CBL-mediated targeting of CIPKs facilitates the decoding of calcium signals emanating from distinct cellular stores

Abstract

SUMMARYDuring adaptation and developmental processes cells respond through nonlinear calcium-decoding signaling cascades, the principal components of which have been identified. However, the molecular mechanisms generating specificity of cellular responses remain poorly understood. Calcineurin B-like (CBL) proteins contribute to decoding calcium signals by specifically interacting with a group of CBL-interacting protein kinases (CIPKs). Here, we report the subcellular localization of all 10 CBL proteins from Arabidopsis and provide a cellular localization matrix of a plant calcium signaling network. Our findings suggest that individual CBL proteins decode calcium signals not only at the plasma membrane and the tonoplast, but also in the cytoplasm and nucleus. We found that distinct targeting signals located in the N-terminal domain of CBL proteins determine the spatially discrete localization of CBL/CIPK complexes by COPII-independent targeting pathways. Our findings establish the CBL/CIPK signaling network as a calcium decoding system that enables the simultaneous specific information processing of calcium signals emanating from different intra-and extracellular stores, and thereby provides a mechanism underlying the specificity of cellular responses.

Tonoplast calcium sensors CBL2 and CBL3 control plant growth and ion homeostasis through regulating V-ATPase activity in Arabidopsis

“…Not only the expression pattern, but the subcellular localization of CBL-type calcium sensors is also believed to reflect spatial specification of Ca 2+ signaling [8,28]. In this study, we confirmed that CBL2 and CBL3 are specifically associated with the tonoplast in Arabidopsis cells, consistent with an earlier study using N. benthamiana [28].…”

“…In this study, we confirmed that CBL2 and CBL3 are specifically associated with the tonoplast in Arabidopsis cells, consistent with an earlier study using N. benthamiana [28]. We also further demonstrated that the N-terminal 19-aa of the CBL2 protein defines a tonoplast targeting sequence (TTS) that is necessary and sufficient for subcellular localization to the vacuolar membrane.…”

“…However, preliminary yeast two-hybrid assays failed to identify strong interactions between CBL2/3 and any of the known subunits (Supplementary information, Figure S11). Thus, a more inviting scenario is that the calcium sensors CBL2 and CBL3, like other CBLs, activate downstream CIPKs and recruit these kinases to the vacuole membrane [8,28], followed by phosphorylation of particular V-ATPase subunits and activation of the enzyme complex. Through yeast two-hybrid assays, we found that CBL2 and CBL3 shared several interacting CIPKs (Supplementary information, Figure S12), but none of these cipk single mutants showed similar phenotypes as the cbl2 cbl3 double mutant according to our preliminary experiments.…”

“…While CBL1 and CBL9 harbor motifs for N-myristoylation and S-acylation that anchor them to the plasma membrane and target their function towards regulation of transporters [21][22][23]30], several other CBL members, including CBL2, CBL3, CBL6 and CBL10, contain variable N-terminal extensions that target the proteins specifically to the vacuolar membranes [20,28]. As discussed above, substantial progress has been made in characterizing plasma membrane-targeted CBL proteins such as CBL1, CBL4 and CBL9.…”

“…Distinct subcellular localizations of CBL proteins facilitate the localization of their targets including CIPKs, and thereby determine the functional specificity of each CBL [28,29]. While CBL1 and CBL9 harbor motifs for N-myristoylation and S-acylation that anchor them to the plasma membrane and target their function towards regulation of transporters [21][22][23]30], several other CBL members, including CBL2, CBL3, CBL6 and CBL10, contain variable N-terminal extensions that target the proteins specifically to the vacuolar membranes [20,28].…”