Abstract
The Really Interesting New Gene (RING)-type E3 ligase, Keep on Going (KEG) plays a critical role in Arabidopsis growth after germination and the connections between KEG and hormone signaling pathways are expanding. With regards to abscisic acid (ABA) signaling, KEG targets ABA-responsive transcription factors abscisic acid insensitive 5, ABF1 and ABF3 for ubiquitination and subsequent degradation through the 26S proteasome. Regulation of E3 ligases through self-ubiquitination is common to RING-type E3 ligases and ABA promotes KEG self-ubiquitination and degradation. ABA-mediated degradation of KEG is phosphorylation-dependent; however, upstream signaling proteins that may regulate KEG stability have not been characterized. In this report, we show that CBL-Interacting Protein Kinase (CIPK) 26 can phosphorylate KEG in vitro. Using both in vitro and in planta degradation assays we provide evidence which suggests that the kinase activity of CIPK26 promotes the degradation of KEG. Furthermore, we found that the kinase activity of CIPK26 also influences its own stability; a constitutively active version is more stable than a wild type or a kinase dead version. Our results suggest a reciprocal regulation model wherein an activated and stable CIPK26 phosphorylates KEG to promote degradation of the E3.
Highlights
The ubiquitin proteasome system (UPS) is responsible for the degradation of numerous proteins and regulates a wide range of cellular events
In an attempt to identify the kinase involved in Keep on Going (KEG) phosphorylation, we examined the ability of the CIPK26, a known interactor (Lyzenga et al, 2013; Pauwels et al, 2015), to directly phosphorylate the E3
We found that His-Flag-CIPK26N displayed self-phosphorylation and was able to phosphorylate the HisFlag-KEGRK (Figure 1A)
Summary
The ubiquitin proteasome system (UPS) is responsible for the degradation of numerous proteins and regulates a wide range of cellular events. Ubiquitin-dependent proteolysis plays an indispensable role in the regulation of plant hormone production, perception, signal transduction and output (Santner and Estelle, 2010; Liu and Stone, 2011; Lyzenga and Stone, 2012). Central to the ubiquitination pathway are the ubiquitin ligases (E3) that govern target selection. They facilitate transfer of ubiquitin molecules onto the selected protein prior to degradation by the 26S proteasome. Keep on Going (KEG) is a single subunit Really Interesting New Gene (RING)-type E3 that plays an essential role during early plant development.
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