Do calcineurin B-like proteins interact independently of the serine threonine kinase CIPK23 with the K+ channel AKT1? Lessons learned from a ménage à trois.

Calcineurin B-like proteins (CBLs) function as membrane-anchored Ca sensors that, when activated, recruit a specific set of Ser-Thr kinases, calcineurin B-like protein-interacting protein kinases (CIPKs), to their sites of action (for review, see Luan et al., 2009; Kudla et al., 2010). The CBL-CIPK network, and its roles in regulating ion transport and cellular homeostasis, have drawn considerable attention over the past decade. Arabidopsis (Arabidopsis thaliana) encodes 10 CBL and 26 CIPK proteins, many of which have been reported to show distinct and selective interactions among these complementary partners. This selectivity would allow for a complex interplay of different CBLCIPK combinations that, in turn, could encode different stimuli through spatiotemporal regulation of downstream signaling cascades. Several CBL-CIPK complexes have been identified that connect Ca sensing with different physiological responses through a range of target proteins. Of these, the best known example is the salt overly-sensitive (SOS) pathway, which comprises the interaction of CBL4 (SOS3) with CIPK24 (SOS2). CBL4-CIPK24 binding recruits the kinase to the plasma membrane, where it activates the SOS1 H/Na antiporter to drive Na export and reduce toxic sodium levels from the cytosol (Zhu et al., 1998). CBL-CIPK pairing plays a complementary role in K nutrition through the activation of the K channel AKT1, which mediates in K uptake by the roots: a forward-genetic screen for mutants sensitive to low potassium levels showed that loss of CIPK23 function impaired growth under K-limiting conditions (Xu et al., 2006). In this case, direct interaction of the kinase with CBL1 or CBL9 recruited CIPK23 to the plasma membrane, where it phosphorylated AKT1 (Xu et al., 2006; Cheong et al., 2007). To date, studies of CBLand CIPK-dependent signaling have focused primarily on the interaction of the kinase with its target protein and on CIPK pairing with its cognate CBL protein(s). There is little known of the roles (if any) for the CBL proteins beyond their recruitment of the soluble CIPK proteins to one or another membrane surface. We ascribe this gap in knowledge first and foremost to difficulties associated with the yeast two-hybrid (Y2H) approach on which evidence of interaction is primarily based, for example, in the use of the C-terminal cytosolic domain of the channel in analysis of the AKT1-CIPK-CBL network (Li et al., 2006; Xu et al., 2006; Lee et al., 2007). Here, we draw attention to the consequences and often neglected limitations of the Y2H method in work with membrane proteins (for review, see Van Criekinge and Beyaert, 1999; Coates and Hall, 2003). Most important, Y2H methods necessitate nuclear localization of the interacting partners in order to activate reporter gene expression. Hence, membrane proteins need to be truncated to include only soluble domains that are small enough to pass through the nuclear pore. As a result, Y2H assays often are carried out after first eliminating large segments of the protein(s) of interest and, potentially, important interaction sites. Other methodical difficulties have frequently included the omission of data verifying protein expression, streaking of yeast rather than using exact dilution series, and the inherent flaw of most Y2H vector sets: the inability to control expression levels that could increase stringency and signal-to-noise ratios. The mating-based split-ubiquitin system (SUS) in yeast offers a number of substantial advantages over the Y2H approach (Johnsson and Varshavsky, 1994; Stagljar et al., 1998; Grefen et al., 2009; Dünkler et al., 2012), and we commend it as the method of choice for work with integral membrane proteins and proteins that are membrane anchored. The SUS method enables the use of full-length membrane proteins, thus overcoming the most significant limitations of Y2H. SUS assays make use of the ubiquitin protein, split between two halves, with each half fused to one of the proteins of interest. The bait protein incorporates the C-terminal half of ubiquitin fused with a transcription factor, and the prey protein is fused to the N-terminal half of the ubiquitin, which is mutated (NubI to NubG) to prevent spontaneous association. Interaction of the bait and prey leads to reassembly of the ubiquitin moiety, its cleavage by ubiquitin-specific proteases, and release of the transcription factor, which then diffuses to the nucleus, where it activates reporter genes for auxotrophy selection and quantitative enzymatic assays (see Fig. 2C below). The bait protein construct is driven * Corresponding author; e-mail [email protected]. www.plantphysiol.org/cgi/doi/10.1104/pp.112.198051