA was evident (Figure 5A). Following release into mitosis, a portion of NIMA became enriched

in subnuclear regions that were often beginning DNA condensation (Figure 5B). As cells progressed further into mitosis, NIMA remained in the nucleus with metaphase cells occasionally displaying staining on what appeared to be spindle microtubules (Figure 5C). This spindle-like staining for NIMA persisted as cells entered anaphase and telophase with staining also becoming evident at the spindle poles later in mitosis (Figures 5D and 5E). Colocalization of NIMA with spindle microtubules and the spindle pole bodies was confirmed by dual staining for the NIMA-HA and either ␣-tubulin for microtubules or ␥-tubulin for spindle pole bodies (data not shown). Similar localization patterns for NIMA through mitosis were obtained in a strain containing nimA5-HA as the only version of nimA when cells were released from the nimA5 arrest point or in asynchronous cells (data not shown). This demonstrates that the dynamic location of NIMA seen through mitosis is not an artifact of slight overexpression and that the HA-tagged version of NIMA used is functional. Nuclear Localization of NIMA Is Tightly Correlated with Ser-10 Phosphorylation of Histone H3 The subnuclear localization of NIMA and the onset of Ser-10 phosphorylation of histone H3 both occurred prior to the formation of the mitotic spindle within the nucleus. Thus, we decided to investigate if there was any relationship between the localization of the NIMA kinase and the phosphorylation of histone H3. We examined cells from 2 to 5 min following release from the G2 nimT23 arrest point in order to obtain a high proportion of cells that had entered mitosis but had not begun spindle formation. Dual immunofluorescent staining for NIMA-HA and Ser-10-phosphorylated histone H3 indicated that the localization of NIMA more closely correlated with that of the phosphorylated histone H3 epitope than with DAPI (Figure 6). Moreover, there was a strong Figure 5. Cell Cycle–Dependent Localization of NIMA during Mitosis Cells of a strain containing an extra copy of nimA-HA (CDS46) were arrested in G2 at the nimT23cdc25 arrest point of 42⬚C before downshift to 32⬚C to allow synchronous release into mitosis. Cells were fixed and stained with DAPI for DNA and an anti-HA antibody to localize NIMA-HA. (A–E) Localization of NIMAHA in representative cells at the indicated stages of mitosis. Bar, 5 ␮m.

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Figure 6. Nuclear Accumulation of NIMA and the Onset of Histone H3 Ser-10 Phosphorylation Are Tightly Correlated Cells as in Figure 5 were fixed and stained with DAPI for DNA, anti-HA to localize NIMAHA and antiphosphorylated-Ser-10 histone H3. Bar, 5 ␮m.

correlation between the two events with 95% ⫾ 6% of premetaphase cells with chromatin-like NIMA staining displaying mitotic levels of histone H3 phosphorylation. NIMA Can Phosphorylate Histone H3 Ser-10 In Vitro While the above data indicate that NIMA may play a role in chromatin condensation by promoting Ser-10 phosphorylation of histone H3, it is unclear if it does so directly or by regulating the activity of another kinase(s) or phosphatase. In order to determine if NIMA could potentially be the mitotic histone H3 kinase, we determined if NIMA could phosphorylate histone H3 in vitro. The histone H3 kinase activity of NIMA immunoprecipitates was assessed at time points following release from the nimT23 arrest point (Figure 7A). This indicated that NIMA was able to phosphorylate histone H3 in vitro, displaying a pattern of activity toward this substrate through mitosis identical to the nonphysiological ␤-casein substrate (Figure 7A). To determine if phosphorylation of histone H3 by NIMA was occurring on Ser-10, we performed kinase assays as above and immunoblotted for histone H3 phosphorylated on Ser-10. Bacterially expressed NIMA is active as a kinase and was used for these assays to eliminate the possibility of associating kinases being present. Under these conditions, NIMA was able to phosphorylate histone H3 at Ser-10 with the phosphoepitope being detectable as soon as the kinase was added to the reaction (Figure 7B). Levels of Ser-10phosphorylated histone H3 were further increased by 15 min by which time the reaction was complete (Figure 7B). No immunoreactive bands were produced if either NIMA or histone H3 was omitted from the reaction even after incubation for 60 min (Figure 7B). To provide further evidence that NIMA is able to phosphorylate Ser-10 on histone H3, we examined the ability of NIMA to phosphorylate a peptide containing the first 20 amino acids of the N-terminal of histone H3 (Hendzel et al., 1997). As a negative control, we used a peptide containing an alanine in place of serine at position 10 (hist H3 S10A). Quantitation indicated that NIMA was able to phosphorylate the unmodified peptide almost 30 times more effectively than the hist H3 S10A peptide (Figure 7C) even though this peptide still contains three other phosphorylatable residues. Similar results were obtained

using NIMA immunoprecipitated from nocodazole arrested cell extracts 20 min following release from the nimT23cdc25 arrest point (data not shown). These data indicate that NIMA is able to phosphorylate histone H3 Ser-10 in vitro and together with the above data suggest that NIMA is an in vivo mitotic histone H3 kinase. Discussion Mitotic entry in A. nidulans requires the activities of the NIMA and NIMXcdc2 kinases (Osmani et al., 1991b; Ye et al., 1995). These two kinases each require the activity of the other for full mitotic function. Full activation of NIMA requires multiple phosphorylations that are carried out by NIMXcdc2/NIMEcyclinB (Ye et al., 1995), while the nuclear localization of NIMXcdc2/NIMEcyclinB is dependent upon NIMA activity (Wu et al., 1998). Thus, the interplay between these two kinases is important in the orchestration of mitotic events such as spindle formation and chromosome condensation. Here we have established that NIMA may play a more direct role in chromosome dynamics through histone H3 phosphorylation. NIMA Is a Mitotic Histone H3 Kinase Histone H3 Ser-10 phosphorylation occurs downstream of nimA activation following release from the nimA5 arrest point in late G2. Evidence that NIMA directly functions in the pathway leading to histone H3 phosphorylation comes in part from overexpression experiments. Specifically, ectopic expression of NIMA is able to promote histone H3 Ser-10 phosphorylation and, moreover, overcome normal cell cycle constraints and induce histone H3 Ser-10 phosphorylation in S phase–arrested cells. Histone H3 is an integral component of nucleosomal chromatin, thus necessitating that the Ser-10 kinase be nuclear at the G2/M transition. We have previously demonstrated that NIMXcdc2/NIMEcyclinB becomes nuclear at the G2/M transition and that this localization depends on NIMA activity (Wu et al., 1998). However, NIMA-induced histone H3 Ser-10 phosphorylation can occur at the restrictive temperature for the nimX3cdc2 mutation, indicating that cdc2 kinase activity is not directly involved in H3 phosphorylation, supporting studies in other systems (Guo et al., 1995). We demonstrate here that NIMA

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NIMA substrate preferences of Phe-Arg-Xaa-Ser/Thr (Lu et al., 1994) and Arg-Phe-Arg-Arg-Ser-Arg-Arg-Met-Ile (Songyang et al., 1996), NIMA is able to phosphorylate histone H3 at Ser-10 in vitro. To date, no in vivo substrates for NIMA have been identified, and several factors may influence the substrate preference for NIMA in vivo. Clearly, the nucleosomal histone H3 environment is markedly different from conditions used in vitro. NIMA is highly regulated upon mitotic entry becoming hyperphosphorylated and, as demonstrated here, enriched in the nuclear compartment. While it is at present unclear what state of phosphorylation NIMA is in at the time of histone H3 Ser-10 phosphorylation, these posttranslational modifications may potentiate interaction with basic proteins such as histone H3. Proteins interacting with NIMA or with its substrates may also regulate its ability to phosphorylate substrates. One candidate is the prolyl-isomerase pin1 that was isolated in human cells as a NIMA-interacting protein (Lu et al., 1996) and that has similarly been isolated from A. nidulans using the yeast two-hybrid system (Crenshaw et al., 1998).

Figure 7. NIMA Phosphorylates Histone H3 at Ser-10 In Vitro Cells of a nimT23cdc25 strain (CDS46) were grown to early log phase at 30⬚C (log) prior to rapid temperature shift to 42⬚C for 3 hr to arrest cells in G2. Cells were then released synchronously into mitosis by rapid temperature downshift to 32⬚C in the presence or absence of nocodazole and samples taken and analyzed as indicated. (A) ␤-casein and histone H3 kinase activity in NIMA immunoprecipitates. (B) Immunoblotting for Ser-10-phosphorylated histone H3 following kinase assays using bacterially expressed NIMA as indicated. (C) Relative ability of bacterially expressed NIMA to phosphorylate peptides containing the first 20 amino acids of the histone H3 N-terminal either unmodified (Hist H3) or with alanine in place of serine at position 10 (Hist H3 S10A). Assays were performed in triplicate with standard deviation indicated by error bars.

itself is nuclear throughout the majority of mitosis and that at the time of histone H3 phosphorylation there is a high correlation between NIMA chromatin-like staining and phosphorylated histone H3. This correlation suggests that NIMA plays an intimate role in Ser-10 phosphorylation. Although the histone H3 site Thr-Ala-Arg-Lys-Ser10Thr-Gly-Gly-Lys differs from the previously defined

Both NIMXcdc2 and NIMA Kinase Activities Are Required for Chromosome Condensation It is becoming increasingly clear that the compartmentalization of kinases and phosphatases and their substrates is a major form of cell cycle regulation. Although histone H3 Ser-10 phosphorylation induced by overexpression of NIMA can occur in the absence of NIMXcdc2 activity, NIMXcdc2 is still likely to be important for the regulation of histone H3 phosphorylation. We have shown here that nuclear localization of NIMA occurs downstream of the nimA5 or nimT23cdc25 arrest points, indicating that both of these kinase activities are required for this event at mitosis. In the case of overexpression of NIMA, it is likely that the requirement for NIMXcdc2 activity for the nuclear localization of NIMA is negated by high levels of NIMA expression. Chromatin condensation promoted by NIMA overexpression often leads to atypical nuclear morphologies and fragmented nuclei (Osmani et al., 1988). This is in part because chromatin condensation is induced at inappropriate points in the cell cycle in the majority of cells under these conditions, perhaps when other mitotic regulators are not activated. Evidence from Xenopus demonstrates that cdc2 is involved in the phosphorylation of components of the 13S condensin complex, which is also required for chromosome condensation. Interestingly, kinases other than cdc2 appear to be required to phosphorylate Xenopus 13S condensin to mitotic levels (Kimura et al., 1998). Thus, proper mitotic chromosome condensation is regulated by multiple kinase pathways, with NIMA playing a role as a mitotic histone H3 kinase. Given that NIMA plays a role in mitotic phosphorylation of histone H3, it is interesting that defects in nimA prevent all aspects of mitosis, not just this phosphorylation. This is most likely because there is a requirement for NIMA and cdc2/cyclin B to help promote each other’s mitotic-promoting functions. For instance, lack of NIMA does not prevent activation of cdc2/cyclin B as a kinase but does prevent its location to the nucleus (Wu et al., 1998). Therefore, although NIMA may play multiple roles in promoting mitotic events by phosphorylating mitoticspecific substrates such as histone H3, it also plays a wider role at the G2/M transition by regulating other

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mitotic regulators required for entry into mitosis. For these reasons, the G2 arrest caused by lack of nimA is unlikely to be a direct consequence of the inability of NIMA to phosphorylate histone H3. The Localization of NIMA Suggests It Has Numerous Mitotic Functions The cell cycle–specific localization of NIMA to the spindle microtubules and spindle pole bodies following metaphase suggests that NIMA has functional roles during mitosis at these sites. The most obvious role would be regulation of microtubule dynamics to promote spindle elongation at metaphase and beyond. Supporting this, overexpression of full-length NIMA causes a transient increase in the spindle mitotic index, with cells often displaying long, telophase-like spindles (Osmani et al., 1988; Osmani and Ye, 1996). The localization of NIMA to the spindle pole bodies is particularly intriguing given that human NEK2 localizes to the centrosome, where it appears to play a role in the splitting of the centrosome (Fry et al., 1998). However, the timing of NIMA spindle pole body association is not consistent with it playing a role in the initial separation of the spindle pole bodies. It will be of interest to determine if NIMA and NEK2 (or other NEKs) have yet undefined functional similarities, given the major structural differences between the centrosomes and spindles pole bodies. NIMA localized to the spindle pole bodies in anaphase and telophase could potentially play a role in the separation of the spindle poles at these stages of mitosis. Components of the anaphase-promoting complex/cyclosome (APC/C) localize to microtubuleorganizing centers during mitotic exit (Mirabito and Morris, 1993; Tugendreich et al., 1995; Kurasawa and Todokoro, 1999). Therefore, another attractive hypothesis is that NIMA may regulate the activity of APC/C components in a manner similar to the polo mitotic kinase (Charles et al., 1998; Descombes and Nigg, 1998; Shirayama et al., 1998). In this way, NIMA may turn on its own destruction, which is required for correct mitotic exit (Pu and Osmani, 1995). NIMA-Like Kinases in Higher Eukaryotes The highly conserved nature of the nucleosome and of the potential role of histone H3 phosphorylation in chromosome condensation would suggest that the mitotic histone H3 kinase might also be highly conserved. To date, no bona fide mitotic histone H3 kinases have been reported. Although no members of the NIMArelated kinases have been proven to play a role in chromosome condensation, murine Nek2 has been located to meiotic chromosomes (Rhee and Wolgemuth, 1997), and overexpression of NIMA in Xenopus and human cells and NIMA or Fin1 in fission yeast leads to abnormal chromatin condensation (O’Connell et al., 1994; Lu and Hunter, 1995; Krien et al., 1998). Additionally, dominantnegative versions of NIMA cause a G2 arrest in human cells. Given this, it is tempting to speculate that a yet unidentified kinase(s) may carry out the mitotic phosphorylation of histone H3 at Ser-10 in higher eukaryotes and that this kinase may be a NIMA-related kinase. In conclusion, we have demonstrated that NIMA functions in the pathway leading to histone H3 Ser-10 phosphorylation during mitosis. Histone H3 phosphorylation correlates with chromosome condensation in A. nidulans, and we have characterized the localization of NIMA

through mitosis and found it in the right place at the right time to carry out the phosphorylation of histone H3. NIMA also localizes to the mitotic spindle and spindle pole bodies in anaphase and telophase, suggesting that it may play a role in the regulation of these structures as well. Finally, we demonstrate that NIMA can phosphorylate histone H3 Ser-10 in vitro, suggesting that it is a mitotic histone H3 kinase. This may help to explain its role in chromosome condensation and its ability to induce premature chromatin condensation in other eukaryotic systems as well. Experimental Procedures Aspergillus nidulans Strains Strains used in this study were R153 (pyroA4; wA3), CDS46 (nimT23; pyrG89; pyr4⫹; one extra copy nimA-4xHA; nicA2; chaA1), LPW51 (pyrG89; pyr4⫹; nimA5-4xHA; wA2; cnxE16; choA1; sC12; yA2; chaA1), SO54 (nimA5; wA2), SO65 (nimX3; wA3; pyroA4; riboA1), RP2 (nimX3; alcA::nimA; wA3; pyroA4; riboA1), and 5C (alcA::nimA; fwA1; benA22; pabaA1). CDS46 was generated by transformation of SO182 (nimT23; pyrG89; nicA2; chaA1) with the plasmid pLW39 containing a version of nimA C-terminally tagged with four copies of the HA epitope using standard techniques. Single-copy integration of pLW39 in CDS46 was confirmed by Southern blotting. The strain LPW51 was generated by transformation of SO6 (nimA5; wA2; pyrG89; cnxE16; choA1; sC12; yA2; chaA1) with the plasmid pLW42 containing an N-terminally truncated version of nimA5 C-terminally tagged with four copies of the HA epitope using standard techniques. This generated a strain containing a C-terminally HA-tagged version of nimA5 and an N-terminally truncated, untagged nimA5 that is not expressed due to lack of a promoter as confirmed by Western blotting using anti-NIMA and anti-HA antibodies.

General Techniques Media and general techniques for culture of A. nidulans, DAPI staining for chromosome mitotic index, protein extraction, immunoprecipitation, immunofluorescence, bacterial expression of NIMA, and protein kinase assays were as previously described (Osmani et al., 1987, 1991a, 1991b, 1994; Oakley and Osmani, 1993; Ye et al., 1995). Antibodies for immunofluorescence and immunoblotting were as described previously apart from the anti-phospho histone H3 mitosis marker (Upstate Biotechnology), anti-HA clone 3F10 (Roche Molecular Biochemicals), and anti-rat Texas red X (Molecular Probes) antibodies. The anti-␥-tubulin antibody was a kind gift from Dr. Berl Oakley (Ohio State University). Extracts for Western blotting were prepared by boiling 5 mg of freeze-dried mycelia ground to a fine powder in 200 ␮l of 2⫻ sample buffer also containing 6 M urea. Assays for histone H3 kinase activity were carried out using 5 ␮g of histone H3 (Roche Molecular Biochemicals) per reaction in place of ␤-casein. Kinase assays using peptide substrates were performed as described (Lu et al., 1993) using either the unmodified histone H3 peptide corresponding to the histone H3 amino-terminal amino acids 1–20 (A1RTKQTARKS10TGGKAPRKQL20C; Hist H3) or the same peptide with alanine in place of serine 10 (Hist H3 S10A).

Acknowledgments We would like to thank all members of the Osmani laboratory and Dr. Xiang Ye (Lilly Research Laboratories, Ely Lilly, Indianapolis, Indiana) for their interest and input into this work, Dr. Berl Oakley (Department of Genetics, The Ohio State University, Columbus, Ohio) for providing the anti-␥-tubulin antibody, and Dr. David Allis (Department of Biochemistry and Molecular Genetics, University of Virginia Health Science Center, Charlottesville, Virginia) for helpful discussions and sharing of unpublished data. This work was supported by NIH grant GM 42564.

Received March 22, 2000; revised July 18, 2000.

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