The combinatorial effect of several mechanistic and structural features inherent to the CBL–CIPK signaling system contributes to its ability to allow specific information processing in signal–response coupling. This versatility of the CBL–CIPK signaling system is achieved through its flexible network-like character in combination with an array of ‘specificity factors’ mutually affecting one another. Such specificity factors include, for example, specific gene expression, differential Ca2+ binding and differential cellular localization of CBL–CIPK complexes (Batistič & Kudla, 2004, 2009). Moreover, preferential complex formation between certain CBLs and CIPKs enables focused signal transmission of signals from the calcium sensor proteins to the kinases (Albrecht et al., 2001).
Like many other Ca2+-binding proteins, CBLs form two globular structures, which are separated by a short linker domain (Nagae et al., 2003). Although each CBL protein harbors four EF-hand motifs, principally enabling these proteins to bind to at most four Ca2+ ions, no CBL protein contains four canonical EF-hand motifs. Five CBL proteins from A. thaliana (CBL2, CBL3, CBL4, CBL5 and CBL8) possess no canonical EF hand, while CBL6, CBL7 and CBL10 have one and CBL1 and CBL9 contain two EF hands with a canonical sequence motif (Batističet al., 2004). In all other EF-hand motifs, sequence variations have been observed that occur in different combinations distributed over the four EF hands. Therefore, this variation in EF-hand composition probably results in differential Ca2+-binding efficiencies of individual CBL proteins. This likely allows differential decoding of several distinct Ca2+ signals occurring in parallel in the cell. Moreover, these biochemical differences may also affect the Ca2+ dependence of the CBL–CIPK interaction. However, to what extent Ca2+ binding to CBLs alters the CIPK interaction, or to what extent the CIPK interaction influences the Ca2+-binding affinity of CBLs, needs to be further investigated in detailed side-by-side studies. Nevertheless, in vitro experiments investigating the interaction of CBL1 and CIPK1 revealed a dependence of complex formation on the presence of micromolar concentrations of Ca2+ (Shi et al., 1999). By contrast, the interaction of CBL4/SOS3 with CIPK24/SOS2 occurs independently of Ca2+ but influences the Ca2+-binding ability of CBL4 (Halfter et al., 2000; Sánchez-Barrena et al., 2007). The requirement for further experimental verification is also underlined by the development of our understanding regarding the function of the first EF hand. Because of a 2 amino acid extension in the binding loop of the EF-hand motif, this EF hand in all CBLs was predicted not to be able to bind Ca2+ (Luan et al., 2002). However, crystal structure analyses of CBL2 and CBL4 have shown that, despite the additional amino acids, EF1 is able to bind Ca2+ (Nagae et al., 2003; Sánchez- Barrena et al., 2005, 2007). Moreover, in the non-CIPK-interacting state, CBL2 binds two Ca2+ ions via EF1 and EF4, whereas when bound to CIPK14 all four EF hands are occupied by Ca2+ ions (Nagae et al., 2003; Akaboshi et al., 2008). By contrast, CBL4 already possesses four Ca2+ ions when not interacting with a CIPK and binds only two Ca2+ ions upon interaction with CIPK24 (Sánchez-Barrena et al., 2005, 2007). These results not only demonstrate that functional predictions based on sequence analyses need careful experimental verification but also reveal that the CBL–CIPK interaction may also influence the Ca2+-binding capacity of CBLs.