Journal of Experimental Botany, Vol. 53, No. 369, pp. 765-767,
April 1, 2002
© 2002 Oxford University Press
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
1 Gene Research Center, Tokyo University of Agriculture and Technology, Fuchu-shi, Tokyo 183-8509, Japan
2 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
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 self-recognition 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 serine/threonine 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 SRK9 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 employed for the screening of a stigma cDNA expression library of B. campestris (S9/S9) described previously (Matsushita et al., 2001). Phage plaques on agar plates were overlaid with nitrocellulose membranes soaked with isopropyl ß-D-thiogalactopyranoside and further incubated for the expression of cDNA products. The membranes containing the induced proteins were then incubated with [
-32P]ATP 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%).
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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 increased about 10-fold. This increase was not affected by KN-62 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 Bc-NDPK (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.
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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.
Acknowledgments
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.
Notes
5 To whom correspondence should be addressed. Fax: +81423608830. E-mail: nyunoya{at}cc.tuat.ac.jp 
References
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