Download Isolation of a cDNA for a nucleoside diphosphate kinase capable of

Journal of Experimental Botany, Vol. 53, No. 369, pp. 765–767, April 2002
GENE NOTE
Isolation of a cDNA for a nucleoside diphosphate kinase capable
of phosphorylating the kinase domain of the self-incompatibility
factor SRK of Brassica campestris
Yasuhiko Matsushita1, Tatsuya Suzuki1, Ryo Kubota1, Masako Mori1, Hiroko Shimosato1, Masao Watanabe2, Toshiaki Kayano3,
Takeshi Nishio4 and Hiroshi Nyunoya1,5
1
Gene Research Center, Tokyo University of Agriculture and Technology, Fuchu-shi, Tokyo 183-8509, Japan
Faculty of Agriculture, Iwate University, Morioka 020-8550, Japan
3
National Institute of Agrobiological Sciences, Tukuba 305-8602, Japan
4
Faculty of Agriculture, Tohoku University, Sendai, 981-8555, Japan
2
Received 25 September 2001; Accepted 21 November 2001
Abstract
SRK is a plant receptor kinase involved in the self-incompatibility
system of Brassica species. During a cDNA screening for
the phosphoproteins from a stigma expression library, a clone
encoding the nucleoside diphosphate kinase III (Bc-NDPK III) was
obtained. After in vitro phosphorylation assays with recombinant
proteins, Bc-NDPK III contained mostly phosphoserine. By
contrast, the kinase domain of SRK contained phosphoserine
and phosphothreonine, both of which were significantly increased
by the addition of Bc-NDPK III in the presence of an SRK inhibitor KN-62. The result suggested the possible involvement of
Bc-NDPK III in the signal transduction pathway through SRK.
Key words: Brassica campestris, nucleoside diphosphate kinase,
self-incompatibility factor, SRK.
In higher plants, different types of self-recognition systems are
operating during pollination to avoid self-fertilization. The selfrecognition signal is generated by the interaction between
pollen ligand and stigma receptor proteins and transduced by
a receptor kinase to induce incompatibility response (Brugière
et al., 2000). The SRK (S-locus receptor kinase) gene was
molecularly cloned from Brassica species and shown to have an
intrinsic serineuthreonine kinase activity (Stein and Nasrallah,
1993). In an in vitro assay system using a recombinant SRK
protein, the authors observed that SRK protein could interact
with several phosphoproteins extracted from stigma cells (data
not shown). To elucidate the phosphorylation cascade involved
in the self-recognition mechanism, screening for cDNA clones
encoding the stigma phosphoproteins was undertaken.
The SRK 9 cDNA derived from Brassica campestris (synonym
B. rapa) was used. Plasmid DNA of pCRII-SRK9 (Watanabe
et al., 1994) was provided for the construction of a series of
expression plasmids. The final plasmid pGEXfB-His-SRK9KD
was used for the expression of glutathione S-transferase
(GST)-fused kinase domain of SRK9 (SRK-KD). A solid-phase
phosphorylation method (Fukunaga and Hunter, 1997) was
5
employed for the screening of a stigma cDNA expression library
of B. campestris (S9uS9) described previously (Matsushita et al.,
2001). Phage plaques on agar plates were overlaid with nitrocellulose membranes soaked with isopropyl b-D-thiogalactopyranoside and further incubated for the expression of cDNA
products. The membranes containing the induced proteins were
then incubated with wc-32PxATP and the recombinant kinase
SRK-KD, allowing the cDNA screening for SRK substrates in
addition to other kinases with autophosphorylation activity.
Phosphorylation-positive clones were identified from the autoradiograms. Phage DNA prepared from each phage clone was
converted to the corresponding expression plasmid for the
analysis of each cDNA product (Fukunaga and Hunter, 1997).
One of the clones showing autophosphorylation activity
revealed an ORF highly homologous to nucleoside diphosphate
kinases (NDPK; EC 2.7.4.6) from various organisms. Since
plant type III enzymes gave the highest scores, the gene for this
clone was designated Bc-NDPK III. The nucleotide sequence
was submitted to the DDBJ database (AB029400). Figure 1
shows an alignment with three classes of NDPKs from
Arabidopsis thaliana and Spinacia oleracea. A putative matured
protein beginning with Ala-42 of Bc-NDPK III was highly
homologous to At-NDPK 3 (94% identity) and So-NDPK
III (86%), while it was only moderately homologous to
At-NDPK 2 (53%), At-NDPK 1 (54%), So-NDPK II (55%),
and So-NDPK I (57%).
The GST-fused Bc-NDPK III protein (Bc-NDPK) was
expressed and subjected to an in vitro phosphorylation assay
either separately or in combination with purified SRK-KD. The
separate assays (Fig. 2, left and right panels) indicated that both
SRK-KD and Bc-NDPK had autophosphorylation activity
by themselves. The specific activity of autophosphorylation of
Bc-NDPK was much higher than that of SRK-KD. Addition of
a kinase inhibitor KN-62 (Seikagaku Co., Tokyo) resulted in a
decrease in the level of autophosphorylation of SRK-KD but
not of Bc-NDPK. When the two proteins were combined, the
phosphorylation level of Bc-NDPK was not altered (middle
panel). By contrast, the phosphorylation level of SRK-KD
To whom correspondence should be addressed. Fax: q81 42 360 8830. E-mail: [email protected]
ß Society for Experimental Biology 2002
766
Matsushita et al.
Fig. 1. Amino acid sequence comparison among various NDPKs. The deduced amino acid sequence of Bc-NDPK III is aligned with NDPK sequences
of A. thaliana (At-NDPK 3, AF044265; At-NDPK 2, AF017640; At-NDPK 1, AF058391) and S. oleracea (So-NDPK III, S60363 wPIRx; So-NDPK II,
D11465; So-NDPK I, D10659). Amino acid residues conserved among four or more NDPKs were shaded. Arrowhead shows a putative amino
terminus of matured protein that was reported for So-NDPK III. Asterisk shows the putative autophosphorylation sites.
Fig. 2. Phosphorylation of SRK-KD by Bc-NDPK. The purified SRK-KD (1 mg) was assayed either by itself or combined with Bc-NDPK in 50 ml of
kinase reaction buffer containing 20 mM PIPES (pH 7.5), 10 mM MgCl2, 2 mM MnCl2, 0.67 mM ATP, and 740 kBq wc-32Px ATP (168 TBq mmol 1).
The assays were performed with increasing amounts of KN-62 (0, 1, 10, 25, 50, and 100 mM) as indicated with solid triangles above the lanes. The
upper panels are the autoradiograms and the lower ones are protein staining with Coomassie blue. The positions of SRK-KD (65 kDa) and Bc-NDPK
(43 kDa) are indicated by arrows.
Self-incompatibility factor SRK of Brassica campestris 767
increased about 10-fold. This increase was not affected by KN62 indicating that the autophosphorylation activity of SRK-KD
was not involved. It was concluded that SRK-KD was
phosphorylated by Bc-NDPK. It was confirmed that the GST
moiety of the fusion protein was not phosphorylated by BcNDPK (data not shown). Phosphoamino acid analysis indicated
that the autophosphorylated SRK-KD contained both phosphoserine and phosphothreonine, while autophosphorylated
Bc-NDPK contained mostly phosphoserine (data not shown).
No changes in the phosphoamino acid composition were
observed in the combined assay.
The signal transduction pathway downstream of the receptor
kinase SRK is largely unknown. Although ARC1 was reported
to be phosphorylated by SRK (Gu et al., 1998), the phosphorylation cascade may involve multiple factors that are yet to be
determined. Regulatory roles of NDPK have been reported for
light signal transduction in higher plants. The NDPKs of pea
and rice were found to be phosphorylated upon irradiation of
red light after being grown in the dark (Hamada et al., 1996,
1999). Activated phytochrome was shown to bind to NDPK 2 of
Arabidopsis (Choi et al., 1999). It would be an intriguing
possibility that Bc-NDPK III might have some role in the
phosphorylation cascade through the receptor kinase SRK.
Acknowledgements
This work was supported by Grants-in-Aid for Special Research on
Priority Areas (Genetic Dissection of Sexual Differentiation and
Pollination Process in Higher Plants) to HN from the Ministry of
Education, Science, Culture and Sports, Japan.
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