FIGURE 4.

The target site of CIPK-mediated phosphorylation is conserved in Arabidopsis CBL proteins. A, LC-multistage activation spectrum showing phosphorylation of CBL10 Ser²³⁷. Fragmentation of the precursor (m/z 1006.9353, doubly charged) was performed by collision-induced dissociation (CID). The major b-type and y-type ions as well as ions corresponding to the neutral loss of phosphoric acid (-P) are indicated in the spectrum and in the corresponding amino acid sequence depicted above the spectrum. The phosphorylation site is marked by an asterisk. The +80-Da mass shift observed for b8 to b15 and y10 to y15 in addition the occurrence of corresponding neutral loss ions revealed the phosphorylation of Ser²³⁷. B, ClustalW alignment of the Arabidopsis CBL C-terminal region. The conserved phosphorylation site is indicated by an arrow. Semi-conserved, conserved, and fully conserved amino acid residues are indicated by dots, double dots, and asterisks below the alignment, respectively. C, compared with CBL wild-type (WT) proteins, mutation of the corresponding phosphorylation site in CBL1 (Ser²⁰¹), CBL4 (Ser²⁰⁵), and CBL10 (Ser²³⁷) to Ala prevented phosphorylation by CIPK24 in in vitro kinase assays. D, CIPK24 did not phosphorylate CBL6, which lacks the conserved Ser/Thr residue in the C terminus. Autoradiograph of in vitro phosphorylation assays in the absence (−) or presence (+) of CIPK24 with 50 ng of CBL4 (left) and CBL6 proteins (right). CBL4 as the positive control was phosphorylated by CIPK24.