Arabidopsis calcineurin B-like proteins differentially regulate phosphorylation activity of CBL-interacting protein...

Calcium (Ca²⁺) is a versatile and ubiquitous second messenger in all eukaryotes including plants. In response to various stimuli, cytosolic calcium concentration ([Ca²⁺]_cyt) is increased, leading to activation of Ca²⁺ sensors including Arabidopsis calcineurin B-like proteins (CBLs). CBLs interact with CBL-interacting protein kinases (CIPKs) to form CBL-CIPK complexes and transduce the signal downstream in the signalling pathway. Although there are many reports on the regulation of downstream targets by CBL-CIPK module, knowledge about the regulation of upstream components by individual CIPKs is inadequate. In the present study, we have carried out a detailed biochemical characterization of CIPK9, a known regulator of K⁺ deficiency in Arabidopsis, with its interacting CBLs. The present study suggests that CIPK9 specifically interacts with four CBLs, i.e. CBL1, CBL2, CBL3 and CBL9, in yeast two-hybrid assays. Out of these four CBLs, CBL2 and CBL3, specifically enhance the kinase activity of CIPK9, while the CBL1 and CBL9 decrease it as examined by in vitro kinase assays. In contrast, truncated CIPK9 (CIPK9ΔR), without the CBL-interacting regulatory C-terminal region, is not differentially activated by interacting CBLs. The protein phosphorylation assay revealed that CBL2 and CBL3 serve as preferred substrates of CIPK9. CBL2- and CBL3-CIPK9 complexes show altered requirement for metal cofactors when compared with CIPK9 alone. Moreover, the autophosphorylation of constitutively active CIPK9 (CIPK9T178D) and less active CIPK9 (CIPK9T178A) in the presence of CBL2 and CBL3 was further enhanced. Our study suggests that CIPK9 differentially phosphorylates interacting CBLs, and furthermore, the kinase activity of CIPK9 is also differentially regulated by specific interacting CBLs.