Abstract
The calcium/calmodulin-dependent protein kinase (CCaMK) is regulated by free Ca2+ and Ca2+-loaded calmodulin. This dual binding is believed to be involved in its regulation and associated physiological functions, although direct experimental evidence for this is lacking. Here we document that site-directed mutations in the calmodulin-binding domain of CCaMK alters its binding capacity to calmodulin, providing an effective approach to study how calmodulin regulates CCaMK in terms of kinase activity and regulation of rhizobial symbiosis in Medicago truncatula. We observed that mutating the tryptophan at position 342 to phenylalanine (W342F) markedly increased the calmodulin-binding capability of the mutant. The mutant CCaMK underwent autophosphorylation and catalyzed substrate phosphorylation in the absence of calcium and calmodulin. When the mutant W342F was expressed in ccamk-1 roots, the transgenic roots exhibited an altered nodulation phenotype. These results indicate that altering the calmodulin-binding domain of CCaMK could generate a constitutively activated kinase with a negative role in the physiological function of CCaMK.
Highlights
Calcium/calmodulin (Ca2+/CaM)-mediated signaling plays important roles in sensing and transducing environmental stimuli and developmental cues (Du and Poovaiah, 2005; Poovaiah et al, 2013), plant immune responses (Du et al, 2009) and mutualistic plant–microbe interactions (Gleason et al, 2006; Yuan et al, 2017)
CCaMK is nuclear localized and lies downstream of the cellular calcium spiking response that occurs in the symbiosis pathway
After activation by Ca2+/CaM, CCaMK phosphorylates CYCLOPS or IPD3 in Medicago truncatula, which are required for symbiosis (Yano et al, 2008)
Summary
Calcium/calmodulin (Ca2+/CaM)-mediated signaling plays important roles in sensing and transducing environmental stimuli and developmental cues (Du and Poovaiah, 2005; Poovaiah et al, 2013), plant immune responses (Du et al, 2009) and mutualistic plant–microbe interactions (Gleason et al, 2006; Yuan et al, 2017). One important effector protein of this Ca2+/CaMmediated signaling in plants is the Ca2+/CaM-dependent protein kinase, CCaMK (Patil et al, 1995). CCaMK is nuclear localized and lies downstream of the cellular calcium spiking response that occurs in the symbiosis pathway. CCaMK has been well studied because of its unique protein structure, and due to its critical role in the symbiotic interaction between leguminous plants and bacteria and/or fungi (Patil et al, 1995; Ramachandiran et al, 1997; Harper et al, 2004).
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