Abstract
Calcium-dependent protein kinases (CDPKs) are Ca(2+) sensors that regulate diverse biological processes in plants and apicomplexans. However, how CDPKs discriminate specific substrates in vivo is still largely unknown. Previously, we found that a potato StCDPK5 is dominantly localized to the plasma membrane and activates the plasma membrane NADPH oxidase (RBOH; for respiratory burst oxidase homolog) StRBOHB by direct phosphorylation of the N-terminal region. Here, we report the contribution of the StCDPK5 N-terminal variable (V) domain to activation of StRBOHB in vivo using heterologous expression system in Nicotiana benthamiana. Mutations of N-terminal myristoylation and palmitoylation sites in the V domain eliminated the predominantly plasma membrane localization and the capacity of StCDPK5 to activate StRBOHB in vivo. A tomato SlCDPK2, which also contains myristoylation and palmitoylation sites in its N terminus, phosphorylated StRBOHB in vitro but not in vivo. Functional domains responsible for activation and phosphorylation of StRBOHB were identified by swapping regions for each domain between StCDPK5 and SlCDPK2. The substitution of the V domain of StCDPK5 with that of SlCDPK2 abolished the activation and phosphorylation abilities of StRBOHB in vivo and relocalized the chimeric CDPK to the trans-Golgi network, as observed for SlCDPK2. Conversely, SlCDPK2 substituted with the V domain of StCDPK5 localized to the plasma membrane and activated StRBOHB. These results suggest that the V domains confer substrate specificity in vivo by dictating proper subcellular localization of CDPKs.
Highlights
Substrate specificity of Calcium-dependent protein kinases (CDPKs) involved in diverse physiological processes is largely unknown
Myristoylation and Palmitoylation in StCDPK5 Are Required for the Plasma Membrane Localization and Phosphorylation of StRBOHB in Vivo—CDPKs consist of a V domain comprising highly variable amino acid sequences, a K domain that phosphorylates a substrate, a J domain that acts as an autoinhibitor in a pseudo-substrate fashion, and a C domain, including Ca2ϩsensing EF-hand motifs (Fig. 1A)
We reported that the N-terminal V domain of StCDPK5, including myristoylation and palmitoylation sites, confers its proper subcellular localization, resulting in interaction with and phosphorylation of StRBOHB in vivo
Summary
Substrate specificity of CDPKs involved in diverse physiological processes is largely unknown. SlCDPK2 substituted with the V domain of StCDPK5 localized to the plasma membrane and activated StRBOHB These results suggest that the V domains confer substrate specificity in vivo by dictating proper subcellular localization of CDPKs. Protein kinases regulate almost all aspects of cell life through phosphorylation of Ser, Thr, and Tyr. Protein phosphorylation is known to affect protein functions such as enzymatic activity, stabilization, subcellular localization, and interactions with other biomolecules [1]. CDPKs are composed of an N-terminal variable (V) domain, a protein kinase (K) domain, an autoinhibitory junction (J) domain, and a calmodulin-like (C) domain including four EF-hand motifs and are activated via conformational changes triggered by the binding of Ca2ϩ to the C domain
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