title: The variable domain of a plant calcium-dependent protein kinase (CDPK) confers subcellular localization and substrate recognition for NADPH oxidase.
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.
authors.affiliation: Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan; The Sainsbury Laboratory, Norwich Research Park, Norwich NR4 7UH, United Kingdom.
authors.forename: Shuta
authors.initials: S
authors.lastname: Asai
authors.affiliation: Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan.
authors.forename: Tatsushi
authors.initials: T
authors.lastname: Ichikawa
authors.affiliation: Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan.
authors.forename: Hironari
authors.initials: H
authors.lastname: Nomura
authors.affiliation: Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan; Iwate Biotechnology Research Center, 22-174-4 Narita, Kitakami, Iwate 024-0003, Japan.
authors.forename: Michie
authors.initials: M
authors.lastname: Kobayashi
authors.affiliation: Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan; Department of Horticultural Sciences, University of Florida, Gainesville, Florida 32611-0690.
authors.forename: Yusuke
authors.initials: Y
authors.lastname: Kamiyoshihara
authors.affiliation: Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan.
authors.forename: Hitoshi
authors.initials: H
authors.lastname: Mori
authors.affiliation: The Sainsbury Laboratory, Norwich Research Park, Norwich NR4 7UH, United Kingdom.
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Kadota
authors.affiliation: The Sainsbury Laboratory, Norwich Research Park, Norwich NR4 7UH, United Kingdom.
authors.forename: Cyril
authors.initials: C
authors.lastname: Zipfel
authors.affiliation: The Sainsbury Laboratory, Norwich Research Park, Norwich NR4 7UH, United Kingdom.
authors.forename: Jonathan D G
authors.initials: JDG
authors.lastname: Jones
authors.affiliation: Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan. Electronic address: hyoshiok@agr.nagoya-u.ac.jp.
authors.forename: Hirofumi
authors.initials: H
authors.lastname: Yoshioka
mesh_terms: D002118:Calcium / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D018506:Gene Expression Regulation, Plant; D018551:Solanum lycopersicum / Q000201:enzymology / Q000235:genetics; D018613:Microscopy, Confocal; D009154:Mutation; D019255:NADPH Oxidases / Q000378:metabolism*; D010766:Phosphorylation; D057865:Plant Immunity; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism*; D017382:Reactive Oxygen Species; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D016897:Respiratory Burst; D015398:Signal Transduction; D011198:Solanum tuberosum / Q000201:enzymology / Q000235:genetics; D013379:Substrate Specificity; D014026:Nicotiana / Q000378:metabolism*; D017871:Calcium-Calmodulin-Dependent Protein Kinases
chemical_list: D002118:Calcium; D019255:NADPH Oxidases; D010940:Plant Proteins; D011494:Protein Kinases; D017382:Reactive Oxygen Species; D011994:Recombinant Proteins; D017871:Calcium-Calmodulin-Dependent Protein Kinases
keywords: Calcium-dependent Protein Kinase; NADPH Oxidase; Phosphorylation; Plant Defense; Plant Immunity; Reactive Oxygen Species (ROS); Signal Transduction