The pK7FWG2-R destination vector (Smit et al., 2005) was altered to remove theGFPfusion tag and replace the 35S promoter with a 1-kb nativeCCaMKpromoter usingSpeI-HindIII sites (Gleason et al., 2006), resulting in a construct with the native promoter driving the expression ofCCaMK, with noGFPfusion tag, and with constitutiveDsRedexpression THY1 as a herb transformation marker. domains compared with those ofCaMsuggest thatCCaMKis managed in the inactive state at basal calcium concentrations and is activated viaCaMbinding during calcium oscillations. This work provides a model for decoding calcium oscillations that uses differential calcium binding affinities to create a robust molecular switch that is responsive to calcium concentrations associated with both the basal state and with oscillations. == INTRODUCTION == Calcium ions (Ca2+) play an essential and ubiquitous Vatalanib (PTK787) 2HCl role as secondary messengers in many signaling pathways (Kudla et al., 2010). Decoding Ca2+signaling via phosphorylation has been attributed to the action of several plant-specific proteins, including the Ca2+-dependent protein kinases (CDPKs;Harper et al., 2004) and the combined action of the calcineurin B-like proteins (CBLs) andCBL-interacting protein kinases (Batistic and Kudla, 2004).CBLsbind Ca2+via EF-hand domains but rely on their interactingCBL-interacting protein kinases for any kinase domain name to phosphorylate their targets (Batistic and Kudla, 2004). By contrast,CDPKscan directly phosphorylate their targets in response to Ca2+binding since they possess EF-hands as well as a kinase domain name (Harper et al., 2004). In animal systems, the Ca2+decoding properties of calmodulin (CaM)dependent protein kinases, such as CaMK-II, have been strongly established (e.g., during neuronal impulses). These proteins possess aCaMbinding domain name, which allows the protein to respond to Ca2+viaCaM(De Koninck and Schulman, 1998). Plants also possessCaM-responsive protein kinases, including a Ca2+/CaM-dependent protein kinase Vatalanib (PTK787) 2HCl (CCaMK), which was first recognized in lily (Lilium longiflorum;Patil et al., 1995), is present in legumes (Lvy et al., 2004;Mitra et al., 2004) and rice (Oryza sativa;Godfroy et al., 2006;Banba et al., 2008;Gutjahr et al., 2008), and functions during symbiosis signaling in these species. Phylogenetic analyses have also identifiedCCaMKin mosses, liverworts, and hornworts but not in users of the Brassicaceae (Lvy et al., 2004;Wang et al., 2010), which lack mycorrhizal associations.CCaMKhas the exceptional ability among herb and animal proteins to bind Ca2+through two different mechanisms, either directly via EF-hand domains or indirectly through aCaMbinding domain (Patil et al., 1995). In legumes, symbiosis signaling is initiated by the belief of diffusible signals released from your symbiotic microorganisms: lipochitooligosaccharides called Nod and Myc factors released by rhizobial bacteria or mycorrhizal fungi, respectively (Lerouge et al., 1990;Maillet et al., 2011). Nod factor is perceived by the herb via LysM receptor-like kinases (Broghammer et al., 2012), leading to the activation of the common symbiosis signaling pathway, for which Ca2+oscillations are central (Ehrhardt et al., 1996;Oldroyd and Downie, 2006;Singh and Parniske, 2012). Many components of the symbiosis signaling pathway have been characterized in relation to the generation or belief of Ca2+oscillations (Wais et al., 2000;Walker et al., 2000;Miwa et al., 2006) such that the nuclear-localizedCCaMKhas been situated immediately downstream Vatalanib (PTK787) 2HCl of Ca2+(Wais et al., 2000;Lvy et al., 2004), along with an interacting partner, CYCLOPS (Messinese et al., 2007;Yano et al., 2008) and the transcription factors NSP1 and NSP2 (Kal et al., 2005;Smit et al., 2005;Heckmann et al., 2006). The domains present inCCaMKand its genetic position suggest a role for this protein in decoding symbiotic Ca2+oscillations. A number of gain-of-function mutations exist inCCaMK, and these give rise to spontaneous nodulation in the absence of rhizobia (Gleason et al., 2006;Tirichine et al., 2006). Spontaneous nodulation was observed by expressing just the kinase domain name alone or by the mutation of the Thr at position 271 (Thr-271), a major Ca2+-dependent autophosphorylation site Vatalanib (PTK787) 2HCl ofCCaMK(Gleason et al., 2006;Tirichine et al., 2006). The constitutive activity of Thr-271 mutants, or their comparative in other species (Gleason et al., 2006;Tirichine et al., 2006;Yano et al., 2008;Hayashi et al., 2010;Madsen et al., 2010;Takeda et al., 2012), reveals the importance of autophosphorylation for the regulation ofCCaMK. A model for the activation ofCCaMKhas been proposed based on work withCCaMKfrom lily (Sathyanarayanan et al., 2000): Ca2+binding to the EF-hands ofCCaMKpromotes autophosphorylation, which in turn promotesCaMbinding, relieving autoinhibition of the kinase and thus promoting substrate phosphorylation. This model implies a two-step activation ofCCaMKby Ca2+(Poovaiah et al., 2013).Shimoda et al. (2012)recently suggested that a hydrogen-bond network promoted by Thr-271 was important for the suppression ofCCaMKand that phosphorylation at Thr-271 would break the hydrogen-bond network and thus allow activation of the protein. However, these models fail to explain why both phosphoablative and phosphomimetic mutations at Thr-271 give rise to spontaneous nodulation in legumes (Gleason et al., 2006;Tirichine et al., 2006;Yano et al., 2008;Hayashi et al., 2010;Madsen et al., 2010;Takeda et al., 2012). Linking the molecular mechanism ofCCaMKto the actual.