Arabidopsis CIPK26 interacts with KEG, components of the ABA signalling network and is degraded by the ubiquitin-proteasome system

Wendy J. Lyzenga; Hongxia Liu; Andrew Schofield; Alexandria Muise-Hennessey; Sophia L. Stone

doi:10.1093/jxb/ert123

Arabidopsis CIPK26 interacts with KEG, components of the ABA signalling network and is degraded by the ubiquitin-proteasome system

Wendy J. Lyzenga, Hongxia Liu, Andrew Schofield, Alexandria Muise-Hennessey, Sophia L. Stone

Medicine

Research output: Contribution to journal › Article › peer-review

134 Citations (Scopus)

Abstract

The RING-type E3 ligase, Keep on Going (KEG), is required for early seedling establishment in Arabidopsis thaliana. Post-germination, KEG negatively regulates abscisic acid (ABA) signalling by targeting Abscisic Acid Insensitive 5 (ABI5) for ubiquitination and subsequent degradation. Previous reports suggest that the role of KEG during early seedling development is not limited to regulation of ABI5 abundance. Using a yeast two-hybrid screen, this study identified Calcineurin B-like Interacting Protein Kinase (CIPK) 26 as a KEG-interacting protein. In vitro pull-down and in planta bimolecular fluorescence complementation assays confirmed the interactions between CIPK26 and KEG. In planta experiments demonstrated that CIPK26 was ubiquitinated and degraded via the 26S proteasome. It was also found that turnover of CIPK26 was increased when KEG protein levels were elevated, suggesting that the RING-type E3 ligase is involved in targeting CIPK26 for degradation. CIPK26 was found to interact with the ABA signalling components ABI1, ABI2, and ABI5. In addition, CIPK26 was capable of phosphorylating ABI5 in vitro. Consistent with a role in ABA signalling, overexpression of CIPK26 increased the sensitivity of germinating seeds to the inhibitory effects of ABA. The data presented in this report suggest that KEG mediates the proteasomal degradation of CIPK26 and that CIPK26 is part of the ABA signalling network.

Original language	English
Pages (from-to)	2779-2791
Number of pages	13
Journal	Journal of Experimental Botany
Volume	64
Issue number	10
DOIs	https://doi.org/10.1093/jxb/ert123
Publication status	Published - Jul 2013

Bibliographical note

Funding Information:
The authors would like to thank Dr Russ Finley (Wayne State University) for yeast two-hybrid vectors, Dr Yuhai Cui (Agriculture and Agri-Food Canada) for the BiFc vectors and Dr Sonia Gazzarrini (University of Toronto) for providing the yeast two-hybrid cDNA library. We would also like to thank Judy Booth for critical reading of the manuscript. This project was supported in part by a Natural Science and Engineering Research Council of Canada (NSERC) grant and post-graduate scholarship to S.L.S and W.J.L, respectively.

ASJC Scopus Subject Areas

Physiology
Plant Science

PubMed: MeSH publication types

Journal Article
Research Support, Non-U.S. Gov't

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10.1093/jxb/ert123

Cite this

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title = "Arabidopsis CIPK26 interacts with KEG, components of the ABA signalling network and is degraded by the ubiquitin-proteasome system",

abstract = "The RING-type E3 ligase, Keep on Going (KEG), is required for early seedling establishment in Arabidopsis thaliana. Post-germination, KEG negatively regulates abscisic acid (ABA) signalling by targeting Abscisic Acid Insensitive 5 (ABI5) for ubiquitination and subsequent degradation. Previous reports suggest that the role of KEG during early seedling development is not limited to regulation of ABI5 abundance. Using a yeast two-hybrid screen, this study identified Calcineurin B-like Interacting Protein Kinase (CIPK) 26 as a KEG-interacting protein. In vitro pull-down and in planta bimolecular fluorescence complementation assays confirmed the interactions between CIPK26 and KEG. In planta experiments demonstrated that CIPK26 was ubiquitinated and degraded via the 26S proteasome. It was also found that turnover of CIPK26 was increased when KEG protein levels were elevated, suggesting that the RING-type E3 ligase is involved in targeting CIPK26 for degradation. CIPK26 was found to interact with the ABA signalling components ABI1, ABI2, and ABI5. In addition, CIPK26 was capable of phosphorylating ABI5 in vitro. Consistent with a role in ABA signalling, overexpression of CIPK26 increased the sensitivity of germinating seeds to the inhibitory effects of ABA. The data presented in this report suggest that KEG mediates the proteasomal degradation of CIPK26 and that CIPK26 is part of the ABA signalling network.",

author = "Lyzenga, {Wendy J.} and Hongxia Liu and Andrew Schofield and Alexandria Muise-Hennessey and Stone, {Sophia L.}",

note = "Funding Information: The authors would like to thank Dr Russ Finley (Wayne State University) for yeast two-hybrid vectors, Dr Yuhai Cui (Agriculture and Agri-Food Canada) for the BiFc vectors and Dr Sonia Gazzarrini (University of Toronto) for providing the yeast two-hybrid cDNA library. We would also like to thank Judy Booth for critical reading of the manuscript. This project was supported in part by a Natural Science and Engineering Research Council of Canada (NSERC) grant and post-graduate scholarship to S.L.S and W.J.L, respectively.",

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AU - Lyzenga, Wendy J.

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AU - Schofield, Andrew

AU - Muise-Hennessey, Alexandria

AU - Stone, Sophia L.

N1 - Funding Information: The authors would like to thank Dr Russ Finley (Wayne State University) for yeast two-hybrid vectors, Dr Yuhai Cui (Agriculture and Agri-Food Canada) for the BiFc vectors and Dr Sonia Gazzarrini (University of Toronto) for providing the yeast two-hybrid cDNA library. We would also like to thank Judy Booth for critical reading of the manuscript. This project was supported in part by a Natural Science and Engineering Research Council of Canada (NSERC) grant and post-graduate scholarship to S.L.S and W.J.L, respectively.

PY - 2013/7

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N2 - The RING-type E3 ligase, Keep on Going (KEG), is required for early seedling establishment in Arabidopsis thaliana. Post-germination, KEG negatively regulates abscisic acid (ABA) signalling by targeting Abscisic Acid Insensitive 5 (ABI5) for ubiquitination and subsequent degradation. Previous reports suggest that the role of KEG during early seedling development is not limited to regulation of ABI5 abundance. Using a yeast two-hybrid screen, this study identified Calcineurin B-like Interacting Protein Kinase (CIPK) 26 as a KEG-interacting protein. In vitro pull-down and in planta bimolecular fluorescence complementation assays confirmed the interactions between CIPK26 and KEG. In planta experiments demonstrated that CIPK26 was ubiquitinated and degraded via the 26S proteasome. It was also found that turnover of CIPK26 was increased when KEG protein levels were elevated, suggesting that the RING-type E3 ligase is involved in targeting CIPK26 for degradation. CIPK26 was found to interact with the ABA signalling components ABI1, ABI2, and ABI5. In addition, CIPK26 was capable of phosphorylating ABI5 in vitro. Consistent with a role in ABA signalling, overexpression of CIPK26 increased the sensitivity of germinating seeds to the inhibitory effects of ABA. The data presented in this report suggest that KEG mediates the proteasomal degradation of CIPK26 and that CIPK26 is part of the ABA signalling network.

AB - The RING-type E3 ligase, Keep on Going (KEG), is required for early seedling establishment in Arabidopsis thaliana. Post-germination, KEG negatively regulates abscisic acid (ABA) signalling by targeting Abscisic Acid Insensitive 5 (ABI5) for ubiquitination and subsequent degradation. Previous reports suggest that the role of KEG during early seedling development is not limited to regulation of ABI5 abundance. Using a yeast two-hybrid screen, this study identified Calcineurin B-like Interacting Protein Kinase (CIPK) 26 as a KEG-interacting protein. In vitro pull-down and in planta bimolecular fluorescence complementation assays confirmed the interactions between CIPK26 and KEG. In planta experiments demonstrated that CIPK26 was ubiquitinated and degraded via the 26S proteasome. It was also found that turnover of CIPK26 was increased when KEG protein levels were elevated, suggesting that the RING-type E3 ligase is involved in targeting CIPK26 for degradation. CIPK26 was found to interact with the ABA signalling components ABI1, ABI2, and ABI5. In addition, CIPK26 was capable of phosphorylating ABI5 in vitro. Consistent with a role in ABA signalling, overexpression of CIPK26 increased the sensitivity of germinating seeds to the inhibitory effects of ABA. The data presented in this report suggest that KEG mediates the proteasomal degradation of CIPK26 and that CIPK26 is part of the ABA signalling network.

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Arabidopsis CIPK26 interacts with KEG, components of the ABA signalling network and is degraded by the ubiquitin-proteasome system

Abstract

Bibliographical note

ASJC Scopus Subject Areas

PubMed: MeSH publication types

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