Structural basis of the regulatory mechanism of the plant CIPK family of protein kinases controlling ion homeostasis and abiotic stress

Abstract

Plant cells have developed specific protective molecular machinery against environmental stresses. The family of CBL-interacting protein kinases (CIPK) and their interacting activators, the calcium sensors calcineurin B-like (CBLs), work together to decode calcium signals elicited by stress situations. The molecular basis of biological activation of CIPKs relies on the calcium-dependent interaction of a self-inhibitory NAF motif with a particular CBL, the phosphorylation of the activation loop by upstream kinases, and the subsequent phosphorylation of the CBL by the CIPK. We present the crystal structures of the NAF-truncated and pseudophosphorylated kinase domains of CIPK23 and CIPK24/SOS2. In addition, we provide biochemical data showing that although CIPK23 is intrinsically inactive and requires an external stimulation, CIPK24/SOS2 displays basal activity. This data correlates well with the observed conformation of the respective activation loops: Although the loop of CIPK23 is folded into a well-ordered structure that blocks the active site access to substrates, the loop of CIPK24/SOS2 protrudes out of the active site and allows catalysis. These structures together with biochemical and biophysical data show that CIPK kinase activity necessarily requires the coordinated releases of the activation loop from the active site and of the NAF motif from the nucleotide-binding site. Taken all together, we postulate the basis for a conserved calciumdependent NAF-mediated regulation of CIPKs and a variable regulation by upstream kinases.signaling | ion transport | abiotic stress C ell perception of extracellular stimuli is followed by a transient variation in cytosolic calcium concentration. Plants have evolved to produce the specific molecular machinery to interpret this primary information and to transmit this signal to the components that organize the cell response (1-4). The plant family of serine/threonine protein kinases PKS or CIPKs (hereinafter CIPKs) and their activators, the calcium-binding proteins SCaBPs or CBLs (hereinafter CBLs) (5, 6) function together in decoding calcium signals caused by different environmental stimuli. Available data suggest a mechanism in which calcium mediates the formation of stable CIPK-CBL complexes that regulate the phosphorylation state and activity of various ion transporters involved in the maintenance of cell ion homeostasis and abiotic stress responses in plants. Among them, the Arabidopsis thaliana CIPK24/SOS2-CBL4/SOS3 complex activates the Na + /H + antiporter SOS1 to maintain intracellular levels of the toxic Na + low under salt stress (7-9), the CIPK11-CBL2 pair regulates the plasma membrane H + -ATPase AHA2 to control the transmembrane pH gradient (10), the CIPK23-CBL1/9 (11, 12) regulates the activity of the K + transporter AKT1 to increase the plant K + uptake capability under limiting K + supply conditions (12, 13), and CIPK23-CBL1 mediates nitrate sensing and uptake by phosphorylation of the nitrate transporter CHL1 (14). Together these findings show that underst...

The Kinase Activity of Calcineurin B-like Interacting Protein Kinase 26 (CIPK26) Influences Its Own Stability and that of the ABA-regulated Ubiquitin Ligase, Keep on Going (KEG)

“…Alternatively, downregulation of CIPK26 may be achieved through selective dephosphorylation. In addition, activation of CIPK kinases incorporates binding of Ca 2+ sensing CBL proteins to the self-inhibitory NAF motif, activation loop phosphorylation by upstream kinases and phosphorylation of the CBL by the CIPK (Chaves-Sanjuan et al, 2014; Sanyal et al, 2015). Developmental phases or abiotic stress may promote CIPK26 activation, self-phosphorylation and subsequent stabilization.…”

Tonoplast CBL–CIPK calcium signaling network regulates magnesium homeostasis in Arabidopsis

“…Interaction between CBLs and CIPKs is mediated by the regulatory C-terminal region of CIPKs and is required for full activation of the kinase activity (9)(10)(11). Although CIPKs appear to be soluble in the cytosol, CBL proteins are largely associated with the cellular membranes through their N-terminal motifs that are subject to lipid modifications (12).…”

Identification and characterization of CBL and CIPK gene families in eggplant (Solanum melongena L.)

“…CBL proteins bind calcium through four EF-hand domains (Sanchez-Barrena et al 2005) and interact specifically with a group of CIPKs to relay the perceived calcium signal Kudla et al 1999;Shi et al 1999). Commonly, CIPK proteins comprise a unique C-terminal regulatory domain with a conserved NAF or FISL motif (Albrecht et al 2001;Guo et al 2001), and a conserved N-terminal kinase domain with a putative activation loop between the DEF and APE residues Chaves-Sanjuan et al 2014). The highly conserved NAF/FISL motif in the C-terminal domain of CIPKs is the pivotal point in regulating the CBL-CIPK interaction (Albrecht et al 2001).…”