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.

Two Distinct Families of Protein Kinases Are Required for Plant Growth under High External Mg2+ Concentrations in Arabidopsis

“…These observations suggested that CIPK26/3/9/23 is unlikely to play a key role in ABA signaling during the vegetative growth stage and unlikely to be involved in modulating subclass III SnRK2s activity under water deficit stress conditions. Conversely, CIPK26 overexpressors showed a slightly ABA-hypersensitive phenotype during the germination stage, and it was suggested that CIPK26 could function as a positive regulator of plant responses to ABA (Lyzenga et al, 2013). The discrepancy between our results and the previous report could, at least partially, be explained by a difference in growth stages.…”

“…S2B), where it physically interacts with AREB/ABFs Yoshida et al, 2014a), it is likely that SRK2D regulates AREB/ABFs in the nucleus to induce osmotic stress-responsive gene expression and other ABA-related responses independent of CIPK26. Conversely, a previous study reported that CIPK26 localizes to both the cytoplasm and the nucleus and physically interacts with ABI5 mainly in the nucleus (Lyzenga et al, 2013). Another report showed that CIPK26 predominantly localizes in the cytoplasm, partially localizes in the nucleus, and physically interacts with plasma membranelocalized RBOHF (Drerup et al, 2013).…”

“…In this study, we revealed a novel role of subclass III SnRK2s in plant growth under high external Mg 2+ concentrations. Previous studies on CIPK26/3/9/23 have characterized the diverse and distinct functions of each of these CIPK genes (Kim et al, 2003;Li et al, 2006;Xu et al, 2006;Cheong et al, 2007;Pandey et al, 2007;Drerup et al, 2013;Kimura et al, 2013;Lyzenga et al, 2013). Other than the individual functions of CIPK26, CIPK3, CIPK9, and CIPK23, it seems that these CIPK genes also have some overlapping functions in planta, because CIPK26/3/9/23 formed a monophyletic group in the phylogenetic analysis (Supplemental Fig.…”

The RING E3 Ligase KEEP ON GOING Modulates JASMONATE ZIM-DOMAIN12 Stability

“…We treated the cells with 50 mM JA for 1 min. Although we could not find peptides for JAZ12 or ABI5/ABFs, we did find peptides of the known KEG target protein CIPK26, a calcineurin B-like interacting protein kinase (Lyzenga et al, 2013).…”

“…KEG is known to play a role in ABA signaling and interacts with the bZIP transcription factors ABI5, ABF1, and ABF3 through the ankyrinrepeat domain and with the kinase CIPK26 (Stone et al, 2006;Chen et al, 2013;Lyzenga et al, 2013). KEG promotes the proteasome-mediated degradation of these proteins in order to repress ABA signaling.…”