A Point Mutation in Nucleoside Diphosphate Kinase Results in a Deficient Light Response for Perithecial Polarity in Neurospora crassa*

Yasunobu OguraDagger, Yusuke YoshidaDagger, Naoto Yabe, and Kohji Hasunuma§

From the Kihara Institute for Biological Research and Graduate School of Integrated Science, Yokohama City University, 641-12 Maioka-cho, Totsuka-ku, Yokohama 244-0813, Japan

Received for publication, December 18, 2000, and in revised form, March 26, 2001


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In Neurospora crassa, the phosphorylation of nucleoside diphosphate kinase (NDK)-1 is rapidly enhanced after blue light irradiation. We have investigated the function of NDK-1 in the blue light signal transduction pathway. A mutant called psp (phosphorylation of small protein) shows undetectable phosphorylation of NDK-1 and is defective in light-responsive regulation of perithecial polarity. Sequencing analysis of ndk-1 cDNA by reverse transcription-polymerase chain reaction revealed that proline 72 of ndk-1 was replaced with histidine in psp. The mutation ndk-1P72H resulted in accumulation of normal levels of mRNA and of about 25% of NDK-1P72H protein compared with that of wild type as determined by Western blot analysis. The ectopic expression of cDNA and introduction of genomic DNA of wild type ndk-1 in psp (ndk-1P72H) suppressed the reduction in accumulation and phosphorylation of NDK-1 and the light-insensitive phenotype. These findings demonstrated that the phenotype of psp was caused by the ndk-1P72H mutation. Biochemical analysis using recombinant NDK-1 and NDK-1P72H indicated that the P72H substitution in NDK-1 was responsible for the decrease in phosphotransfer activities, 5% of autophosphorylation activity, and 2% of Vmax for protein kinase activity phosphorylating myelin basic protein, compared with those of wild type NDK-1, respectively.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Development and morphogenesis during the life cycles of fungi, as well as plants, are greatly affected by environmental stimuli such as light (1-3). Recently, the nature of the photoreceptors in fungi and plants has been investigated. In Arabidopsis thaliana, the photoreceptors (4, 5) are phytochromes (6), cryptochromes (7, 8), phototropin (7, 9), and zeaxanthin (7). However, the light signal transduction downstream of these photoreceptors has remained to be elucidated unambiguously (10).

The filamentous fungus Neurospora crassa shows several biological responses to blue light, such as a mobilization of ions in the mycelia within minutes after illumination (11, 12), initiation of carotenoid synthesis in the mycelium (13), promotion of conidial and protoperithecial development (14, 15), positive phototropism of perithecial beaks (16), regulation of perithecial polarity (the position of the beak formed on the perithecium) (17), and resetting of the circadian rhythm of conidiation (18). The N. crassa mutants white collar-1 (wc-1)1 and white collar-2 (wc-2) lack all of these blue light responses (19, 20). Isolation and characterization of wc-1 and wc-2 genes revealed that both of them have a zinc finger DNA-binding motif, a PAS PERIOD, ARNT, and SIM dimerization domain, and a glutamine-rich transcriptional activation domain (21, 22). WC proteins are proposed to form heterodimers through their PAS domain (23). Furthermore, WC-1 has the LOV (light, oxygen, or voltage) domain found in blue light receptor phototropin (7). These results suggest that WC proteins perceive blue light and regulate the transcription of the blue light-responsive genes. However, it is difficult to explain ion mobilization by WC proteins (12). Furthermore, phosphorylation of WC-1 is increased after blue light irradiation (24), which appears to be an important part of this pathway. However, except for protein kinase C, no candidate for a protein kinase mediating the Neurospora blue light signaling has been suggested (25).

We developed a system to analyze the light-responsive phosphorylation of proteins in vitro utilizing the mycelial membrane fraction. The phosphorylation of a 15-kDa protein increased specifically after blue light irradiation in the wild type, but not in either wc-1 or wc-2. However, mixing the membrane fractions from wc-1 and wc-2 restored the increase in the phosphorylation. These results suggested that the 15-kDa protein was involved in blue light signal transduction downstream of WC proteins (26). This protein was purified and identified as nucleoside diphosphate kinase (NDK) (27). The characteristics of an N. crassa mutant known as psp suggested that it is a mutant allele of ndk-1. Phosphorylation of NDK-1 is undetectable in psp (see Fig. 1A), and the psp mutant is known to be defective in light-responsive regulation of perithecial polarity (17), suggesting that phosphorylation of NDK-1 is critical for the photoresponse.

NDK is a ubiquitous enzyme that catalyzes the transfer of the gamma -phosphate group of nucleoside 5'-triphosphate to nucleoside 5'-diphosphate to form nucleoside 5'-triphosphate (28). Recent studies suggest that NDK is involved in various signal transduction pathways such as suppression of tumor metastasis (29), modulation of muscarinic K+ channels (30), transcriptional activation of proto-oncogenes (31), development of the wing disc cells in Drosophila melanogaster (32), and sexual development in Schizosaccharomyces pombe (33). In plants, the phosphorylation of NDK in pea, PNDK1, was stimulated by red light, and the stimulation was reversed by subsequent irradiation with far-red light (34). PNDK1 also showed protein-phosphorylation activity (35). In A. thaliana, NDPK2 is hypothesized to be a photo-signaling component because of the partial defect in photo-dependent cotyledon opening and greening in the T-DNA insertional disruptant of NDPK2 (36). Several roles for NDK in signal transduction have been proposed: transcriptional activation (31, 37), supply of GTP to G-proteins (38), regulation of muscarinic K+ channels (30), interaction with other signaling proteins (36, 39), and phosphorylation of proteins (40). NDK-1 has been shown to be able to phosphorylate itself and other proteins (27). Based on its protein-phosphorylation activity, NDK-1 may be involved in phosphorelay signaling.

In the present study, we have investigated the roles of NDK-1 in the light signal transduction pathway through the gamma -phosphotransferring activity from nucleoside 5'-triphosphate to nucleoside 5'-diphosphate and the phosphotransfer activity to other proteins.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

N. crassa Strains and Growth Conditions-- The N. crassa wild type strains 74OR23-1A (FGSC987) and 74OR8-1a (FGSC988) were obtained from the Fungal Genetics Stock Center (Kansas City, KS). The mutants psp A and psp a (renamed ndk-1P72H in this paper) were isolated previously (17). Culture was carried out in Vogel's minimal medium with 2% sucrose at 25 °C. Protoperithecia of 74OR8-1a, ndk-1P72H a, and the transformants were developed in darkness at 25 °C on synthetic crossing medium for 5 days and fertilized with the 74OR23-1A conidia. After 3 days in darkness, they were placed in a box with a slit 4-cm wide and illuminated from the side under a 12-h light/12-h dark regime for 2 weeks. Perithecia were observed and photographed through dissecting microscopy.

Northern Blot Analysis-- N. crassa wild type 74OR23-1A and ndk-1P72H were grown for 2 days. The mycelia were harvested by filtration and powdered with a pestle and mortar in liquid nitrogen. Total RNA was prepared by the small scale method of RNA extraction (41). 5 µg of total RNA was separated by glyoxal gel electrophoresis and transferred to a piece of Gene Screen Plus membrane (PerkinElmer Life Science). Hybridization with 32P-labeled ndk-1 probe was carried out overnight at 42 °C in hybridization buffer containing 50% formamide, 10% dextran sulfate, 1% SDS, and 1 M NaCl. Radioisotopic signals were visualized by autoradiography using X-Omat AR film (Eastman Kodak Co.).

Protein Experiments-- Mycelia grown for 36 h in 1 ml of Vogel's minimal medium were dried on filter paper and ground by KONTES pellet pestle in microcentrifuge tubes containing 120 µl of protein extraction buffer (25 mM Na-Pipes, pH 6.3, 0.25 mM EDTA, 0.25 M sucrose, 0.01 mM leupeptine, and 0.5 mM phenylmethylsulfonyl fluoride). Crude extract was obtained as supernatant after centrifugation at 10,000 × g for 10 min. An appropriate amount of the crude extract containing 300 µg of protein was separated by SDS-polyacrylamide (12.5%) gel electrophoresis and electrically transferred onto a piece of polyvinylidene difluoride membrane. The membrane was incubated with anti-NDK-1 antiserum raised in rabbit using purified NDK-1. Antibodies were detected with a VECTASTAIN Elite ABC kit (Vector Laboratories, Inc.). For the protein phosphorylation experiment, the crude extract was incubated with [gamma -32P]ATP for 15 s at 4 °C in buffer containing 20 mM Na-Pipes, pH 6.3, 0.1 mM EDTA, 0.1 M NaCl, 1.5 mM MgCl2, and 0.1% Triton X-100 before SDS-PAGE (17).

RT-PCR-- The sequences of oligodeoxyribonucleotide primers, designed from the DNA sequence of the untranslated regions of the 5' and 3' termini of the ndk-1 transcript, were as follows: Ndk-1F, 5'-AACCTATACTCCCTCACC-3'; and Ndk-1R, 5'-CGGAACAGACATGATACC-3'. Total RNA prepared from wild type 74OR23-1A, as well as psp (ndk-1P72H) A and a, was treated with RNase-free DNase I (Roche Molecular Biochemicals). 1 µg of total RNA and 50 pmol of Ndk-1R primer were denatured in a 10-µl reaction buffer (75 mM KCl, 50 mM Tris-HCl, pH 8.3, and 3 mM MgCl2) for 2 min at 95 °C. They were annealed for 1 h at 55 °C. The RNA solution was mixed with a 4-ml reaction mixture (10 mM dithiothreitol, 0.5 mM each of dNTP, 28 units of RNase inhibitor, and 100 units of MoMuLV reverse transcriptase) and incubated for 1 h at 55 °C to extend single-stranded cDNA. Double-stranded cDNA of ndk-1 was specifically amplified by PCR with 0.1 µM each of Ndk-1F and Ndk-1R primers using a 1-µl RT reaction mixture in a 10-µl working solution (50 mM KCl, 10 mM Tris-HCl, 1.5 mM MgCl2, 0.2 mM each of dNTP and 0.2 units of Taq polymerase). PCR was performed at 94 °C (30 s), 55 °C (30 s), and 72 °C (1 min) for 30 cycles with a final 7 min of extension at 72 °C. Amplified products were subcloned into the EcoRV site of pBluescriptIISK+ (Stratagene) and sequenced by the dideoxy chain termination method.

N. crassa Transformation-- An N. crassa ndk-1P72H a strain was transformed with a plasmid containing ndk-1 genomic DNA (pBARNDK-1) or a plasmid for ectopic expression of ndk-1 cDNA (pTBNDK-1) according to Vollmer and Yanofsky (42). Plasmid pBARNDK-1 was constructed by insertion of a 4.3-kilobase pair genomic DNA fragment containing ndk-1 into pBARKS1, which carried the glufosinate (BASTA; Hoechst) resistance gene (bar) as a selection marker (43). For ectopic expression of cDNA in N. crassa, we constructed a plasmid pTREB, which carried bar for selection and the promoter and terminator regions of Aspergillus nidulans trpC for ectopic expression (44). This plasmid has a multicloning site between the trpC promoter and terminator for SmaI, EcoRI, EcoRV, and ClaI. The ndk-1 fragment digested with EcoRI and ClaI, ~600 bp, was ligated into pTREB to construct pTBNDK-1. The transformants were selected on sorbose-BASTA medium (Vogel's salt mixture without NH4NO3, 0.5% proline, 2% sucrose, 2% sorbose, 1.5% agar, 0.005% BASTA (for basal medium) and 1 M sorbitol (for top medium)).

Expression and Purification of Recombinant Proteins-- Two oligo-DNA primers, ESTndk1-5t (5'-AGGATCCAACCAAGGAGCA-3'), which was designed to have a BamHI site at the 5' terminus and to delete the start codon (ATG) and Ndk-1ER (5'-GCGAAGCTTACTCGAAG-3'), were used for PCR amplification of ndk-1 and ndk-1P72H. PCR products were subcloned into the EcoRV site of pBluescriptIISK+. ndk-1 and ndk-1P72H cDNA were cut out by BamHI and SmaI double digestion and were ligated into pEST-1 (Stratagene), which contained a glutathione S-transferase (GST) gene in the 5'-region of a multicloning site (pESTNDK-1 and pESTP72H). S. pombe SP-Q01 (Stratagene) was transformed with pESTNDK-1 or pESTP72H. Transformants were cultured in Edinburgh minimal medium (45) for 48 h at 30 °C. Cells were centrifuged, and the pellet was resuspended in an equal volume of phosphate-buffered saline (10 mM Na2HPO4, 1.8 mM KH2PO4, 140 mM NaCl, and 2.7 mM KCl). An equal volume of glass beads was added, and the cells were crashed by 20 cycles of a set of 1 min of vortexing and 1 min of chilling on ice. After centrifugation, the supernatant was mixed with GST-Sepharose (Amersham Pharmacia Biotech) and stood for 30 min at room temperature. The resin was washed 10 times with PBS. GST fusion protein was eluted with an equal volume of elution buffer containing 50 mM Tris-HCl, pH 9.6, and 10 mM reduced glutathione. The protein concentration was calculated from the strength of Coomassie Brilliant Blue R-250 staining after electrophoresis by the NIH image program.

Analysis of Enzymatic Activities-- The kinetic constants for NDK activity were determined by a coupled pyruvate kinase-lactate dehydrogenase assay essentially according to Agarwal et al. (46) at 25 °C. In this assay, 10 ng of purified recombinant protein was used. The concentrations of dTDP used were 0.1, 0.2, 0.3, 0.4, and 0.5 mM. The kinetic constants were calculated from the double reciprocal plots. The activity of GST-NDK-1 and GST-NDK-1P72H to phosphorylate myelin basic protein (MBP; lot number HDIBO1; Life Technologies, Inc.) was analyzed essentially according to a previous study (27). The mixture, containing 10 ng of GST-NDK-1 or GST-NDK-1P72H, MBP (1.35, 2.7, 5.4, 8.1, or 10.8 µM), 50 mM Hepes, pH 7.5, 1 mM dithiothreitol, and 10 mM MgCl2, was incubated for 1 min at 25 °C after addition of 0.2 MBq of [gamma -32P]ATP (110 MBq/pmol) at a final concentration of 0.04 mM. After the electrophoresis, a part of the gel containing MBP was cut out. The radioisotopic activity of this piece of gel was measured by the Cerenkov ray method with a liquid scintillation counter (Aloka). The autophosphorylation activity of GST-NDK-1 or GST-NDK-1P72H was assayed by use of 10 ng of the protein in the above reaction mixture. The reaction (20 µl) was started by adding 0.1 nM [gamma -32P]ATP on ice, and after 10 s the reaction was stopped by adding SDS sample buffer. The following procedure was the same as described above.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Proline 72 of NDK-1 Is Replaced with Histidine in psp-- No phosphorylation of NDK-1 was detected in the crude extracts from the mutant psp (ndk-1P72H) (see Fig. 1A and Ref. 17). This result suggested that the sequence of ndk-1 was changed to reduce the accumulation of transcript or to reduce the protein activity. We first examined the levels of ndk-1 mRNA and NDK-1 protein in psp (ndk-1P72H). Northern blot analysis showed no significant difference in the accumulation of ndk-1 mRNA between psp (ndk-1P72H) and wild type (Fig. 1B). In contrast, Western blot analysis using anti-NDK-1 antiserum indicated that about 25% of NDK-1 protein was accumulated in psp (ndk-1P72H) compared with that in wild type (Fig. 1C) from three independent experiments. The amounts of NDK-1 proteins from wild type and mutant were estimated by the densitometric assay of the Western blot. From these results, we speculated that an amino acid substitution in NDK-1 itself affected its stability or activity. To examine this hypothesis, the nucleotide sequence of ndk-1 cDNA, which was amplified from psp (ndk-1P72H) by RT-PCR, was determined. Nucleotide sequences in the 5'- and 3'-untranslated regions of ndk-1 cDNA were used as primers for RT-PCR. A fragment whose length corresponded to the predicted ndk-1 cDNA, ~580 bp, was amplified (Fig. 1D). Ten clones were isolated from each strain, wild type and psp (ndk-1P72H) A and a. In the psp (ndk-1P72H), there were three nucleotide replacements in ndk-1. The first was in the 3'-untranslated region, the second was silent, and the third was a replacement of cytosine with adenosine, causing the replacement of proline 72 with histidine, whereas the sequence of cDNA from wild type was identical to that of ndk-1.


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Fig. 1.   Molecular analysis of ndk-1P72H. N. crassa wild type (WT) and mutant strains were grown for 48 h and subjected to procedures for the preparation of crude extract or total RNA. The crude extract was separated by 12.5% SDS-PAGE and transferred onto a piece of polyvinylidene difluoride membrane. A, before SDS-PAGE, the crude extract was assayed for protein phosphorylation (see "Experimental Procedures"). The gel stained with Coomassie Brilliant Blue R-250 was autoradiographed to visualize the phosphorylated protein. B, total RNA was separated on 1.2% agarose gel, transferred onto a piece of membrane, and hybridized with ndk-1 DNA labeled with 32P. Ethidium bromide staining of 17 S rRNA is shown in the lower panel as a control. C, Western blotting of the crude extract with anti-NDK-1 antiserum. Each sample contained an equal amount of protein (300 µg). The antibodies were visualized by the ABC staining method. D, the fragment amplified by RT-PCR was separated on 1.5% agarose gel and stained with ethidium bromide. Arrows with 360 and 580 bp indicate cDNA amplified by RT-PCR. See "Experimental Procedures" for details.

A 360-bp fragment was also amplified by RT-PCR (Fig. 1D). The nucleotide sequence of this fragment was identical to that of ndk-1 except that the fragment lacked 219 nucleotides, from the 28th to 246th base from the putative translation start site (data not shown). The deduced amino acid sequence of this fragment was identical to that of ndk-1 except that it lacked 73 amino acids in the N-terminal region. This transcript was designated as Tnk-1 (Truncated ndk-1). The fact that Tnk-1 of the psp (ndk-1P72H) mutant contains two nucleotide replacements (data not shown) suggested that Tnk-1 was the alternative transcript of ndk-1. The physiological role of this transcript, however, remains unclear, because the amount of Tnk-1 transcript was quite low compared with that of ndk-1 (Fig. 1D), and no translation product was detectable by Western blot analysis.

The Phenotype of psp (ndk-1P72H) Is Complemented by Wild Type NDK-1-- To examine whether the point mutation in ndk-1 (ndk-1 cDNA was fused with the promotor of trpC) caused the phenotype of psp (ndk-1P72H), a genomic DNA fragment containing wild type ndk-1 or the trpC-promoter::ndk-1 chimeric gene for cDNA ectopic expression was introduced into psp (ndk-1P72H). Homokaryotic transformants were isolated by more than seven rounds of colony selection on a medium containing BASTA. The integration of fragments into the transformants was confirmed by Southern blot analysis (data not shown). Two independent lines containing the extra genomic DNA fragment of ndk-1 (PGN-1 and 2) and three independent lines containing the trpC::ndk-1 chimeric construct (PCN-1, 2, and 3) were isolated. The abundance of NDK-1 in the transformants was examined by Western blot analysis. The transformants were able to accumulate as much NDK-1 as the wild type did (Fig. 2A). Phosphorylation of NDK-1 was also examined. The ability to phosphorylate NDK-1 itself was recovered in all of the transformants (Fig. 2B). In PGN-2, more NDK-1 was recovered than in psp (ndk-1P72H) but less than in other transformants and the wild type (Fig. 2A, lane 4). These results and the results in Fig. 1, A and C, showed that the point mutation in ndk-1 caused less NDK-1 accumulation of about 25% and almost no activity of NDK-1 phosphorylation in psp (ndk-1P72H) compared with those in wild type (Fig. 2, lane 2).


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Fig. 2.   Molecular analysis of ndk-1 transformants. Extragenic ndk-1 genomic DNA (PGN-1 and 2) and the trpC::ndk-1 chimeric construct (PCN-1, 2, and 3) were introduced into ndk-1P72H. The crude extract was prepared from wild type, ndk-1P72H, and the ndk-1 transformants that were grown for 48 h and was separated by 12.5% SDS-PAGE. A, Western blot analysis of NDK-1. After SDS-PAGE, proteins were transferred to a piece of polyvinylidene difluoride membrane and incubated with anti-NDK-1 antiserum. Hybridized antibodies were visualized by the ABC staining method. B, phosphorylation of NDK-1 was examined as in Fig. 1A.

The perithecial polarities of the ndk-1 transformants were observed. The probabilities that the beaks would form at the top or at some other position on the perithecia are shown in Fig. 3. In darkness, the positioning of the perithecial beak was nondirectional when any of the strains were used as a protoperithecial parent. When white light was illuminated from one direction parallel to the surface of the solid medium, the beaks were formed preferentially at the top of the perithecia in the wild type. In psp (ndk-1P72H), the positioning of the beaks was nondirectional even when white light was illuminated from one direction, as in darkness. In psp (ndk-1P72H) transformants carrying wild type ndk-1, beaks formed at the top of the perithecia under unidirectional light. Fig. 4 shows typical perithecia of wild type, psp (ndk-1P72H), and PGN-2 strains grown in darkness or under unidirectional light. These results indicate that the phenotype of the psp mutant is caused by the ndk-1P72H mutation and that NDK-1 plays a role in the light-responsive regulation of perithecial polarity. Here the psp mutation was determined to be ndk-1P72H.


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Fig. 3.   Light-induced polarity of perithecia in wild type, ndk-1P72H, and the ndk-1 transformants. Wild type (WT) 74OR8-1a, ndk-1P72H a, PGN-1, PGN-2, PCN-1, PCN-2, and PCN-3 were used as protoperithecial mother strains. 3 days after the cross with wild type 74OR23-1A conidia, unilateral light was irradiated parallel to the solid medium with a 12-h light and 12-h dark regime at 25 °C (open box). Control cultures were placed in continuous darkness (shaded box). The position of beaks, upward or sideward, of perithecia, which were formed separately, was observed by dissecting microscopy. The average values for the percentage of the upward-formed beaks against total beaks observed and their standard errors (represented by bars) were calculated from three independent experiments. On the right side of the figure, the total number of perithecia scored after triplicate assays is presented under n.


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Fig. 4.   Perithecial polarity of wild type, ndk-1P72H, and the ndk-1 transformants. White light was provided parallel to the surface of the solid medium (from left in the photographs). Perithecia were observed and photographed through a dissecting microscope. WT, wild type.

Autophosphorylation and Protein Kinase Activities of NDK-1P72H Are Reduced-- Kinetic constants for NDK activity and protein-phosphorylation activity of NDK-1 or NDK-1P72H were determined using purified recombinant proteins. HisNDK-1 and HisNDK-1P72H showed comparable gamma -phosphotransferring activity for CDP, GDP, UDP, dCDP, dGDP, and dTDP in the thin-layer chromatography assay (data not shown). Therefore dTDP was used as a typical phosphate acceptor of gamma -phosphotransferring activity to determine the kinetic constants for the production of dTTP. As shown in Table I, neither the Michaelis constant (Km) that referred to dTDP as a substrate nor Vmax differed significantly between HisNDK-1 and HisNDK-1P72H; the Km and Vmax values were 0.28 ± 0.04 mM (dTDP) and 0.10 ± 0.09 nmol dTTP/min/mg of protein with HisNDK-1 and 0.29 ± 0.12 mM (dTDP) and 0.12 ± 0.10 nmol dTTP/min/mg of protein with HisNDK-1P72H, respectively. Similar results were also obtained by use of GST-NDK-1 and GST-NDK-1P72H. These findings indicated that the nucleotide-phosphorylation activity of NDK-1 was not affected by the P72H substitution. In contrast, the autophosphorylation and protein kinase activities of HisNDK-1P72H were greatly reduced compared with those of HisNDK-1 (data not shown). However, it is difficult to examine this in detail, because the electrophoretic mobility of HisNDK-1 (18 kDa) and MBP (18.5 kDa) is very similar. For this reason, we constructed GST-NDK-1 (NDK-1P72H) fusion protein (44 kDa), whose electrophoretic mobility is quite different from that of MBP.

                              
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Table I
Km and Vmax values of fusion proteins of NDK-1 and NDK-1P72H for gamma -phosphotransferring activity for dTDP
The values were averages of three independent experiments with standard errors. Vmax is the maximum velocity of the enzyme at 25°C.

Autophosphorylation of GST fusion proteins was examined by autoradiography (Fig. 5A). Although equal amounts of proteins were used (determined by Western blotting), there was about 5% of 32P-phosphorylated GST-NDK-1P72H compared with that in GST-NDK-1. This indicates that the ability of NDK-1P72H to phosphorylate itself was greatly reduced. Next, the activity to phosphorylate MBP was examined. GST fusion proteins were incubated with [gamma -32P]ATP and various concentrations of MBP (1.35, 2.7, 5.4, 8.1, or 10.8 µM) and separated by SDS-PAGE. Autoradiography shows that less [32P]phospho-MBP is produced when incubated with GST-NDK-1P72H (Fig. 5B). The kinetic constants were determined by the double-reciprocal plot of the concentration of MBP and the radioisotopic activity of [32P]phospho-MBP (Table II). Km and Vmax values were 7.3 ± 2.6 µM (MBP) and 0.48 ± 0.18 nmol/min/mg of protein ([32P]phospho-MBP) with GST-NDK-1 and 4.9 ± 0.1 µM (MBP) and 0.01 ± 0.00 nmol/min/mg of protein ([32P]phospho-MBP) with GST-NDK-1P72H, respectively. The Vmax value with GST-NDK-1P72H was about 2% of that with GST-NDK-1. These results revealed that the P72H mutation affected the activities of NDK-1 to phosphorylate both itself and MBP.


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Fig. 5.   Protein phosphorylation activities of recombinant proteins. A, the autophosphorylation of GST-NDK-1 and GST-NDK-1P72H was performed as in Fig. 1A except that 100 ng of purified GST-NDK-1 or GST-NDK-1P72H was used instead of crude extract. Western blotting with anti-NDK-1 antiserum (upper panel) and autophosphorylation activity visualized by autoradiography on x-ray film (lower panel) are shown. B, with the various concentrations of MBP (1.35, 2.7, 5.4, 8.1, and 10.8 µM), 10 ng of GST-NDK-1 or GST-NDK-1P72H was incubated for 1 min at 25 °C (see "Experimental Procedures"). The reaction mixtures were separated by SDS-PAGE, and the gel dried after staining with Coomassie Brillant Blue R-250 was exposed to x-ray film.

                              
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Table II
Km and Vmax values of fusion proteins of NDK-1 and NDK-1P72H for phosphotransfer activity for MBP
The values were averages of three independent experiments with standard errors.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We report here that the P72H substitution in NDK-1 causes partial reduction in accumulation of NDK-1 and significant loss of the activity to phosphorylate itself and MBP. This proline residue is highly conserved among the NDK genes reported to date (27) and is thought to be located between the alpha 2-helix and the beta 3-sheet from x-ray structural analyses of NDK in Dictyostelium discoideum (47, 48). These effects result in light insensitivity for perithecial polarity. In transformants of either construct containing wild type ndk-1 genomic DNA or cDNA, NDK-1 was adequately accumulated and phosphorylated as in the wild type, although in the PGN-2 strain, NDK-1 was accumulated less than in the wild type or other transformants (Fig. 2). However, the amount of NDK-1 in PGN-2 was sufficient, because there was no significant difference in the phosphorylation of NDK-1 compared with the wild type and other transformants (Fig. 2). This difference may be caused by the position of the integrated transgene in the N. crassa chromosome. The recovery of NDK-1 accumulation and phosphorylation in the transformants, even in PGN-2, results in the normal light response of perithecial polarity that is defective in psp (ndk-1P72H). This shows that the psp mutation was an allele of the ndk-1 locus and indicated that NDK-1 played a role in the regulation of perithecial polarity in response to light.

WC-1 and WC-2 are the key components in the blue light signaling pathway in N. crassa (21, 22). They are putative transcription factors and form a heterocomplex that was suggested to function as a signal transducer (23). The WC complex is also proposed to be a photoreceptor based on its sequence similarity in the LOV domain with Arabidopsis blue light receptor phototropin (NPH1) (7). WC-1 is phosphorylated, and this phosphorylation increases in response to light treatment (24). This fact suggests the involvement of a protein phosphorylation in the blue light signaling pathway. However, the protein kinases that mediate the light signaling have not been identified in N. crassa, though protein kinase C is suspected to play a role (25). Because the increase in the level of NDK-1 phosphorylation after blue light irradiation is very rapid, but not detected in wc mutants, the protein-phosphorylation activity of NDK-1 would contribute to the early steps of the light signaling downstream of WC proteins. Light response of perithecial polarity was also defective in wc-1 and wc-2, whereas other light-insensitive phenotypes observed in wc mutants, such as phototropism of perithecial beak, were not observed in ndk-1P72H. This fact suggests that various light-response phenomena were regulated by different pathways downstream of WC proteins and that NDK-1 was involved in one such pathway regulating perithecial polarity.

There was no significant difference in the level of ndk-1 mRNA accumulation between wild type and ndk-1P72H mutant, but Western analysis revealed that about 25% of NDK-1P72H protein was accumulated in the crude extract when compared with that of wild type. This result suggests that some post-translational regulation could play an essential role for NDK-1 accumulation. In a fish hepatocyte cell line, PLHC-1, NDK was co-purified with molecular chaperone HSC70 as an accessory protein (39). It could be possible that the region around the proline 72 was responsible for the contact with proteins such as molecular chaperons regulating the stability or folding of NDK-1. We are now underway on isolating proteins interacting with NDK-1 by affinity purification and by two-hybrid screening.

The reduction in Vmax for the phosphorylation of MBP by GST-NDK-1P72H shows that the P72H substitution affects not only autophosphorylation but also protein kinase activities of NDK-1. This result indicates that autophosphorylation and protein kinase activities occur via different mechanisms from that of nucleotide-phosphorylating activity of NDK-1. Therefore, the region around the proline 72 is proposed to contribute to the proper access to the substrate protein. Thus, the total decrease in the NDK-1 protein kinase activity, caused synergistically by less protein accumulation (25%) and less Vmax (2%) in the activity, could lead Neurospora to the loss of response to light.

We speculated that NDK-1 plays a crucial role in an unidentified pathway including autophosphorylation and protein kinase activities that regulate perithecial polarity in response to light. The biochemical activity of NDK-1P72H was reduced in terms of autophosphorylation and protein kinase activities but not nucleotide-phosphorylation activity. Therefore, the putative role of NDK-1 in signal transduction providing GTP to G-protein, e.g. regulating G-protein-coupled ion channels (30), may not be affected by the mutation, ndk-1P72H. However, we could consider that before providing GTP to G-protein, NDK-1 might also elicit signal through the autophosphorylation and protein kinase activities, indicating that there may be a hitherto unknown signal transduction pathway regulated by these two activities of NDK-1. Further understanding of the role of NDK-1 in the signaling pathway may require investigation of the proteins that interact with NDK-1, such as a protein recognizing the phosphorylated NDK-1 and a protein phosphorylated by NDK-1.

    ACKNOWLEDGEMENTS

We are grateful to D. D. Perkins for critical reading and to W. R. Briggs for critical reading and discussion of the manuscript. We thank C. Yanofsky, N. Kimura, K. Oda, and K. Ichimura for helpful discussions, C. Aoyagi for help in RNA experiments, H. Inoue, C. Ishii, and S. Hatakeyama for help in Neurospora transformation, and S. Hirose for technical assistance.

    FOOTNOTES

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Contributed equally to this work.

§ To whom correspondence should be addressed. Tel.: 81-45-820-1903; Fax: 81-45-820-1901; E-mail: kohji@yokohama-cu.ac.jp.

Published, JBC Papers in Press, April 3, 2001, DOI 10.1074/jbc.M011381200

    ABBREVIATIONS

The abbreviations used are: wc (or WC), white collar; NDK, nucleoside diphosphate kinase; RT, reverse transcription; PCR, polymerase chain reaction; Pipes, 1,4-piperazinediethanesulfonic acid; PAGE, polyacrylamide gel electrophoresis; bp, base pair(s); GST, glutathione S-transferase; MBP, myelin basic protein.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Kendrick, R. E., and Kronenberg, G. H. M. (eds) (1994) Photomorphogenesis in Plants , 2nd Ed. , Kluwer Academic Publishers, Netherlands
2. Meyerowitz, E. M., and Somerville, C. R. (eds) (1994) Arabidopsis , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
3. Horwitz, B. A., and Berrocal, T. G. M. (1995) Bot. Acta 110, 360-368
4. Whitelam, G. C., and Devlin, P. F. (1998) Plant Physiol. Biochem. 36, 125-133[CrossRef]
5. Batschauer, A. (1998) Planta 206, 479-492[CrossRef][Medline] [Order article via Infotrieve]
6. Quail, P. H. (1997) BioEssays 19, 571-579[Medline] [Order article via Infotrieve]
7. Briggs, W. R., and Huala, E. (1999) Annu. Rev. Cell Dev. Biol. 15, 33-62[CrossRef][Medline] [Order article via Infotrieve]
8. Ahmad, M., Jarillo, J. A., Smirnova, O., and Cashmore, A. R. (1998) Mol. Cell 1, 939-948[Medline] [Order article via Infotrieve]
9. Christie, J. M., Reymond, P., Powell, G. K., Bernasconi, P., Raibekas, A. A., Liscum, E., and Briggs, W. R. (1998) Science 282, 1698-1701[Abstract/Free Full Text]
10. Lasceve, G., Leymarie, J., Olney, M. A., Liscum, E., Christie, J. M., Vavasseur, A., and Briggs, W. R. (1999) Plant Physiol. 120, 605-614[Abstract/Free Full Text]
11. Potapova, T. V., Levina, N. N., Belozerskaya, T. A., Kritsky, M. S., and Chailakhian, L. M. (1984) Arch. Microbiol. 137, 262-265
12. Belozerskaya, T. A., and Potapova, T. V. (1993) Exp. Mycol. 17, 157-169[CrossRef]
13. Harding, R. W., and Turner, R. V. (1981) Plant Physiol. 68, 745-749
14. Lauter, F.-R., and Russo, V. E. A. (1991) Nucleic Acids Res. 19, 6883-6886[Abstract]
15. Degli Innocenti, F., and Russo, V. E. A. (1984) J. Bacteriol. 159, 757-761[Medline] [Order article via Infotrieve]
16. Harding, R. W., and Melles, S. (1984) Plant Physiol. 72, 996-1000
17. Oda, K., and Hasunuma, K. (1997) Mol. Gen. Genet. 256, 593-601[CrossRef][Medline] [Order article via Infotrieve]
18. Sargent, M. L., and Briggs, W. R. (1967) Plant Physiol. 42, 1504-1510
19. Perkins, D. D., Radford, A., Newmeyer, D., and Bjorkman, M. (1982) Microbiol. Rev. 46, 426-570
20. Nelson, M. A., Morelli, G., Carattoli, A., Romano, N., and Macino, G. (1989) Mol. Cell. Biol. 9, 1271-1276[Medline] [Order article via Infotrieve]
21. Ballario, P., Vittorioso, P., Margrelli, A., Talora, C., Cabibbo, A., and Macino, G. (1996) EMBO J. 15, 1650-1657[Abstract]
22. Linden, H., and Macino, G. (1997) EMBO J. 16, 98-109[Abstract/Free Full Text]
23. Ballario, P., Talora, C., Galli, D., Linden, H., and Macino, G. (1998) Mol. Microbiol. 29, 719-729[CrossRef][Medline] [Order article via Infotrieve]
24. Talora, C., Franchi, L., Linden, H., Ballario, P., and Macino, G. (1999) EMBO J. 18, 4961-4968[Abstract/Free Full Text]
25. Arpaia, G., Cerri, F., Baima, S., and Macino, G. (1999) Mol. Gen. Genet. 262, 314-322[CrossRef][Medline] [Order article via Infotrieve]
26. Oda, K., and Hasunuma, K. (1994) FEBS Lett. 345, 162-166[CrossRef][Medline] [Order article via Infotrieve]
27. Ogura, Y., Yoshida, Y., Ichimura, K., Aoyagi, C., Yabe, N., and Hasunuma, K. (1999) Eur. J. Biochem. 266, 709-714[Abstract/Free Full Text]
28. Parks, R. E., Jr., and Agarwal, R. P. (1973) in The Enzymes (Boyer, P. D., ed) , pp. 307-333, Academic Press, NY
29. Steeg, P. S., Bevilacqua, G., Pozzatti, R., Liotta, L. A., and Sobel, M. E. (1988) Cancer Res. 48, 6550-6554[Abstract]
30. Otero, A. S., Doyle, M. B., Hartsough, M. T., and Steeg, P. S. (1999) Biochim. Biophys. Acta 1449, 157-168[Medline] [Order article via Infotrieve]
31. Postel, E. H., Berberich, S. J., Flint, S. J., and Ferrone, C. A. (1993) Science 261, 478-480[Medline] [Order article via Infotrieve]
32. Biggs, J., Hersperger, E., Steeg, P. S., Liotta, L. A., and Shearn, A. (1990) Cell 63, 933-940[Medline] [Order article via Infotrieve]
33. Izumiya, H., and Yamamoto, M. (1995) J. Biol. Chem. 270, 27859-27864[Abstract/Free Full Text]
34. Tanaka, N., Ogura, T., Noguchi, T., Hirano, H., Yabe, N., and Hasunuma, K. (1998) J. Photochem. Photobiol. 45, 113-121[CrossRef]
35. Ogura, T., Tanaka, N., Yabe, N., Komatsu, S., and Hasunuma, K. (1999) Photochem. Photobiol. 69, 397-403
36. Choi, G., Yi, H., Lee, J., Kwon, Y.-K., Soh, M. S., Shin, B., Luka, Z., Hahn, T.-R., and Song, P.-S. (1999) Nature (London) 401, 610-613[CrossRef][Medline] [Order article via Infotrieve]
37. Zimmermann, S., Baumann, A., Jaekel, K., Marbach, I., Engelberg, D., and Frohnmeyer, H. (1999) J. Biol. Chem. 274, 17017-17024[Abstract/Free Full Text]
38. Kimura, N., and Shimada, N. (1983) J. Biol. Chem. 258, 2278-2283[Abstract/Free Full Text]
39. Leung, S.-M., and Hightower, L. E. (1997) J. Biol. Chem. 272, 2607-2614[Abstract/Free Full Text]
40. Freije, J. M. P., Blay, P., MacDonald, N. J., Manrow, R. E., and Steeg, P. S. (1997) J. Biol. Chem. 272, 5525-5532[Abstract/Free Full Text]
41. Sokolovsky, V., Kaldenhoff, R., Ricci, M., and Russo, V. E. A. (1990) Fungal Genet. Newsl. 37, 41-43
42. Vollmer, S. J., and Yanofsky, C. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 4869-4873[Abstract]
43. Pall, M. L., and Brunelli, J. P. (1993) Fungal Genet. Newsl. 40, 59-62
44. Mullaney, E. J., Hamer, J. E., Roberti, K. A., Yelton, M. M., and Timberlake, W. E. (1985) Mol. Gen. Genet. 199, 37-45[Medline] [Order article via Infotrieve]
45. Moreno, S., Klar, A., and Nurse, P. (1991) Methods Enzymol. 194, 795-823[Medline] [Order article via Infotrieve]
46. Agarwal, R. P., Robinson, B. R. E., and Parks, J. (1978) Methods Enzymol. 51, 376-386[Medline] [Order article via Infotrieve]
47. Dumas, C., Lascu, I., Moréra, S., Glaser, P., Fourme, R., Wallet, V., Lacombe, M.-L., Véron, M., and Janin, J. (1992) EMBO J. 11, 3203-3208[Abstract]
48. Moréra, S., LeBras, G., Lascu, I., Lacombe, M. L., Véron, M., and Janin, J. (1994) J. Mol. Biol. 243, 873-890[CrossRef][Medline] [Order article via Infotrieve]


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