Update on Calcium-Dependent Protein Kinases Calcium-Dependent Protein Kinases: Hubs in Plant Stress Signaling and Development

Calcium ions (Ca) have long been recognized as a major, conserved second messenger in eukaryotic signal transduction. In plants, the multigene family of calciumdependent protein kinases (CDPKs) encodes structurally conserved, unimolecular calcium sensor/protein kinase effector proteins. CDPKs are ideal candidates for perceiving intracellular changes in Ca concentration and translating them into specific phosphorylation events to initiate further downstream signaling processes. Accordingly, CDPKs have been predominantly characterized in rapid abiotic stress and immune signaling responses, with little focus upon CDPK function in long-term adaptive processes or plant development. The long-awaited identification of in vivo/in planta CDPK targets, including their respective phosphorylation sites, allows the elucidation of CDPK function on a molecular and biochemical level. Moreover, the resolution of kinase-specific phosphorylation patterns within a target protein provides mechanistic evidence suggesting that CDPKs act as signaling hubs in plant stress signaling and development. The CDPK gene family comprises Ser/Thr protein kinases organized in four subgroups (Harmon et al., 2000; Cheng et al., 2002); families of varying size have been characterized in Arabidopsis (Arabidopsis thaliana; 34), rice (Oryza sativa; 29), wheat (Triticum aestivum; 20), maize (Zea mays; 35), and poplar (Populus trichocarpa; 20; Harmon et al., 2000; Asano et al., 2005; Li et al., 2008; Ma et al., 2013; Zuo et al., 2013). CDPKs have a conserved molecular structure, consisting of a variable N-terminal domain, fused to a Ser/Thr kinase domain, and a CDPK activation domain (CAD). The CAD is formed from a pseudosubstrate region (sometimes referred to as the “inhibitory junction domain”) and a Ca-binding domain, highly homologous to calmodulin (also described as a calmodulin-like domain; Fig. 1A). The calcium-binding domain of a canonical CDPK contains up to four elongation factor (EF) hand motifs. These are organized in N-terminal and C-terminal EF lobes and may display different Ca-binding affinities (Harper et al., 2004; Harper and Harmon, 2005; Boudsocq and Sheen, 2013; Liese and Romeis, 2013). Based on this modular structure, a model for CDPK activation was proposed in which Ca binding to the CAD induces conformational changes, leading to the release of the pseudosubstrate segment from the active site (Fig. 1B). This results in kinase activity, enabling CDPK autophosphorylation as well as the phosphorylation of target substrates (Liese and Romeis, 2013). With the recent availability of crystal structures from apicomplexan CDPKs, encompassing the kinase and CAD, this model has been refined (Wernimont et al., 2010, 2011). In the absence of Ca, the autoinhibitory region and the N-terminal lobe of the Ca-binding domain form two extended a-helices that block the active site, and likely bury the N-terminal part, of the kinase. In addition, one may speculate that the N-terminal variable domain (which is absent in the crystal structure of apicomplexan CDPKs) may be hidden, so that it has no access to the CDPK target. Upon Ca binding, the structure of the helices is broken down, inducing a rotation of the entire regulatory domain around the kinase domain, probably releasing the N-terminal variable domain. It has been proposed that both the apoand calcium-bound forms of the enzyme are stabilized by distinct contact sites between the kinase and regulatory domains (Wernimont et al., 2010). It is tempting to speculate that, in particular, some of the amino acids involved in forming these contact sites may be subjected to posttranslational modifications during the activation process (Liese and Romeis, 2013), modulating the stability of the active or inactive form. CDPKs have been shown to function in many different aspects of plant biology, from environmental stress signaling upon the perception of abiotic and biotic stress stimuli to hormone-regulated processes in development (Cheng et al., 2002; Ludwig et al., 2004; Asano et al., 2012; Boudsocq and Sheen, 2013). While previously, CDPK characterization often relied on the biochemical activation proteins or the transcriptional induction of the respective genes in a given signaling response, recent studies draw a more comprehensive picture of CDPK function, including in vivo/in planta data concerning kinase/substrate interactions. Despite this significant progress, elucidating the impact of biochemical regulation on the in vivo function of a 1 This work was supported by the Deutsche Forschungsgemeinschaft within the Priority Programme SPP1212, research unit FOR964, and the CROPTIMISE consortium (grant no. 0315959A) within the Plant 2030 initiative from the German Federal Ministry of Education and Research. * Address correspondence to romeis@zedat.fu-berlin.de. www.plantphysiol. org/cgi/doi/10.1104/pp.113. 222539