Phosphorylation of Calcineurin B-like (CBL) Calcium Sensor Proteins by Their CBL-interacting Protein Kinases (CIPKs) Is Required for Full Activity of CBL-CIPK Complexes toward Their Target Proteins

Abstract

Calcineurin B-like proteins (CBLs) represent a family of calcium sensor proteins that interact with a group of serine/threonine kinases designated as CBL-interacting protein kinases (CIPKs). CBL-CIPK complexes are crucially involved in relaying plant responses to many environmental signals and in regulating ion fluxes. However, the biochemical characterization of CBL-CIPK complexes has so far been hampered by low activities of recombinant CIPKs. Here, we report on an efficient wheat germ extract-based in vitro transcription/translation protocol that yields active full-length wild-type CIPK proteins. We identified a conserved serine residue within the C terminus of CBLs as being phosphorylated by their interacting CIPKs. Remarkably, our studies revealed that CIPK-dependent CBL phosphorylation is strictly dependent on CBL-CIPK interaction via the CIPK NAF domain. The phosphorylation status of CBLs does not appear to influence the stability, localization, or CIPK interaction of these calcium sensor proteins in general. However, proper phosphorylation of CBL1 is absolutely required for the in vivo activation of the AKT1 K ؉ channel by CBL1-CIPK23 and CBL9-CIPK23 complexes in oocytes. Moreover, we show that by combining CBL1, CIPK23, and AKT1, we can faithfully reconstitute CBL-dependent enhancement of phosphorylation of target proteins by CIPKs in vitro. In addition, we report that phosphorylation of CBL1 by CIPK23 is also required for the CBL1-dependent enhancement of CIPK23 activity toward its substrate. Together, these data identify a novel general regulatory mechanism of CBL-CIPK complexes in that CBL phosphorylation at their flexible C terminus likely provokes conformational changes that enhance specificity and activity of CBL-CIPK complexes toward their target proteins.

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

“…Our results showed that although the phosphomimicking mutation T168D greatly enhanced the kinase activity of CIPK24/ SOS2ΔC, the corresponding T190D mutation produced little effect on CIPK23ΔC. These data correlate well with those obtained with full-length proteins expressed by using a wheat germ extract-based in vitro transcription/translation protocol (13). In these experiments, the catalytic efficiency (k cat /K m ) was roughly eightfold lower for CIPK23 than for CIPK24/SOS2.…”

“…The required phosphorylation of the C-terminal region of CBLs by CIPKs to achieve fully functional activation of the CBL-CIPKs complexes may be central to understand why the moderate NAF motif-mediated stimulation of the in vitro CIPK23 (Fig. 2) is absolutely required for in vivo activation of AKT1 K + channel (13). This data additionally supports the idea that CBL phosphorylation may affect the structure of the CBL-CIPKs complexes and consequently, their activity.…”

“…This data additionally supports the idea that CBL phosphorylation may affect the structure of the CBL-CIPKs complexes and consequently, their activity. Besides, an additional layer of CIPK regulation comes from the cofactor preference of Mn 2+ compared with Mg 2+ (13,27,28). The fact that one of the metal ion binding sites of CBL4/SOS3 displays higher affinity for Mn 2+ than for Ca 2+ suggests that CBLs could act as a carrier for this cofactor or, alternatively, could buffer the availability of free Mn 2+ to prevent a constitutive activation of the CIPKs (56).…”

“…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)(8)(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 understanding the molecular mechanisms underling CIPKs function provides opportunities to increase plant tolerance to abiotic stress and to improve plants for human benefit.…”

The Kinase CIPK23 Inhibits Ammonium Transport in Arabidopsis thaliana

“…CBL1 binding recruits CIPK23 to the plasma membrane and enables autophosphorylation of the kinase . Autophosphorylation and CBL1 binding lead to complete activity of the kinase (Hashimoto et al, 2012). The phosphorylation of AKT1 is essential for its activation at low external potassium concentrations.…”