Abstract
Plant growth largely depends on the maintenance of adequate intracellular levels of potassium (K+). The families of 10 Calcineurin B-Like (CBL) calcium sensors and 26 CBL-Interacting Protein Kinases (CIPKs) of Arabidopsis (Arabidopsis thaliana) decode the calcium signals elicited by environmental inputs to regulate different ion channels and transporters involved in the control of K+ fluxes by phosphorylation-dependent and -independent events. However, the detailed molecular mechanisms governing target specificity require investigation. Here, we show that the physical interaction between CIPK23 and the noncanonical ankyrin domain in the cytosolic side of the inward-rectifier K+ channel AKT1 regulates kinase docking and channel activation. Point mutations on this domain specifically alter binding to CIPK23, enhancing or impairing the ability of CIPK23 to regulate channel activity. Our data demonstrate the relevance of this protein-protein interaction that contributes to the formation of a complex between CIPK23/CBL1 and AKT1 in the membrane for the proper regulation of K+ transport.
Highlights
Potassium (K+) serves important roles in plants for the control of cellular pH, regulation of membrane electric potentials, cell turgor, and as a co-factor in essential metabolic processes including protein synthesis (Leigh and Wyn Jones, 1984; Rodriguez-Navarro, 2000; Pardo, 2010; Cherel and Gaillard, 2019; Ragel et al, 2019)
Our results show that the ankyrin repeat (ANK) domain of AKT1 functions as a platform for docking of the catalytic domain of CIPK23, and that this interaction contributes to the activation of the channel by promoting the formation of a productive CBL-Interacting Protein Kinases (CIPKs)-AKT1 complex
We tested the functionality of the mutated forms of the channel leading to AKT1(-) and AKT1(+). Both mutated channels behaved as the wild-type channel, indicating that the mutated proteins were expressed and that the change in the polarity of the ANK domain did not affect the transport mechanism of the channel (Fig. 4). These results show that the ANK domain is not essential for AKT1 function or stability and suggests that it would be mostly involved in the recruitment of regulatory proteins, e.g. CIPK23, to the vicinity of the channel
Summary
Potassium (K+) serves important roles in plants for the control of cellular pH, regulation of membrane electric potentials, cell turgor, and as a co-factor in essential metabolic processes including protein synthesis (Leigh and Wyn Jones, 1984; Rodriguez-Navarro, 2000; Pardo, 2010; Cherel and Gaillard, 2019; Ragel et al, 2019). Several ion transporters mediating K+ fluxes in plants have been characterized, and the knowledge of their activity in response to environmental stresses and the molecular mechanisms underlying their regulation has been central for understanding the adaptation to K+. Starvation (Cherel et al, 2014; Ragel et al, 2015; Lefoulon et al, 2016; Behera et al., 2017), high salinity (Aleman et al, 2014), drought (Maierhofer et al, 2014), and to pathogen attack or herbivore-mediated wounding (Forster et al, 2019). Functional GORK1 and AKT1 are tetramers whose subunits display an N-terminal transmembrane domain that is homologous to the animal VG Shaker channels, and a large cytoplasmic domain that consists of a cyclic nucleotide binding homologus domain (CNBHD), followed by an ankyrin repeat (ANK) domain and a characteristic family-conserved (KHA) motif at the C-terminal end (Daram et al, 1997; Pilot et al., 2003; Jegla et al, 2018). Depending on the K+ status, the protein phosphatases AIP1 and ABI2 would revert this process and dephosphorylate AKT1 and GORK1, respectively (Lee et al., 2007; Lan et al, 2011; Lefoulon et al, 2016)
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