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
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ABSTRACT |
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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.
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
In the present study, we have investigated the roles of NDK-1 in the
light signal transduction pathway through the 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
[ 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
[ 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.
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).
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.
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
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
[ 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 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.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-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.
-phosphotransferring activity from nucleoside 5'-triphosphate to nucleoside 5'-diphosphate and the phosphotransfer activity to other proteins.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-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).
-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 [
-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
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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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.
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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.
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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.
-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
-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.
Km and Vmax values of fusion proteins of NDK-1 and
NDK-1P72H for -phosphotransferring activity for dTDP
-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.
View larger version (56K):
[in a new window]
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.
Km and Vmax values of fusion proteins of NDK-1 and
NDK-1P72H for phosphotransfer activity for MBP
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2-helix and the
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.
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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* 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.
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
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ABBREVIATIONS |
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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.
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REFERENCES |
---|
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---|
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 |
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 |
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 |
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 |
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 |
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 |
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 |
38. |
Kimura, N.,
and Shimada, N.
(1983)
J. Biol. Chem.
258,
2278-2283 |
39. |
Leung, S.-M.,
and Hightower, L. E.
(1997)
J. Biol. Chem.
272,
2607-2614 |
40. |
Freije, J. M. P.,
Blay, P.,
MacDonald, N. J.,
Manrow, R. E.,
and Steeg, P. S.
(1997)
J. Biol. Chem.
272,
5525-5532 |
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] |