Journal of Experimental Botany, Vol. 52, No. 354, pp. 181-182,
January 2001
© 2001 Oxford University Press
Gene Notes |
The AKT3 potassium channel protein interacts with the AtPP2CA protein phosphatase 2C
1 Vakgroep Moleculaire Genetica and Departement Plantengenetica, Vlaams Interuniversitair Instituut voor Biotechnologie (VIB), Universiteit Gent, K.L. Ledeganckstraat 35, B-9000 Gent, Belgium
2 Department of Biosciences, Division of Genetics, PO Box 56, FIN-00014 University of Helsinki, Finland
Received 21 July 2000; Accepted 4 October 2000
Abstract
The AKT3 potassium channel protein was identified as a strongly interacting partner of the Arabidopsis thaliana protein phosphatase 2C (AtPP2CA) in a yeast two-hybrid screen. A deletion analysis indicated that the catalytic domain of AtPP2CA was essential for the interaction with AKT3. Furthermore, the related PP2C phosphatase ABI1 did not interact with AKT3 in yeast.
Key words: Arabidopsis thaliana, potassium channel, PP2C, yeast two-hybrid screening.
Protein phosphatases 2C (PP2Cs) are essential components of the abscisic acid (ABA) signal transduction pathway. Mutation in the phosphatases 2C ABI1 and ABI2 of Arabidopsis thaliana confers insensitivity to externally applied ABA for seed dormancy, germination, stomatal closure, drought rhizogenesis, and ABA-induced gene expression. The pleiotropic responses in the abi1-1 and abi2-1 mutants position these phosphatases early in the ABA signal transduction pathway (Merlot and Giraudat, 1997
). ABI1 and ABI2 phosphatases fulfil partly redundant functions, but at the same time they seem to control different steps in the ABA signalling cascade (Pei et al., 1997). Two additional PP2Cs in Arabidopsis are implicated in ABA signalling (AtPP2CA and AtP2C-HA) (Kuromori and Yamamoto, 1994; Rodriguez et al., 1998), suggesting that multiple control points are affected by this class of enzymes (Grill and Himmelbach, 1998). However, substrates of PP2Cs have not been identified thus far. As part of a study on the signalling mechanism of AtPP2CA, a yeast two-hybrid screen was performed by using AtPP2CA as bait.
The AtPP2CA cDNA was amplified from an Arabidopsis thaliana (L.) Heynh. cDNA library (Minet et al., 1992) with the forward primer 5'-AGTTTTGCTGCAGATCATATGGCTGGG-3' and the reverse primer 5'-ATTATTA-ACTGCAGGCTTGATTATTCC-3', and cloned into the pAS2-1 GAL4 DNA-binding domain vector (Clontech, Palo Alto, CA) as NdeI-PstI fragment (pASPP2C). The GAL4 activation domain::cDNA fusion library originated from 3-week-old green vegetative tissue of A. thaliana, ecotype Columbia (Clontech). The Saccharomyces cerevisiae reporter strain HF7c was co-transformed with 250 µg pASPP2C and 500 µg DNA of the library by using the lithium acetate method (Gietz et al., 1992). The transformation mix was plated on histidine-lacking (His-) selection medium supplemented with 5 mM 3-amino-1,2,4-triazole (Sigma, St Louis, MO). The activation domain plasmids were isolated from the colonies that were able to grow on His- selective plates. Inserts were amplified by PCR and digested with AluI. Plasmids with distinct restriction patterns were retransformed together with the bait to HF7c cells and tested for histidine auxotrophy (His+). Positive interactions between the hybrid constructs were reconfirmed in the yeast Y190 host strain by the ß-galactosidase (LacZ) colony-lift filter assay (Matchmaker yeast protocols handbook; Clontech). Plasmids from His+ and LacZ+ colonies were isolated and cDNAs encoding four different proteins were identified by sequence analysis of inserts.
One of the encoded proteins with the strongest ß-galactosidase activity was identified as the inward-rectifying potassium (
) channel protein AKT3 (Cao et al., 1995; Ketchum and Slayman, 1995). The isolated cDNA encoded a truncated protein (amino acids 319–787) that lacked the N-terminus with the putative membrane spanning segments. channels play a role in potassium uptake and in controlling the membrane potential. AKT3 is expressed predominantly in phloem (Marten et al., 1999). It is currently unknown whether ABA and/or PP2Cs control this channel activity. However, the activity of K+ channels in guard cells is known to be modulated by ABA, and PP2C (ABI1) has been implicated downstream of ABA in this process (Armstrong et al., 1995). ABA regulation of K+ channels is not restricted to the guard cells and recently, regulation of K+ channel activity by ABA in stelar root cells has been reported (Roberts, 1998). The data presented here show that AtPP2CA, a PP2C implicated in ABA signalling, interacts with phloem-localized channel protein AKT3 in a yeast two-hybrid system, suggesting that the AKT3 channel protein is a substrate of the AtPP2CA protein phosphatase 2C. Moreover, it could be shown that the catalytic domain of AtPP2CA is required for the binding to AKT3, since deletion of part of the catalytic domain (
220–339 amino acids) completely abolished interaction of AtPP2CA with AKT3 (Fig. 1rpar;.
|
PP2Cs in Arabidopsis are largely redundant with respect to many physiological responses (Merlot and Giraudat, 1997
). It has been demonstrated that AtPP2CA can substitute for ABI1 in the regulation of ABA-induced gene expression when ectopically expressed in maize protoplasts (Sheen, 1998). To test whether ABI1 also interacts with AKT3 in the two-hybrid system, the GAL4 DNA-binding domain vector pAS2-1 containing ABI1 cDNA (pASABI1) or AtPP2CA cDNA (pASPP2C) was co-transformed to yeast together with the pGAD10 GAL4 activation domain vector that contained the isolated AKT3 cDNA (pGADAKT3). Histidine auxotrophy and ß-galactosidase activity were observed only in yeast co-transformed with AtPP2CA and AKT3 cDNAs (Fig. 1
). This result is indicative of the specificity of interaction between AtPP2CA and AKT3 and suggests that AtPP2CA and ABI1 are not functionally equivalent with respect to the binding and control of AKT3. In summary, the protein phosphatase 2C AtPP2CA specifically interacts in a yeast two-hybrid system with the K+ channel protein AKT3 and this interaction is aborted when part of the catalytic domain of AtPP2CA is deleted. This result is the first that identifies a substrate of PP2C implicated in ABA signalling, and shows that plant K+ channels may be directly dephosphorylated by type 2C protein phosphatases.
Acknowledgments
The Arabidopsis Biological Resource Center and the DNA Stock Center are acknowledged for providing the Arabidopsis thaliana cDNA EST clone 64D5T7, and M Minet and F Lacroute for providing the A. thaliana cDNA library. The authors thank the sequencing group for the technical assistance with DNA sequencing, L De Veylder for advice on the yeast two-hybrid screen and critical reading of the manuscript, D Geelen for comments on the manuscript, and M De Cock, K Spruyt and S Debruyne for help in preparing it. This work was supported by the Academy of Finland, Biocentrum Helsinki and by grants from the European Union (BIOTECH Program ERB-BIO4-CT96-0101) and the Belgian Administration for Development Cooperation.
Notes
3 Permanent address: Department of Experimental Botany and Genetics, P.J. 
afárik University, Mánesova 23, 04154 Ko
ice, Slovakia. 
4 Present address: Aventis CropScience N.V., Jozef Plateaustraat 22, B-9000 Gent, Belgium.
5 Present address: CropDesign N.V., Technologiepark 3, B-9052 Zwijnaarde, Belgium.
6 To whom correspondence should be addressed. Fax: +32 9 264 5349. E-mail: diinz{at}gengenp.rug.ac.be
References
Armstrong F, Leung J, Grabov A, Brearley J, Giraudat J, Blatt MR.1995. Sensitivity to abscisic acid of guard-cell K+ channels is suppressed by abi1-1, a mutant Arabidopsis gene encoding a putative protein phosphatase. Proceedings of the National Academy of Sciences, USA92, 9520–9524.
Cao Y, Ward JM, Kelly WB, Ichida AM, Gaber RF, Anderson JA, Uozumi N, Schroeder JI, Crawford NM.1995. Multiple genes, tissue specificity and expression-dependent modulation contribute to the functional diversity of potassium channels in Arabidopsis thaliana. Plant Physiology109, 1093–1106.[Abstract]
Gietz D, St Jean A, Woods RA, Schiestl RH.1992. Improved method for high efficiency transformation of intact yeast cells. Nucleic Acids Research20, 1425.
Grill E, Himmelbach A.1998. ABA signal transduction. Current Opinion in Plant Biology1, 412–418.[ISI][Medline]
Ketchum KA, Slayman CW.1995. Isolation of an ion channel gene from Arabidopsis thaliana using the H5 signature sequence from voltage-dependent K+ channels. FEBS Letters378, 19–26.[ISI][Medline]
Kuromori T, Yamamoto M.1994. Cloning of cDNAs from Arabidopsis thaliana that encode putative protein phosphatase 2C and a human Dr1-like protein by transformation of a fission yeast mutant. Nucleic Acids Research22, 5296–5301.
Marten I, Hoth S, Deeken R, Ache P, Ketchum KA, Hoshi T, Hedrich R.1999. AKT3, a phloem-localized K+ channel, is blocked by protons. Proceedings of the National Academy of Sciences, USA96, 7581–7586.
Merlot S, Giraudat J.1997. Genetic analysis of abscisic acid signal transduction. Plant Physiology114, 751–757.[ISI][Medline]
Minet M, Dufour M-E, Lacroute F.1992. Complementation of Saccharomyces cerevisiae auxotrophic mutants by Arabidopsis thaliana cDNAs. The Plant Journal2, 417–422.[ISI][Medline]
Pei Z-M, Kuchitsu K, Ward JM, Schwarz M, Schroeder JI.1997. Differential abscisic acid regulation of guard cell slow anion channels in Arabidopsis wild-type and abi1 and abi2 mutants. The Plant Cell9, 409–423.[Abstract]
Roberts SK.1998. Regulation of K+ channels in maize roots by water stress and abscisic acid. Plant Physiology116, 145–153.
Rodriguez PL, Leube MP, Grill E.1998. Molecular cloning in Arabidopsis thaliana of a new protein phosphatase 2C (PP2C) with homology to ABI1 and ABI2. Plant Molecular Biology38, 879–883.[ISI][Medline]
Sheen J.1998. Mutational analysis of protein phosphatase 2C involved in abscisic acid signal transduction in higher plants. Proceedings of the National Academy of Sciences, USA95, 975–980.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  S. C. Lee, W.-Z. Lan, B.-G. Kim, L. Li, Y. H. Cheong, G. K. Pandey, G. Lu, B. B. Buchanan, and S. Luan A protein phosphorylation/dephosphorylation network regulates a plant potassium channel PNAS, October 2, 2007; 104(40): 15959 - 15964. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Yoshida, N. Nishimura, N. Kitahata, T. Kuromori, T. Ito, T. Asami, K. Shinozaki, and T. Hirayama ABA-Hypersensitive Germination3 Encodes a Protein Phosphatase 2C (AtPP2CA) That Strongly Regulates Abscisic Acid Signaling during Germination among Arabidopsis Protein Phosphatase 2Cs Plant Physiology, January 1, 2006; 140(1): 115 - 126. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W. Zhang, C. Qin, J. Zhao, and X. Wang Phospholipase D{alpha}1-derived phosphatidic acid interacts with ABI1 phosphatase 2C and regulates abscisic acid signaling PNAS, June 22, 2004; 101(25): 9508 - 9513. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 I. Cherel Regulation of K+ channel activities in plants: from physiological to molecular aspects J. Exp. Bot., February 1, 2004; 55(396): 337 - 351. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Flajolet, S. Rakhilin, H. Wang, N. Starkova, N. Nuangchamnong, A. C. Nairn, and P. Greengard Protein phosphatase 2C binds selectively to and dephosphorylates metabotropic glutamate receptor 3 PNAS, December 23, 2003; 100(26): 16006 - 16011. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. P. Gonzalez-Garcia, D. Rodriguez, C. Nicolas, P. L. Rodriguez, G. Nicolas, and O. Lorenzo Negative Regulation of Abscisic Acid Signaling by the Fagus sylvatica FsPP2C1 Plays A Role in Seed Dormancy Regulation and Promotion of Seed Germination Plant Physiology, September 1, 2003; 133(1): 135 - 144. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 I. Cherel, E. Michard, N. Platet, K. Mouline, C. Alcon, H. Sentenac, and J.-B. Thibaud Physical and Functional Interaction of the Arabidopsis K+ Channel AKT2 and Phosphatase AtPP2CA PLANT CELL, May 1, 2002; 14(5): 1133 - 1146. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||