regulatory phosphorylation of bacterial-type pep ......2017/03/31 · 35 r.t.m., and w.c.p.), as...

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Short title: RcCDPK1 phosphorylates bacterial-type PEPC 1

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Corresponding author: William Plaxton, Department of Biology, Queen’s University, 3

Kingston, Ontario, Canada K7L 3N6. 4

Phone: 01-613-533-6150; FAX: 01-613-533-6617; E-mail: [email protected] 5

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Article title: 7

Regulatory Phosphorylation of Bacterial-type PEP Carboxylase by the Ricinus 8

kinase CDPK1 9

Author names and affiliations: 10

Sheng Ying*, Allyson T. Hill, Michal Pyc, Erin M. Anderson, Wayne A. Snedden, 11

Robert T. Mullen, Yi-Min She, and William C. Plaxton 12

Department of Biology, Queen’s University, Kingston, Ontario, Canada K7L 3N6 (S.Y., 13

A.T.H., W.A.S., W.C.P.); Department of Molecular and Cellular Biology, University of 14

Guelph, Guelph, Ontario, Canada N1G 2W1 (M.P., A.M.A., R.T.M.); Centre for Vaccine 15

Evaluation, Biologics and Genetic Therapies Directorate, Health Canada, 251 Sir Frederick 16

Banting Driveway, Tunney's Pasture, Ottawa, Ontario, Canada K1A 0K9 (Y-M. S.); 17

Department of Biomedical and Molecular Sciences, Queen’s University, Kingston, Ontario, 18

Canada K7L 3N6 (W.C.P.) 19

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Summary: 21

RcCDPK1 catalyzes in vivo inhibitory phosphorylation of the bacterial-type PEP 22

carboxylase subunits of the novel heteromeric Class-2 PEPC complex of developing castor 23

beans. 24

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Plant Physiology Preview. Published on March 31, 2017, as DOI:10.1104/pp.17.00288

Copyright 2017 by the American Society of Plant Biologists

www.plantphysiol.orgon May 28, 2020 - Published by Downloaded from Copyright © 2017 American Society of Plant Biologists. All rights reserved.

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Author contributions: 27

S.Y., W.A.S., R.T.M., and W.C.P. designed and supervised this study. S.Y., A.T.H., M.P., 28

E.M.A., and Y-M.S. performed the experiments. S.Y., W.A.S., R.T.M., Y-M.S., and W.C.P. 29

prepared the article. All authors read, contributed, and approved the article. 30

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Funding information: 32

This research was supported by Natural Sciences and Engineering Research Council of 33

Canada (NSERC) Discovery, and Research Tool and Infrastructure grants (to W.A.S., 34

R.T.M., and W.C.P.), as well as the Queen’s and Guelph Research Chair programs (to 35

W.C.P. and R.T.M). 36

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1Present address: 38

Division of Plant Biology, The Samuel Robert Noble Foundation, Ardmore, Oklahoma, USA 39

73401 40

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Corresponding author email: 42

[email protected] 43



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ABSTRACT 44

Phosphoenolpyruvate carboxylase (PEPC) is a tightly controlled cytosolic enzyme situated 45

at a crucial branch point of central plant metabolism. In developing castor oil seeds (COS) 46

(Ricinus communis) a novel, allosterically-desensitized 910-kD Class-2 PEPC hetero-47

octameric complex arises from a tight interaction between 107-kD plant-type PEPC and 48

118-kD bacterial-type (BTPC) subunits. The native Ca2+-dependent protein kinase (CDPK) 49

responsible for in vivo inhibitory phosphorylation of Class-2 PEPC’s BTPC subunit’s at Ser-50

451 was highly purified from COS and identified as RcCDPK1 (XP_002526815) by mass 51

spectrometry. Heterologously expressed RcCDPK1 catalyzed Ca2+-dependent, inhibitory 52

phosphorylation of BTPC at Ser-451 while exhibiting a: (i) pair of Ca2+ binding sites with 53

identical dissociation constants of 5.03 µM, (ii) Ca2+-dependent electrophoretic mobility 54

shift , and (iii) marked Ca2+-independent hydrophobicity. Pull-down experiments 55

established the Ca2+-dependent interaction of GST-RcCDPK1 with BTPC. RcCDPK1-56

Cherry localized to the cytosol and nucleus of tobacco BY-2 cells, but co-localized with 57

mitochondrial-surface associated BTPC-enhanced yellow fluorescent protein when both 58

fusion proteins were co-expressed. Deletion analyses demonstrated that although its N-59

terminal variable domain plays an essential role in optimizing Ca2+-dependent RcCDPK1 60

autophosphorylation and BTPC transphosphorylation activity, it is not critical for in vitro or 61

in vivo target recognition. Arabidopsis thaliana CPK4 (AtCPK4) and soybean CDPKβ are 62

RcCDPK1 orthologs that effectively phosphorylated castor BTPC at Ser-451. Overall, the 63

results highlight a potential link between cytosolic Ca2+ signaling and the post-translational 64

control of respiratory CO2 refixation and anaplerotic photosynthate partitioning in support of 65

storage oil and protein biosynthesis in developing COS. 66

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INTRODUCTION 68

Calcium plays a central role in eukaryotic signal transduction with various Ca2+-sensor 69

proteins being critical transducers of Ca2+-signatures elicited in response to external stimuli 70

or developmental cues. The activation of protein phosphorylation cascades is often the first 71

and most important signalling event triggered by Ca2+ signals (DeFalco et al., 2010). 72

Among Ca2+ sensors, including calmodulin (CaM) and CaM-like proteins (CMLs), Ca2+-73

dependent protein kinases (CDPKs) are unique because they function as catalytic 74

responders that directly transduce Ca2+ signals into protein phosphorylation events that 75

modulate physiological responses (Harper et al., 2004; DeFalco et al., 2010; Boudsocq and 76

Sheen, 2013; Schulz et al., 2013; Simeunovic et al., 2016). This combination of signalling 77

properties likely arose following the early fusion of a protein kinase gene with a CaM gene, 78

and was followed by CDPK diversification into a relatively large multigene family in vascular 79

plants, thus providing a mechanism to decode different Ca2+ signals in a temporal and 80

spatially-specific manner (Harper et al., 2004). Different CDPK isozymes exhibit distinctive 81

tissue and subcellular locations, substrate specificities, and Ca2+ sensitivities. Thus, diverse 82

developmental programs and stress responses are likely controlled by specific CDPKs 83

including hormone-regulated developmental processes, seed development, pollen tube 84

formation, and abiotic and biotic stress signaling (Harper et al., 2004; Boudsocq and 85

Sheen, 2013; Schulz et al., 2013; Simeunovic et al., 2016). Downstream targets of CDPK 86

action include other protein kinases, transcription factors, ion channels and pumps, 87

cytoskeletal proteins, as well as metabolic enzymes such as sucrose synthase, sucrose 88

phosphate synthase, nitrate reductase, and NADPH oxidase (Bachmann et al., 1996; 89

Douglas et al., 1998; Zhang et al., 1999; Kobayashi et al., 2007; Asai et al., 2013; 90

Simeunovic et al., 2016; Almadanim et al., 2017). While no CDPK appears to be an integral 91

membrane protein, most isoforms contain a myristoylation motif at their N-terminus, which 92

for several CDPKs has been shown to be important for their membrane association (Martin 93

and Busconi, 2000; Ito et al., 2010; Asai et al., 2013). However, a major gap in our 94

understanding of plant Ca2+ signaling and CDPK biology in general is that relatively few in 95



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vivo CDPK targets have been identified to date (Liese and Romeis, 2013; Schulz et al., 96

2013; Simeunovic et al., 2016). It is evident that some CDPKs, when purified as 97

recombinant enzymes, promiscuously phosphorylate exogenous proteins in vitro at many 98

different Ser and Thr residues, far beyond proposed CDPK phosphorylation motifs (Harper 99

et al., 2004; Boudsocq and Sheen, 2013; Schulz et al., 2013). 100

The aim of the current study was to identify and characterize the putative CDPK that 101

catalyzes in vivo inhibitory phosphorylation of bacterial-type phosphoenolpyruvate (PEP) 102

carboxylase (BTPC) at Ser-451 in developing castor oil seeds (COS) (Dalziel et al., 2012; 103

Hill et al., 2014). PEP carboxylase (PEPC; EC 4.1.1.31) is a tightly regulated enzyme that 104

has been intensively studied owing to its pivotal role in assimilating atmospheric CO2 105

during C4 and Crassulacean acid metabolism (CAM) photosynthesis. Much attention has 106

also been devoted to PEPC’s essential non-photosynthetic functions, particularly the 107

anaplerotic replenishment of tricarboxylic acid cycle intermediates withdrawn during 108

biosynthesis and nitrogen assimilation (O’Leary et al., 2011b). To fulfill its diverse roles and 109

complex regulation, plant PEPC belongs to a small multi-gene family encoding several 110

plant-type PEPCs (PTPCs), along with a distantly-related BTPC (O’Leary et al., 2011b). 111

PTPC genes encode closely related 100-110-kD polypeptides containing conserved seryl-112

phosphorylation (activatory) and lysyl-monoubiquitination (inhibitory) sites and that typically 113

oligomerize as tetrameric Class-1 PEPCs (Supplemental Fig. S1) (Tripodi et al., 2005; 114

Uhrig et al., 2008b; O’Leary et al., 2011b; Ruiz-Ballesta et al., 2014, 2016). By contrast, 115

plant BTPC genes encode distantly related 116-118-kD polypeptides that are more similar 116

to prokaryotic PEPCs. Purification of native PEPCs from unicellular green algae and then 117

developing castor oil seeds (COS) led to the discovery of unusual high-Mr Class-2 PEPC 118

heteromeric complexes composed of tightly associated PTPC and BTPC subunits, and that 119

are largely desensitized to allosteric effectors relative to Class-1 PEPCs (Supplemental Fig. 120

S1) (Rivoal et al., 2001; Blonde and Plaxton, 2003; Gennidakis et al., 2007; Uhrig et al., 121

2008a; O’Leary et al., 2009). Plant BTPC polypeptides and thus Class-2 PEPC complexes 122

have only been documented in biosynthetically active tissues (i.e., developing seeds and 123



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pollen, immature leaves), as opposed to PTPCs that are constitutively expressed in the 124

cytosol as housekeeping Class-1 PEPCs (Igawa et al., 2010; O’Leary et al., 2011a, 2011b). 125

Although COS Class-1 PEPC localizes diffusely throughout the cytosol, the Class-2 PEPC 126

associates with the outer mitochondrial envelope, an interaction mediated by its BTPC 127

subunits (Park et al., 2012). Class-2 PEPC’s unique kinetic and regulatory properties, and 128

dynamic subcellular targeting to the mitochondrial surface support the hypothesis that it 129

facilitates rapid refixation of respiratory CO2 while sustaining a large anaplerotic flux to 130

replenish tricarboxylic acid cycle C-skeletons withdrawn in support of storage oil and 131

protein biosynthesis in developing COS. 132

Although BTPCs lack the N-terminal seryl phosphorylation motif characteristic of non-133

photosynthetic and C4/CAM photosynthetic PTPCs, BTPC is in vivo phosphorylated at 134

multiple sites during COS development (Uhrig et al., 2008a; O’Leary et al., 2011c; Dalziel 135

et al., 2012). This includes inhibitory phosphorylation at Ser-425 and Ser-451, which both 136

occur within a distinctive ~10-kD intrinsically disordered region not found in PTPCs (Dalziel 137

et al., 2012; O’Leary et al., 2011c). Despite the apparent important role of multisite BTPC 138

phosphorylation in the post-translational control of photosynthate partitioning and 139

anaplerotic C-flux at the PEP branchpoint during COS development, nothing was known 140

about the responsible protein kinases or related signaling pathways. Thus, Hill and 141

coworkers (2014) purified the native BTPC Ser-451 kinase from developing COS by over 142

500-fold and provided kinetic evidence that it belongs to the castor CDPK family. The 143

current study used mass spectrometry (MS) to identify the BTPC Ser-451 kinase as 144

RcCDPK1, one of twenty predicted castor CDPK isozymes (Hill et al., 2014). We also 145

demonstrate the specificity of RcCDPK1 for phosphorylating BTPC at Ser-451, as well as 146

its in vivo interaction with BTPC on the surface of mitochondria. 147



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RESULTS AND DISCUSSION 148

Identification of Castor Bean BTPC Serine-451 Kinase as RcCDPK1 149

Peptides derived from a total tryptic digest of the final BTPC Ser-451 kinase preparation 150

of Hill and coworkers (2014) were sequenced via nanoHPLC tandem MS (LC-MS/MS). 151

Database searches identified numerous proteins (Supplemental Table S1) including a 152

single protein kinase, RcCDPK1 (protein #36 on the list). Reliable identification was 153

achieved by 9 tryptic peptides whose respective sequences precisely matched 154

corresponding regions of RcCDPK1’s predicted sequence (30.2% sequence coverage) 155

(protein accession number: XP_002526815) (Fig. 1). The predicted size of RcCDPK1 is 56-156

kD, a value similar to the native molecular mass of 63-kD estimated for BTPC Ser-451 157

kinase isolated from developing COS (Hill et al., 2014). 158

The deduced RcCDPK1 protein corresponds to a 497 amino-acid polypeptide having 78 159

- 84% sequence identity with the orthologs aligned in Fig. 1; maximal identity (83-84%) was 160

achieved with CPK4 and CPK11 from Arabidopsis thaliana (AtCPK4 and AtCPK11, 161

respectively) and soybean (Glycine max) CDPKβ (GmCDPKβ). RcCDPK1 and its orthologs 162

possess all five modular domains characteristic of CDPKs: an N-terminal variable domain 163

which differs both in sequence and length, a protein kinase catalytic domain, an auto-164

inhibitory junction, a CaM-like domain containing four EF-hand motifs implicated in Ca2+ 165

binding, and a C-terminal variable domain (Fig. 1). RcCDPK1, AtCPK4, and AtCPK11 were 166

determined to be soluble rather than membrane bound (Dammann et al., 2003; Boudsocq 167

et al., 2012; Hill et al., 2014). This is consistent with the absence of a membrane-targeting 168

myristoylation sequence (MGXXXS) at their N-terminus (Fig. 1) that is typical of most 169

CDPKs (Schulz et al., 2013). The closest paralog of RcCDPK1 is RcCDPK2 (70% 170

sequence identity) (Hill et al., 2014), the kinase that in vivo phosphorylates castor sucrose 171

synthase-1 (RcSUS1) at Ser-11 in developing COS (Fedosejevs et al., 2016). 172

As with all known eukaryotic protein kinases, the catalytic kinase domain of plant CDPKs 173

contains a conserved activation loop that begins and ends with Asp-Phe-Gly (DFG) and 174

Ala-Pro-Glu (APE) motifs, respectively (e.g., RcCDPK1 residues 175-201; Fig. 1) (Taylor 175



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and Kornev, 2011; Liese and Romeis, 2013). Stimulus-dependent phosphorylation of a 176

Thr/Ser residue within this activation loop is a prerequisite for activation of most protein 177

kinases (Taylor and Kornev, 2011). It is notable that phosphorylation-dependent regulation 178

within the kinase activation loop does not occur for CDPKs (Liese and Romeis, 2013), as 179

they instead have had the target Thr/Ser substituted with a phosphomimetic Asp or Glu 180

residue; e.g., Asp in the case of RcCDPK1 (i.e. Asp-190) and its orthologs (Fig. 1). 181

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RcCDPK1 is Highly Expressed During Castor Bean Development 183

Many CDPK genes are transcriptionally responsive to developmental and stress stimuli 184

(Harper et al., 2004; Schulz et al., 2013). RcCDPK1 is also differentially expressed in 185

castor plants, with maximal transcript levels occurring in male flowers and mature leaves, 186

and the endosperm and cotyledons of developing COS (Fig. 2A). RcCDPK1’s expression 187

profile in COS endosperm matched the pattern of in vivo BTPC phosphorylation at Ser-451 188

with both peaking at the maturation stage (stage IX) of seed development (Fig. 2B) (Dalziel 189

et al., 2012). However, extractable BTPC Ser-451 kinase activity showed a somewhat 190

dissimilar developmental profile as it peaked at stage VII, but was followed by a marked 191

decrease at stage IX (Hill et al., 2014). These results are consistent with the post-192

translational control of RcCDPK1 during COS development. By comparison, BTPC 193

transcript levels maximized during the middle phase of COS development and showed a 194

pronounced drop at stage IX, prior to becoming undetectable in fully mature COS (Fig. 2B). 195

BTPC’s expression profile parallels that previously obtained via semi-quantitative RT-PCR, 196

and correlate well with relative levels of the encoded 118-kD BTPC polypeptides (p118) 197

(Gennidakis et al., 2007; O’Leary et al., 2011a). Thus, maximal BTPC expression during 198

COS development occurs in advance of the subsequent peaks in RcCDPK1 expression, 199

BTPC Ser-451 kinase activity, and in vivo BTPC phosphorylation at Ser-451 (Fig. 2B) 200

(Dalziel et al., 2012; Hill et al., 2014). 201

RcCDPK1 transcript abundance in the endosperm and cotyledon of developing COS 202

was reduced by over 50% within 48 h after photosynthate supply was eliminated by 203



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excision of intact fruit clusters (i.e., depodding) (Fig. 2A). However, this was not paralleled 204

by a decrease in BTPC Ser-451 kinase activity nor a reduction in BTPC phosphorylation at 205

Ser-451 in the developing endosperm (Dalziel et al., 2012; Hill et al., 2014). This provides 206

further evidence in support of post-transcriptional RcCDPK1 control in developing COS. A 207

similar lack of correlation of AtCPK4 and AtCPK11 transcripts with their respective protein 208

levels and kinase activity was noted following abscisic acid treatment of Arabidopsis (Zhu 209

et al., 2007). 210

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RcCDPK1 Cloning, Heterologous Expression, and Antibody Production 212

Full-length RcCDPK1 as well as its N-terminal variable domain truncation mutant (i.e., 213

∆N-RcCDPK1, lacking the first 29 amino acid residues) were heterologously expressed as 214

N-terminal His6-tagged fusion proteins in Escherichia coli BL21 and purified to apparent 215

homogeneity by Ni2+-affinity chromatography (Fig. 3A). RcCDPK1 and ∆N-RcCDPK1 both 216

demonstrated Ca2+-binding dependent mobility shifts during SDS-PAGE (Fig. 3A), as 217

described previously for several CDPKs including RcCDPK1 orthologs AtCPK4, AtCPK11, 218

and GmCDPKβ (Harmon et al., 1987; Zhu et al., 2007; Boudsocq et al., 2012). 219

For production of RcCDPK1-specific antibodies (anti-RcCDPK1), a 29 amino acid 220

oligopeptide matching RcCDPK1’s N-terminal variable domain was synthesized with an 221

extra N-terminal Cys residue (Supplemental Fig. S2A) in order to enable its conjugation to 222

keyhole limpet hemocyanin prior to rabbit immunization. The affinity-purified anti-RcCDPK1 223

detection limit was 10 ng of the corresponding peptide on dot blots, with the signal being 224

quenched by the blocking parent peptide (Supplemental Fig. S2B). Immunoblots of His6-225

RcCDPK1 cross-reacted with the anti-RcCDPK1, whereas His6-∆N-RcCDPK1 was not 226

detected, as expected (Fig. 3B). The anti-RcCDPK1 appears to be monospecific as it 227

cross-reacted with a single 56-kD immunoreactive polypeptide that co-migrated with the 228

recombinant, full length RcCDPK1 (after cleavage of its His6-tag) on immunoblots of both a 229

phenyl-Sepharose-enriched developing COS extract, as well as the final BTPC Ser-451 230

kinase preparation of Hill and co-workers (2014) that was analyzed by LC-MS/MS in the 231



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present study (Fig. 3B). 232

233

RcCDPK1 Catalyzes Ca2+-Dependent, Inhibitory Phosphorylation of Castor BTPC at 234

Serine-451 235

Kinetic studies of heterologously expressed RcCDPK1 were conducted by monitoring Pi 236

incorporation from unlabeled ATP or [γ-32P]-ATP into the p118/ BTPC subunits of 237

recombinant Class-2 PEPC consisting of a 1:1 stoichiometric ratio of castor BTPC 238

(RcPPC4) and an Arabidopsis PTPC isozyme (AtPPC3) (O’Leary et al., 2009). RcCDPK1 239

readily catalyzed Ca2+-dependent Ser-451 phosphorylation of the p118 BTPC subunits of 240

Class-2 PEPC (Fig. 4A, Fig. 5, and Fig. 6A). Furthermore, and as reported for native BTPC 241

Ser-451 kinase (Hill et al., 2014): (i) RcCDPK1 exhibited a relatively narrow substrate 242

specificity, as it phosphorylated BTPC in addition to histone III-S, but was unable to 243

phosphorylate a S451D phosphomimetic BTPC mutant, dephosphorylated COS PTPC or 244

RcSUS1 (Fig. 5), or a pair of synthetic dephosphopeptides containing sequences flanking 245

BTPC’s Ser-451 phosphosite (Supplemental Fig. S3). This not only illustrates the selectivity 246

of RcCDPK1 for phosphorylating BTPC at Ser-451, but also the importance of the 247

structural context of the interaction. Furthermore, all PTPCs and sucrose synthases 248

examined to date share an orthologous phosphorylation motif near their N-terminus; i.e., Ф-249

5-X-4-basic-3-X-2-X-1-Ser-X+1-X+2-X+3-Ф+4 (where Ф is a hydrophobic residue, X is any amino 250

acid, and subscripts denote residue positions relative to the seryl phosphorylation site) 251

(Winter and Huber, 2000; O’Leary et al., 2011b; Fedosejevs et al., 2014, 2016). This differs 252

from the unique and highly conserved BTPC recognition motif identified for RcCDPK1, 253

namely: Ф-4-X-3-Basic-2-X-1-Ser-X+1-X+2-Basic+3-Ф+4 (Dalziel et al., 2012; Hill et al., 2014) 254

(Supplemental Fig. S3). Our combined results therefore demonstrate the importance of not 255

only the linear sequence flanking BTPC’s Ser-451 site, but also the overall conformation of 256

BTPC subunits within the Class-2 PEPC complex. This provides additional evidence that 257

RcCDPK1 is a relatively specific Ser/Thr protein kinase. 258

Phosphorylation of the BTPC subunits of a PTPC-inactive (R644A) Class-2 PEPC 259



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mutant (O’Leary et al., 2009) by RcCDPK1 inhibited its PEPC activity by 55 ±7% (mean 260

±SEM, n = 3 determinations) when assayed under suboptimal conditions (pH 7.3, 1 mM 261

PEP, 10 mM L-malate). This validates our previous studies of a S451D phosphomimetic 262

BTPC mutant, as well as the purified native BTPC Ser-451 kinase of developing COS 263

(Dalziel et al., 2012; Hill et al., 2014). The current results also corroborate our earlier report 264

(Hill et al., 2014) that BTPC Ser-451 kinase activity is subject to additional post-265

translational controls beyond fluctuations in Ca2+ levels, namely inhibition by PEP; i.e., 266

addition of 1-5 mM PEP potently inhibited BTPC phosphorylation at Ser-451 by RcCDPK1 267

(Fig. 4C). PEP appears to directly affect the kinase as similar levels of BTPC Ser-451 268

kinase inhibition occurred when BTPC or histone III-S served as its substrates (Hill et al., 269

2014). By contrast, the Class-1 PEPC (i.e., PTPC) protein kinase of developing COS was 270

activated about 40% by 1 mM PEP (Murmu and Plaxton, 2007). Reciprocal control of COS 271

RcCDPK1 and PTPC protein kinase by PEP provides an intriguing regulatory mechanism 272

whereby a drop in cytosolic PEP levels would diminish overall anaplerotic PEP 273

carboxylation to oxaloacetate; i.e., by simultaneously enhancing inhibitory phosphorylation 274

of Class-2 PEPC’s BTPC subunits at Ser-451, while attenuating phosphorylation-activation 275

of Class-1 PEPC’s PTPC subunits at Ser-11. Reduced cytosolic PEP levels likely occurs 276

following the elimination of photosynthate import that arises during the final stages of COS 277

development or following depodding when vascular connection with the parent plant has 278

been lost; both processes are accompanied by enhanced in vivo BTPC phosphorylation at 279

Ser-451 and complete PTPC dephosphorylation at Ser-11 (Tripodi et al., 2005; Murmu and 280

Plaxton, 2007; Dalziel et al., 2012). 281

RcCDPK1 also exhibited prominent Ca2+-activated autophosphorylation activity (Fig. 282

6B). In vitro autophosphorylation at multiple sites has been widely reported for plant 283

CDPKs and may influence their activity and substrate accessibility (Liese and Romeis, 284

2013). However, the occurrence and functions of in vivo CDPK phosphorylation remain 285

obscure. Autophosphorylation is important for the regulation and/or subcellular localization 286

of eukaryotic protein kinases (Endicott et al., 2012) including at least one plant CDPK, 287



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tobacco CDPK2 (NtCDPK2) (Witte et al., 2010). Both constitutively-phosphorylated 288

residues and stress-induced in vivo phosphorylation, catalyzed by either NtCDPK2 itself or 289

by upstream protein kinases, were reported (Witte et al., 2010). Therefore, an important 290

objective will be to establish RcCDPK1’s (auto)phosphorylation sites, and the impact of 291

site-specific phosphorylation on kinase function. 292

RcCDPK1 orthologs AtCPK4 and GmCDPKβ were also expressed as His6-fusion 293

proteins in E. coli, along with a distantly related Arabidopsis CDPK, AtCPK34. Anti-pSer451 294

based kinase assays established that both orthologs, but not AtCPK34, effectively 295

catalyzed Ca2+-dependent phosphorylation of castor BTPC at Ser-451 (Fig. 4A). It is 296

notable that castor BTPC’s Ser-451 phosphorylation site and its adjacent basophilic CDPK 297

recognition motif (Supplemental Fig. S3) are conserved amongst vascular plant BTPC 298

orthologs, including the Arabidopsis BTPC, AtPPC4 (Hill et al., 2014). Thus, future studies 299

need to determine if AtCPK4 and/or AtCPK11 participate in the in vivo regulatory 300

phosphorylation of AtPPC4, particularly in developing pollen where AtCPK4, AtCPK11, and 301

AtPPC4 are all highly expressed (Harper et al., 2004; Igawa et al., 2010). Extracts 302

prepared from flowers of Col-0 plants but not from atcpk11 mutants showed Ca2+-303

dependent kinase activity against castor BTPC at Ser-451 suggesting that AtCPK11 is 304

functionally as well as structurally homologous to RcCDPK1 (Fig. 4B). 305

306

RcCDPK1 Exhibits a Pair of High-affinity Ca2+-binding sites and Strong Intrinsic 307

Hydrophobicity 308

We next assessed the in vitro Ca2+-binding and hydrophobic properties of RcCDPK1 as 309

these vary among CDPKs and can offer insights into their in vivo roles. RcCDPK1’s binding 310

stoichiometry and affinity for Ca2+ in the presence of Mg2+ was evaluated by isothermal 311

titration calorimetry (Fig. 7A). The data best fit a model that predicted two sets of high 312

affinity Ca2+ binding sites with identical Kd values of 5.03 μM. RcCDPK1’s Kd(Ca2+) values 313

are within the physiological range and compare favorably with K0.5(Ca2+) values of 2.7 μM 314

reported for the native BTPC Ser-451 kinase purified from developing COS (Hill et al., 315



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2014), as well as 3.1 and 4.5 μM determined for RcCDPK1 orthologs AtCPK4 and 316

AtCPK11, respectively (Boudsocq et al., 2012). 317

Calcium-binding to EF-hand containing proteins, including CDPKs, initiates 318

conformational changes in their conserved CaM-related domain that typically results in 319

exposure of hydrophobic clefts believed to be critical to target recognition and interaction 320

(DeFalco et al., 2010). These conformational changes are a hallmark of Ca2+ sensors and 321

have been exploited for their efficient purification via Ca2+-dependent hydrophobic 322

interaction chromatography. Although RcCDPK1 exhibited a Ca2+-dependent 323

electrophoretic mobility shift reminiscent of a classic Ca2+ sensor (Fig. 3A and Fig. 6B), its 324

unusual Ca2+-independent hydrophobicity was demonstrated by 8-anilinonaphthalene-1-325

sulfonic acid-based fluorescence spectroscopy and hydrophobic interaction 326

chromatography using phenyl-Sepharose (Fig. 7B and Supplemental Fig. S4). 8-327

Anilinonaphthalene-1-sulfonic acid exhibits a blue shift and increased fluorescence when it 328

interacts with non-polar surfaces on proteins, whereas Ca2+-dependent binding to phenyl-329

Sepharose has been reported for CaM as well as several, but not all, CDPKs and CMLs. 330

Fluorescence spectroscopy revealed that apo-RcCDPK1 (in contrast to petunia CaM81, a 331

positive control) exhibits considerable hydrophobicity that was unaffected by the addition of 332

up to 1 mM Ca2+ (Fig. 7B). Similarly, the native BTPC Ser-451 kinase from developing COS 333

exhibited tight Ca2+-independent binding to phenyl-Sepharose and could only be eluted 334

from this resin using a buffer containing 10% (v/v) ethylene glycol (i.e., a chaotropic 335

solvent) (Supplemental Fig. S4) or a detergent such as 0.5% (v/v) Triton X-100 (results not 336

shown). Similar results were reported for cytosolic CDPKs that respectively phosphorylate 337

sucrose synthase at Ser-11 in developing COS (Fedosejevs et al., 2016) and nitrate 338

reductase in spinach leaves (Bachmann et al., 1996; Douglas et al., 1998). 339

Calcium-mediated conformational changes in RcCDPK1 were further investigated by 340

probing its secondary structure using far-UV circular dichroism spectroscopy. This 341

indicated that apo-RcCDPK1 was folded in solution, and exhibited a large positive band 342

below 200 nm, with maxima at 191 nm and negative bands with local minima at 209 nm 343



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and 220 nm, typical of proteins having significant α-helical structure (Fig. 7C). The apo- 344

and Ca2+-bound RcCDPK1 spectra were almost identical, indicating little change in 345

secondary structure composition upon Ca2+ binding. Conformational changes associated 346

with Ca2+ binding to RcCDPK1 may trigger a reorientation of helices, as occurs with CaM 347

(Zhang et al., 1995), rather than pronounced changes in helical content. These data 348

demonstrate that high-affinity Ca2+ binding by RcCDPK1 induces predominately tertiary but 349

not secondary structural changes associated with increased surface hydrophobicity as 350

exhibited by canonical Ca2+ sensors such as CaM (Defalco et al., 2010). 351

The strong intrinsic hydrophobicity, and lack of increased hydrophobicity upon exposure 352

to Ca2+ distinguishes RcCDPK1 from CaM and most CMLs examined to date. This 353

indicates that while Ca2+ is clearly important for regulating RcCDPK1 activity (Fig. 4A and 354

Fig. 6B) (Hill et al., 2014), it may do so through structural changes that lead to relief of 355

autoinhibition rather than exposure of hydrophobic target-interacting regions. Given the 356

relatively large number of CDPK isozymes in a given plant, such unique biochemical 357

features may reflect the diversity needed to ensure specificity of activity, especially 358

whenever multiple CDPKs are co-expressed. 359

360

RcCDPK1 is Targeted to the Cytosol and Nucleus of Tobacco BY-2 Cells, but 361

Relocalizes to the Mitochondrial Surface when Co-expressed with BTPC 362

The subcellular location and potential interaction of fluorescent protein fusions of 363

RcCDPK1 and ∆N-RcCDPK1 with castor BTPC were assessed in tobacco bright yellow-2 364

(BY-2) suspension cells, a well-established model system for protein localization and 365

interaction studies (Brandizzi et al., 2003). Cells were transiently transformed using gene 366

constructs encoding a variety of fluorescent protein-tagged RcCDPK1 and BTPC fusion 367

proteins (Fig. 8A), and imaged by confocal laser-scanning microscopy (CLSM). C-terminal 368

Cherry-tagged versions of RcCDPK1 and ΔN-RcCDPK1, similar to Cherry alone, localized 369

to the cytosol and nucleus of individual, representative transformed BY-2 cells (Fig. 8B to 370

Fig. 8D). Likewise, a RcCDPK1-green fluorescent protein (GFP) fusion, as well as N-371



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15

terminal Cherry-tagged versions of RcCDPK1 and ΔN-RcCDPK1, localized to the cytosol 372

and nucleus in BY-2 cells (Supplemental Fig. S5A to Fig. S5C), indicating that the position 373

and/or type of appended fluorescent protein does not influence the subcellular localization 374

of RcCDPK1 or ΔN-RcCDPK1. Notably, the partial nuclear targeting of RcCDPK1 may be 375

due to its putative nuclear localization signal, -P58KRK61- (Milla et al., 2006), which is 376

conserved in the orthologs aligned in Fig. 1, including AtCPK4 whose GFP-tagged fusion 377

protein was also localized to the cytosol and nucleus (of transgenic Arabidopsis plants) 378

(Dammann et al., 2003). 379

We next studied whether subcellular targeting of RcCDPK1-Cherry was altered upon its 380

co-expression with a castor BTPC-enhanced yellow fluorescent protein (EYFP) fusion. The 381

in vivo association of castor BTPC with the surface of mitochondria in developing COS, as 382

well as following its expression as a EYFP-fusion protein in tobacco BY-2 cells, has been 383

well documented (Park et al., 2012). Likewise, transiently expressed, EYFP-tagged castor 384

BTPC (i.e., p118-EYFP) co-localized with an endogenous mitochondrial marker enzyme 385

(i.e., cytochrome c oxidase) (Supplemental Fig. S5D). When RcCDPK1-Cherry was co-386

expressed with p118-EYFP it partially co-localized with BTPC at aggregated mitochondria 387

(Fig. 8E), which are considered to have coalesced owing to BTPC expression and 388

association (Park et al., 2012). This supports the hypothesis of an in vivo interaction 389

between RcCDPK1 and BTPC, and is reminiscent of the reported occurrence of an 390

unspecified CDPK isoform and multiple phosphoproteins in the outer envelope of 391

mitochondria purified from potato tubers (Pical et al., 1993). Allied control experiments 392

confirmed that the co-localization of RcCDPK1-Cherry with p118-EYFP at aggregated 393

mitochondria (Fig. 8E), was not due to p118-EYFP fluorescence bleed-through during 394

CLSM imaging (Supplemental Fig. S5E). Furthermore, no protein co-localization occurred 395

when p118-EYFP was co-expressed with Cherry alone (Supplemental Fig. S5F), nor when 396

RcCDPK1-Cherry was co-expressed with mito-EYFP, which consisted of the E1α subunit of 397

the COS mitochondrial pyruvate dehydrogenase complex appended to EYFP 398

(Supplemental Fig. S5G). 399



16

16

400

BTPC’s Intrinsically Disordered Region is an RcCDPK1 Interaction Domain 401

When PEPC was first isolated from developing COS, 64-kD BTPC polypeptides were 402

identified by LC-MS/MS and determined to be the binding partner of PTPC subunits in the 403

purified Class-2 PEPC complex (Blonde and Plaxton, 2003). Subsequent research 404

revealed that the native BTPC exists as a p118, but that upon COS extraction it is rapidly 405

cleaved by an endogenous thiol endopeptidase into 54- and 64-kD polypeptides (p54 and 406

p64) (Gennidakis et al., 2007). BTPC’s proteolytic cleavage site (Lys-446/Ile-447) as well 407

as its in vivo Ser-425 and Ser-451 phosphorylation sites all occur within an approximate 408

10-kD intrinsically disordered region, a distinctive feature of green algal and vascular plant 409

BTPCs that mediates their tight interaction with co-expressed PTPC subunits to form the 410

Class-2 PEPC heteromeric complex (O’Leary et al., 2011b; Park et al., 2012). 411

To map the RcCDPK1-interaction domain of COS BTPC, we initially focused our 412

attention on p54 and p64. As with full-length p118-EYFP (Fig. 8E), RcCDPK1-Cherry 413

partially co-localized with p54-EYFP, but not with p64-EYFP, at (aggregated) mitochondria 414

in BY-2 cells (Fig. 8G and 8H). Based on these results, we next tested whether the 415

intrinsically disordered region of BTPC is a potential RcCDPK1-interaction domain. 416

Specifically, RcCDPK1-Cherry was co-expressed with an additional BTPC truncation 417

mutant, namely p40-EYFP (consisting of p54, but without the disordered region; 418

corresponding to BTPC residues 1–329). As shown in Fig. 8I, p40-EYFP localized to the 419

mitochondria, as expected (Park et al., 2012), but failed to relocalize co-expressed 420

RcCDPK1-Cherry from the cytosol to mitochondria. 421

Taken together, these results indicate that the interaction of RcCDPK1 and BTPC/p118 422

is mediated, at least in part, by BTPC’s intrinsically disordered region. Similarly, COS 423

PTPC’s p107 subunits localized to the cytosol in the tobacco BY-2 cells, but relocalized to 424

the mitochondrial surface when co-expressed with BTPC/p118 or with various deletion 425

mutants containing portions of BTPC’s disordered region (Park et al., 2012). Intrinsically 426

disordered regions are widespread amongst different proteins, provide a docking site to 427



17

17

promote protein–protein interactions by recruiting binding partners, and are susceptible to 428

site-specific phosphorylation (Dunker et al., 2005). Castor BTPC is no exception since its 429

Ser-425 and Ser-451 phosphorylation sites occur within its disordered region (O'Leary et 430

al. 2011c; Dalziel et al., 2012). Furthermore, this domain appears to be essential for 431

mediating BTPC’s in vivo interaction with PTPC within a Class-2 PEPC complex (Park et 432

al., 2012), as well as with RcCDPK1 (Fig. 8). 433

434

RcCDPK1 Binding to BTPC is Ca2+-Dependent 435

N-terminal glutathione S-transferase-tagged RcCDPK1 (GST-RcCDPK1) was 436

heterologously expressed and purified, then adsorbed to glutathione beads and incubated 437

with recombinant Class-1 or Class-2 PEPCs followed by affinity chromatography. GST-438

RcCDPK1 bound to the BTPC-containing Class-2 PEPC in a Ca2+-dependent manner (Fig. 439

9). Thus, conformational changes induced by Ca2+ binding not only activate RcCDPK1 (Fig. 440

4A and Fig. 6B) (Hill et al., 2014), but also control the ability of RcCDPK1 to recruit its 441

BTPC substrate. GST-RcCDPK1 specifically associated with the BTPC subunits of Class-2 442

PEPC, since no interaction was detected when it was incubated with purified Class-1 443

PEPC (i.e., a PTPC homotetramer) isolated from AtPPC3-overexpressing E. coli cells (Fig. 444

9). 445

446

The N-terminal Variable Domain is Important for RcCDPK1’s Trans- and 447

Autophosphorylation Activities, but not for its Ca2+-Dependent Binding to BTPC 448

The N-terminal variable domain has been the focus of several CDPK studies examining 449

its role in substrate recognition/binding, subcellular localization, and catalysis. For example, 450

this domain of NtCDPK1 appears to mediate a Ca2+-dependent interaction with its 451

substrate, the transcription factor REPRESSION OF SHOOT GROWTH FACTOR that 452

controls expression of gibberellin biosynthetic genes (Ito et al., 2010). N-terminal variable 453

domain deletion eliminated the in vitro and in vivo interaction of NtCDPK1 with 454

REPRESSION OF SHOOT GROWTH FACTOR, while significantly inhibiting its ability to in 455



18

18

vitro phosphorylate this substrate at Ser-114 (Ito et al., 2010). Similarly, the N-terminal 456

variable domain of: (i) AtCPK32 is necessary but not solely sufficient for its interaction with 457

a substrate AtABF4 (Choi et al., 2005), and (ii) a potato CDPK, StCDPK5, is essential for 458

ensuring its proper subcellular location and consequent discrimination of its substrate, 459

NADPH oxidase (Asai et al., 2013). We approached this problem by expressing ∆N-460

RcCDPK1 as His6-, Cherry-, and GST-tagged fusion proteins. 461

N-terminal variable domain deletion triggered a marked reduction in the ability of Ca2+-462

activated RcCDPK1 to phosphorylate castor BTPC at Ser-451 (Fig. 4A and Fig. 6A). 463

Similarly, parallel autophosphorylation assays revealed that relative autophosphorylation 464

activity of ∆N-RcCDPK1 was about 90% lower than that of the non-truncated RcCDPK1 465

(Fig. 6B). Ca2+ addition enhanced RcCDPK1 autophosphorylation activity by about 50%, 466

but had no obvious impact on ∆N-RcCDPK autophosphorylation. By contrast, deletion of 467

the N-terminal variable domain did not disrupt the apparent in vivo interaction of ∆N-468

RcCDPK1-Cherry with p118-EYFP in tobacco BY-2 cells (Fig. 8F), nor hinder the ability of 469

GST-∆N-RcCDPK1 to specifically interact with its BTPC substrate in a Ca2+-dependent 470

fashion (Fig. 9). Overall, these results indicate that although the N-terminal variable domain 471

of RcCDPK1 is not critical for target recognition, it clearly contributes to fine-tuning kinase 472

activity for optimal Ca2+-dependent phosphorylation of BTPC at Ser-451. 473

474

CONCLUSIONS 475

In summary, we have identified BTPC as a novel target for a specific CDPK isozyme in 476

developing COS, and likely across plant taxa. This suggests an intriguing potential link 477

between cytosolic Ca2+ signaling and the control of carbon partitioning at the critical PEP 478

branchpoint in a biosynthetically active, heterotrophic plant tissue. These results have 479

implications not only for PEPC’s broader role in the control of cytosolic carbohydrate 480

partitioning and anaplerosis, but also for the regulation of the enigmatic BTPC by reversible 481

phosphorylation. It is notable that the developmental pattern of in vivo BTPC 482

phosphorylation at Ser-451 in COS (Dalziel et al., 2012; Hill et al., 2014), as well as the 483



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19

basophilic phosphorylation motif flanking its Ser-451 residue (Supplemental Fig. S3) are 484

distinct from those of COS PTPC and RcSUS1 (Tripodi et al., 2005; Fedosejevs et al., 485

2016). These differences imply specific protein kinase-phosphatase pairings in controlling 486

the phosphorylation status of BTPC, PTPC, versus RcSUS1 in developing COS. Indeed, a 487

low molecular mass (−30 kD), Ca2+-independent, and highly specific PTPC kinase 488

mediates Ser-11 phosphorylation/activation of the PTPC subunits of Class-1 PEPC during 489

COS development (Tripodi et al., 2005; Murmu and Plaxton, 2007). Furthermore, 490

RcCDPK2 phosphorylates RcSUS1 at Ser-11 in developing COS and shows many 491

additional and prominent differences compared to RcCDPK1 including: (i) a relatively non-492

specific in vitro substrate selectivity, including abundant peptide kinase activity, (ii) a 493

significantly lower K0.5(Ca2+) value of about 200 nM, (iii) insensitivity to metabolite effectors 494

including PEP, (iv) partial association with microsomal membranes, and (iv) a completely 495

opposite developmental profile in COS (i.e. RcCDPK2 expression, RcSUS1 Ser-11 kinase 496

activity, and in vivo RcSUS1 phosphorylation at Ser-11 progressively decrease during COS 497

development) (Fedosejevs et al., 2014, 2016). 498

Several attempts at metabolic engineering of PEPC have failed due to a lack of in-depth 499

knowledge of the enzyme’s multifaceted post-translational controls (O’Leary et al., 2011b). 500

Owing to the commercial interest in modifying photosynthate partitioning to agronomically 501

important end-products such as oil or storage proteins during seed development, and the 502

key position of PEPC in controlling this process, our castor bean PEPC research should 503

prove informative for developing innovative strategies for engineering PEPC activity. Given 504

PEPC’s critical position in central plant metabolism, it is perhaps not surprising that the 505

emerging model for regulation of non-photosynthetic PEPCs involves a complex and 506

unique set of post-translational control mechanisms that work hand-in-hand with the well-507

documented regulation of PTPC-containing Class-1 PEPCs (including C4 and CAM 508

photosynthetic PEPCs) by allosteric effectors and reversible phosphorylation 509

(Supplemental Fig. S1) (O’Leary et al., 2011b). These control mechanisms include BTPC’s 510

remarkable role as a catalytic and regulatory subunit of the allosterically-desensitized 511



20

20

Class-2 PEPC heteromeric complex (O’Leary et al., 2009), and RcCDPK1’s Ca2+-512

dependent, inhibitory phosphorylation of the BTPC subunits at Ser-451. A major challenge 513

will be to link diverse post-translational PEPC controls with the in vivo regulation of PEP 514

partitioning to specific metabolic pathways. This now includes consideration of how 515

transient alterations in cytosolic Ca2+ signatures and PEP levels may serve to modulate 516

RcCDPK1 phosphorylation of BTPC at Ser-451 and thereby contributing to the control of 517

anaplerotic PEP flux and respiratory CO2 recycling by Class-2 PEPCs. Given the central 518

importance of protein phosphorylation as a post-translational regulatory mechanism, the 519

diversity of CDPKs in plants, and the lack of in vivo CDPK targets that have been 520

pinpointed to date, our RcCDPK1 and RcCDPK2 studies represent a significant advance 521

that helps to set the stage for future research aimed at exploring the interplay between 522

Ca2+ signaling and the integration and control of plant carbon metabolism. 523

524

MATERIALS AND METHODS 525

Plant Material, Protein Extraction, PEPC Activity Assays, and Protein Concentration 526

Determination 527

Castor (Ricinus communis L.; cv. Baker 296) plants were cultivated in a greenhouse as 528

previously described (O’Leary et al., 2011a). Tissues were rapidly harvested, frozen in 529

liquid N2 and stored at -80 °C until used. Tobacco (Nicotiana tabacum L.) BY-2 suspension-530

cultured cells were maintained as described by Park et al. (2012). 531

A homozygous Arabidopsis mutant line (atcpk11, SALK_054495) was kindly provided by 532

Prof. Jeffrey Harper (University of Nevada). Col-0 and atcpk11 seeds were sown in a 533

standard soil mixture (Sunshine Aggregate Plus Mix 1; SunGro, Vancouver, Canada) and 534

stratified at 4 oC for 3-d. Plants were cultivated in growth chambers at 23 °C (16/8 h 535

photoperiod at 100 µmol m-2 s-1 PAR) and fertilized biweekly by subirrigation with 0.25x 536

Hoagland’s media. Flowers (1 g) from 28-d-old plants were ground to a powder under liquid 537

N2 and homogenized (1:2; w/v) in 50 mM HEPES-KOH (pH 7.3) containing 1 mM EDTA, 1 538

mM EGTA, 10 mM MgCl2, 20% (v/v) glycerol, 5 mM thiourea, 1 mM DTT, 1% (w/v) 539



21

21

polyvinyl(polypyrrolidone), 1 mM 2,2’-dipyridyl disulfide, 1 mM phenylmethylsulfonyl 540

fluoride, 25 mM NaF, 1 mM Na3VO4, and 1 mM Na2MoO4. After centrifugation, the 541

supernatant fluid was subjected to non-radioactive BTPC Ser-451 kinase assays as 542

described below and in the legend for Fig. 4A. 543

PEPC activity was assayed at 25 °C by following NADH oxidation at 340 nm using a 544

Molecular Devices microplate spectrophotometer as previously described (O’Leary et al., 545

2009; Hill et al., 2014). Protein concentrations were routinely determined by Coomassie 546

Blue G-250 dye binding using bovine γ–globulin (Pierce) as the protein standard. 547

548

LC-MS/MS and Protein Identification 549

Proteins in the final BTPC Ser-451 kinase preparation of Hill and co-workers (2014) 550

were reduced with 10 mM DTT, alkylated with 55 mM iodoacetamide, dialyzed against 10 551

mM ammonium bicarbonate, and dried using a CentriVap centrifugal concentrator 552

(Labconco Corp.). Protein digestion was performed using sequencing-grade trypsin 553

(Promega) in 25 mM ammonium bicarbonate at 1:100 ratio of trypsin to protein substrates. 554

Tryptic peptides were dried and reconstituted with 4-µL of 0.1% formic acid, and identified 555

using on-line nanoAcquity UPLC (Waters) coupled with an Orbitrap Fusion Tribrid mass 556

spectrometer (Thermo Fisher Scientific Inc.). The peptides were trapped by a 2G-V/MT 557

Trap symmetry C18 column (5 µm particles, 180 µm i.d. x 20 mm length) for 3 min at a flow 558

rate of 5 µL/min, and separated on a BEH130 C18 analytical column (1.7 µm particles, 100 559

µm i.d. × 100 mm length) at 300 nL/min for 1 h. The mobile phase was set up to a linear 560

gradient from 5-30% solvent B (0.1% formic acid in acetonitrile) over 40 min for peptide 561

elution, followed by flushing with 85% solvent B for 15 min and re-equilibrating the column 562

with solvent A (0.1% formic acid) for 10 min. MS survey scan was acquired with a high 563

resolution of 60,000 at the mass region of m/z 350 – 1800, and MS/MS measurements 564

were performed by collision-induced dissociation (CID) at data-dependent acquisition mode 565

for scanning the top twenty most intense ions at multiply charged states of 2+ to 7+, 566

respectively. Dynamic exclusion was set to 60 s. MS/MS data were searched against the R. 567



22

22

communis protein sequence in the NCBI database using Mascot Server (version 2.5.1, 568

Matrix Science). The search parameters were restricted to tryptic peptides at a maximum of 569

2 missed cleavages. Cysteine carbamidomethylation was designated as a fixed 570

modification, and N-terminal protein acetylation, deamidation of asparagine and glutamine, 571

oxidation of methionine, and phosphorylation of Ser, Thr, and Tyr were considered as 572

variable modifications. Mass tolerances were set up to 10 ppm for Orbitrap MS ions and 573

0.8 Da for ion-trap MS/MS fragment ions. Peptide assignments were filtered by an ion 574

score cut off at 20, and the identified MS/MS spectra were verified manually. 575

576

qRT-PCR 577

Total RNA was extracted using an RNeasy® kit (QIAGEN) following the manufacturer’s 578

protocols. The DNase-treated RNA was used for cDNA synthesis with a QuantiTect reverse 579

transcription kit (QIAGEN). Quantitative PCR was performed using an Applied Biosystems 580

7500 Real-Time PCR system and GoTaq® qPCR Master Mix (Promega). The reaction 581

procedures were as follows: denature at 95 oC for 5 min, followed by 40 cycles of 95 oC for 582

15 s, 60 oC for 15 s, and 72 oC for 34 s. The castor Actin (AY360221) gene (RcActin) was 583

used as an internal control. Primers (Supplemental Table S3) were designed using 584

DNAMAN software (Version 5.0), and results were analyzed with Applied Biosystems 7500 585

Software Version 2.0.1. Relative gene expression was calculated using the relative 2–ΔΔCt 586

method (Livak and Schmittgen, 2001). All the experiments were repeated at least three 587

times using cDNAs prepared from two biological replicates. 588

589

RcCDPK1 Cloning and Heterologous Expression 590

Standard PCR amplification was used to introduce restriction sites for all sub-cloning 591

procedures, and all constructs were confirmed by DNA sequencing. Cloning of a cDNA 592

encoding RcCDPK1 was initiated by designing gene-specific primers (Supplemental Table 593

S2). A full-length cDNA library from stage V developing COS endosperm (Gennidakis et al., 594

2007) was used as the template to amplify a PCR product that was amplified and cloned 595



23

23

into pGEM®-T vector (Promega) for sequencing. Sequence assembling was performed 596

using DNAMAN (Version 5.0). Alignment of RcCDPK1’s deduced amino acid sequence 597

with several of its orthologs was performed using Vector NTI® software (Life Technologies) 598

with default parameters. 599

For heterologous expression of RcCDPK1, its full-length cDNA was subcloned into a 600

pET30a(+) expression vector (Novagen) carrying an N-terminal His6-tag. A second 601

construct encoding the His6-tagged N-terminally truncated RcCDPK1 (residues 30-497; 602

∆N-RcCDPK1) was also produced. For recombinant protein production, both constructs 603

were separately transformed into E. coli (BL21-CondonPlus (DE3)-RIL) (Stratagene), and 604

cultured overnight at 37 oC in 500 mL of LB broth containing 50 µg/mL kanamycin to an 605

A600 of ~0.6. Protein production was induced using 0.4 mM isopropyl-β-D-thiogalactoside 606

for 3 h at 37 oC. Cells (~5 gFW) were harvested by centrifugation, resuspended in 40-mL of 607

ice-cold buffer A (50 mM NaH2PO4, pH 8.0, containing 300 mM NaCl), and lysed at 4 oC by 608

passage through a French Pressure Cell at 18,000 p.s.i. After centrifugation the 609

supernatant fluid was loaded at 1 mL/min onto a column (1.6 x 10 cm) of PrepEase® His-610

tagged High Yield Purification Ni2+-affinity resin (Affymetrix) equilibrated in buffer A. The 611

column was washed with buffer A until the A280 approached baseline and then eluted with 612

buffer A containing 250 mM imidazole. Pooled peak fractions were concentrated to 1 mL 613

with an Amicon Ultra-15 centrifugal filter unit (30-kD cutoff), divided into 25-μL aliquots, 614

frozen in liquid N2 and stored in -80 oC. For cleaving the His6-tag, purified RcCDPK1 was 615

incubated with enterokinase (New England Biolabs) according to the manufacturer’s 616

recommendations. For the generation of GST-RcCDPK1 and GST-∆N-RcCDPK1 the 617

respective constructs were ligated into a pGEX-4T-3 vector (GE Healthcare). The proteins 618

were heterologously expressed in E. coli as described above and purified using PrepEase® 619

Protein Purification Glutathione Agarose 4B (Affymetrix) according to the manufacturer’s 620

instructions. Purified GST-RcCDPK1 and GST-∆N-RcCDPK1 were concentrated and 621

stored as described above. 622

623



24

24

RcCDPK1 Antibody Production and Immunoblotting 624

Rabbit antiserum against RcCDPK1 was produced using a synthetic peptide 625

(SynPeptide Co., Ltd.) designed to match amino acids 1-29 (i.e., the N-terminal variable 626

domain) (Supplemental Fig. S2A), with an additional Cys residue introduced at the N-627

terminus. Purified peptide was coupled to maleimide-activated keyhole limpet hemocyanin 628

(Life Technologies), dialyzed against Pi buffered saline (pH 7.4), filter-sterilized, and 629

emulsified with Titermax Gold (CytRx). Following collection of pre-immune serum, desalted 630

conjugate (750 μg) was injected subcutaneously into a rabbit, and a booster injection (250 631

μg) administered at 28 d. Two weeks after the final injection, blood was collected in 632

Vacutainer tubes (Becton Dickinson) by cardiac puncture. Clotted cells were removed by 633

centrifugation at 1,000 g, and the immune serum frozen in liquid N2 and stored at -80 oC in 634

0.04% (w/v) NaN3. For immunoblotting, anti-RcCDPK1 was affinity-purified against 500 μg 635

of nitrocellulose-bound recombinant RcCDPK1, as described previously (Dalziel et al., 636

2012). Production of anti-PTPC, anti-BTPC and anti-pSer451, SDS-PAGE, immunoblotting, 637

and chromogenic detection of immunoreactive polypeptides were carried out as previously 638

described (Gennidakis et al., 2007; O’Leary et al., 2009; Dalziel et al., 2012). All 639

immunoblots were replicated at least three times with representative results shown in the 640

figures. 641

642

In Vitro Kinase Assays 643

Kinase activity was assayed by monitoring P incorporation from non-radioactive or [γ-644

32P]-labelled ATP into the p118 BTPC subunits of purified, heterologously expressed Class-645

2 PEPC as previously described (Hill et al., 2014). Recombinant CDPKs (250 ng) were 646

routinely incubated with 10 μg of the Class-2 PEPC substrate (corresponding to 5 μg 647

p118/BTPC subunits) in a 25 μL reaction mix containing 50 mM HEPES-KOH (pH 7.3), 10 648

mM MgCl2, 1 mM DTT, 0.1 mM Na3VO4, 0.1 mM Na2MoO4, 10% (v/v) glycerol, and 0.2 mM 649

CaCl2. Reactions were initiated by the addition of 0.2 mM ATP or [γ-32P]ATP (1000 650

cpm/pmol), incubated at 30 °C for up to 20 min, and terminated by addition of SDS-PAGE 651



25

25

sample buffer followed by heating at 100 oC for 3 min. For non-radioactive kinase assays, P 652

incorporation from ATP into p118 was determined by subjecting 10-μL aliquots of kinase 653

assays to SDS-PAGE and immunoblotting with anti-pSer451 and anti-BTPC. 654

For radiometric assays, the level of 32P incorporation from [γ-32P]ATP into p118 was 655

visualized by subjecting 10-μL aliquots of kinase assays to SDS-PAGE and developing 656

Coomassie Blue R-250 stained gels overnight in a phosphorimager cassette (Molecular 657

Dynamics, Piscataway NJ, USA) followed by scanning of the cassette using a Typhoon 658

6000 (GE Healthcare). 32P incorporation was quantified by liquid scintillation counting 659

following H2O2 digestion of excised SDS gel slices containing Coomassie blue R-250 660

stained p118 as previously described (Hill et al., 2014). 661

662

Autophosphorylation Assays 663

RcCDPK1 and ∆N-RcCDPK1 (2 µg each) were separately incubated at 30 oC for 30 min 664

with 0.2 mM [γ-32P]ATP (1000 cpm/pmol) in the following autophosphorylation assay buffer: 665

25 mM HEPES-KOH, pH 7.3, containing 10 mM MgCl2 and 0.2 mM CaCl2 or 5 mM EGTA 666

in a final volume of 10 μL. Reactions were terminated by addition of SDS-PAGE sample 667

buffer and heating at 100 oC for 3 min. The samples were subjected to SDS-PAGE and 668

phosphorimaging as described above. 669

670

Biophysical Studies 671

Isothermal scanning calorimetry was performed at 30 oC on a VP-ITC Microcalorimeter 672

(MicroCal). RcCDPK1 (10 mg in 1.5 mL) was further purified by gel filtration at 0.3 mL/min 673

on a Superdex 200 HiLoad 16/60 column (GE Healthcare) equilibrated with 25 mM 674

HEPES-KOH (pH 7.5) containing 100 mM NaCl, 2 mM EDTA, and 2 mM EGTA. Pooled 675

peak fractions were concentrated to 1 mL using an Amicon Ultra-15 concentrator (30-kD 676

cutoff) and dialyzed against 25 mM HEPES-KOH (pH 7.5) containing 100 mM NaCl and 10 677

mM MgCl2. RcCDPK1 (20 µM) and 10-μL injections (29 injections at 360 s intervals) of 500 678

μM CaCl2 were used in each experiment (with appropriate buffer blanks). Origin 7.0 679



26

26

software (MicroCal) was used to obtain values for stoichiometry (N) and dissociation 680

constants (Kd) and binding-type input parameters were adjusted to obtain the best fitting 681

model for each experiment. 682

Fluorescence spectroscopy was performed using 25 µM RcCDPK1 and 250 μM 8-683

anilinonaphthalene-1-sulfonic acid in 10 mM Tris-HCl (pH 7.5) containing 100 mM KCl and 684

1 mM DTT (with various additions described in Fig. 7B). RcCDPK1’s fluorescence emission 685

spectra were recorded at 23 oC using black Fluotrac-200 96-well microplates on a 686

Spectramax Gemini XS Spectrofluorometer. Petunia CaM81 (15 µM) (Bender et al., 2014) 687

was monitored alongside RcCDPK1 as a positive control for Ca2+-dependent hydrophobic 688

exposure. 689

Far-UV circular dichroism spectra from 182-260 nm were acquired at 23 oC on a 690

Chirascan CD spectrometer using a cylindrical quartz cuvette with a pathlength of 0.1 mm. 691

Samples of 15 μM RcCDPK1 were used for data acquisition as described in Fig. 7C. 692

Spectra from a minimum of 10 replicate scans were averaged and corrected for 693

background. 694

695

Transient Transformation and Imaging of Tobacco BY-2 Suspension Cells 696

Full-length and a 5’-truncated version (corresponding to ΔN-RcCDPK1) of the RcCDPK1 697

cDNA sequence (with or without a stop codon) were amplified via PCR with the appropriate 698

primers (Supplemental Table S2) from pET30a-RcCDPK1 (see above). Resulting DNA 699

fragments were gel-purified and subcloned into pRTL2-Cherry using XmaI and NheI to 700

yield RcCDPK1-Cherry and ΔN-RcCDPK1-Cherry. To generate Cherry-RcCDPK1 and 701

Cherry-ΔN-RcCDPK1, purified fragments were subcloned into pRTL2-Cherry (a transient 702

expression vector containing the 35S cauliflower mosaic virus promoter and the open 703

reading frame of the red fluorescent protein Cherry; Gidda et al., 2011) using BamHI. To 704

construct RcCDPK1-GFP, pGreen35S C-GFP and an RcCDPK1 PCR fragment were 705

digested with BamHI and EcoRV, gel purified, and then ligated. Mito-EYFP was 706

constructed by amplifying the full-length open reading frame encoding the mitochondrial 707



27

27

pyruvate dehydrogenase complex E1-α subunit from a developing COS cDNA library 708

(Gennidakis et al., 2007) using the appropriate oligonucleotide primers (Supplemental 709

Table S2). Amplified products were digested with NcoI and EcoRI and ligated into the 710

corresponding sites in pSAT6-EYFP-C1 (Tzfira et al., 2005). Construction of the BTPC-711

EYFP plasmid was previously reported (Park et al., 2012). All constructs were confirmed by 712

DNA sequencing. 713

Transient (co)transformations of tobacco BY-2 suspension-cultured cells was performed 714

with 2-5 μg of plasmid DNA using a biolistic particle delivery system (Bio-Rad) as described 715

previously (Park et al., 2012). Bombarded cells were incubated for ~6 h to allow for gene 716

expression and protein sorting, fixed in 4% (w/v) formaldehyde, and then either processed 717

for immunostaining or imaged directly using a Leica SP2 CLSM at the Molecular and 718

Cellular Imaging Facility (University of Guelph) as previously described (Park et al., 2012). 719

Primary and secondary antibodies used were rabbit anti-Arabidopsis cytochrome oxidase c 720

subunit II (CoxII) affinity-peptide purified IgGs (Cedarlane Labs) and goat anti-rabbit 721

rhodamine red-X conjugated IgGs (Jackson Immunoresearch Laboratories), respectively. 722

Fluorophore emissions were collected sequentially in all fluorescent protein fusion co-723

expression experiments; single-labeling experiments shown no detectable crossover (i.e., 724

bleed through) at the settings used for data collection. False colorizations of images (i.e., 725

EYFP to green and Cherry to magenta) and merges were generated using ImageJ 726

(http://imagej.nih.gov/ij/), and figure compositions were generated using Adobe Photoshop 727

CS6 and/or Illustrator CS6 (Adobe Systems). All micrographs shown are representative 728

images obtained from at least three independent experiments. 729

730

GST Pull-Down Assays 731

Purified GST-RcCDPK1 or GST-∆N-RcCDPK1 (2 μg each) was incubated with bait 732

proteins (3 μg each) and glutathione agarose beads (40-µL) (Affymetrix) in a binding buffer 733

(25 mM HEPES-KOH (pH 7.0), 100 mM NaCl, 0.05% (v/v) β-mercaptoethanol, 0.1% (v/v) 734

Triton X-100, and 0.1 mM CaCl2 or 2 mM EGTA) for 2 h at 4 °C with end-over-end mixing. 735



28

28

The beads were washed three times with binding buffer, and bound proteins eluted using 736

40-µL of 50 mM Tris-HCl (pH 8.0) containing 20 mM reduced glutathione, and analyzed by 737

SDS-PAGE and immunoblotting as described above. 738

739

Accession Numbers 740

Sequence data from this article can be found in the GenBank/EMBL databases under 741

the following accession numbers: NM_117025 (AtCPK4), NM_103271 (AtCPK11), 742

NM_001248700 (GmCDPKβ), NM_001072396 (OsCPK24), XM_002526769 (RcCDPK1), 743

NM_001112282 (ZmCPK11). 744

745

Supplemental Data 746

The following materials are available in the online version of this article. 747

Supplemental Figure S1. Model illustrating the biochemical complexity of castor bean 748

PEPC. 749

Supplemental Figure S2. Dot-blot assessment of affinity-purified antibodies raised 750

against a synthetic peptide matching RcCDPK1’s N-terminal variable domain. 751

Supplemental Figure S3. RcCDPK1 or ∆N-RcCDPK1 cannot phosphorylate a pair of 752

synthetic peptides containing residues flanking BTPC’s Ser-451 phosphorylation 753

site. 754

Supplemental Figure S4. Phenyl-Sepharose elution profile for native BTPC Ser-451 755

kinase activity from developing COS. 756

Supplemental Figure S5. Various fluorescent protein-tagged versions of RcCDPK1 757

and ∆N-RcCDPK1 localize to the cytosol and nucleus in tobacco BY-2 cells. 758

Supplemental Table S1. Proteins reliably identified by nanoHPLC-MS/MS analysis of 759

a tryptic digest of the final preparation of partially-purified native BTPC Ser-451 760

kinase from developing COS (Hill et al., 2014). 761

Supplemental Table S2. Primers used for cloning and PCR. 762



29

29

763

ACKNOWLEDGMENTS 764

We are grateful to Prof. Steven Huber (Univ. of Illinois) for helpful discussions as well as 765

the gift of heterologous expression plasmids for AtCPK4, AtCPK34, and GmCDPKβ. We 766

are also indebted to Prof. Joonho Park (Seoul National Univ. of Science and Technology) 767

for preparation of the Mito-EYFP construct, Mr. Kim Munro (Queen’s Protein Function 768

Discovery Facility) for assisting with isothermal titration calorimetry and circular dichroism 769

spectroscopy, Ms. Deni Ogunrinde (Dept. of Biomedical and Molecular Sciences, Queen’s 770

Univ.) for assisting with fluorescence spectroscopy, Prof. Jeffrey Harper (Univ. of Nevada) 771

for the gift of atcpk11 knockout seeds, and Prof. Greg Moorhead (Univ. of Calgary) for 772

helpful discussions and encouragement. 773



30

30

FIGURE LEGENDS 774

Figure 1. Alignment of tryptic peptides derived from purified castor bean BTPC Ser-451 775

kinase with the deduced sequence of RcCDPK1 and several orthologs from other plants. 776

LC-MS/MS analysis of the final BTPC Ser-451 kinase preparation of Hill and co-workers 777

(2014) resulted in 9 peptide sequences unique to RcCDPK1 that are underlined with a solid 778

gray line. The protein kinase catalytic domain and 4 EF hand (Ca2+-binding) motifs are 779

overlined with dashed and solid black lines, respectively. The conserved phosphomimetic 780

Asp (D) residue that occurs within the activation loop of RcCDPK1 and its orthologs is 781

highlighted with a bold font (i.e. Asp-190 in the case of RcCDPK1). Black shading indicates 782

identical residues, whereas gray shading denotes residues conserved amongst the six 783

proteins. Gaps, indicated by dashes, were introduced to maximize alignment. Inset: 784

schematic diagram of RcCDPK1 functional domains (NTVD, N-terminal variable domain; 785

AIJ, auto-inhibitory junction; CTVD, C-terminal variable domain). 786

787

Figure 2. Analysis of RcCDPK1 expression. Levels of mRNA were analyzed by qRT-PCR 788

using gene-specific primers. Castor Actin (AY360221) was used as the internal control for 789

normalization. A, Tissue-specific expression of RcCDPK1. Developing and germinating 790

seed tissues were respectively harvested from stage VII and 5 d post-imbibition COS, male 791

flowers were harvested at maturity, whereas female flowers were harvested at 5 d post-792

anthesis (corresponding to proembryo or stage I COS). Abbreviations are: E, endosperm; 793

C, cotyledon; MF, male flower; Int. and P, integument and pericarp of female flowers, 794

respectively; H, hypocotyl; RM, root middle; RT, root tip; Bud, leaf bud; Exp, expanding leaf; 795

Mat, mature leaf; dC and dE, cotyledon and endosperm, respectively, from stage VII 796

developing COS that had been depodded for 48 h. B, The profile of RcCDPK1 transcripts 797

was compared with that of BTPC (RcPPC4; EF634318) in endosperm of developing COS. 798

Stages III, V, VII, IX correspond to heart-shaped embryo, mid-cotyledon, full cotyledon, and 799

maturation stages of endosperm development, respectively; the lane labelled ‘Dry’ 800

designates fully mature COS; n.d., not detected. All values in panels A and B represent 801

means ±SEM of n = 4 determinations using cDNAs prepared from two biological replicates. 802

803



31

31

Figure 3. SDS-PAGE and immunoblot analysis of RcCDPK1 and ∆N-RcCDPK1. 804

A, SDS-PAGE of 2.5 μg each of the final His6-tagged RcCDPK1 and ∆N-RcCDPK1 805

preparations was performed following the addition of 2 mM CaCl2 or 2 mM EGTA to the 806

samples as indicated. The gel was stained with Coomassie Brilliant Blue R-250. 807

B, Immunoblotting following SDS-PAGE was performed with a 1:50 dilution of affinity-808

purified anti-RcCDPK1 peptide antibodies. Protein loading was: His6-RcCDPK1 and His6-809

∆N-RcCDPK1 (50 ng each); ‘RcCDPK1’ represents 75 ng of recombinant RcCDPK1 after 810

incubation with enterokinase to remove its His6-tag; ‘COS Phenyl Sepharose Eluate’ 811

represents 20 µg of BTPC Ser-451 kinase enriched extract from Stage V-VII developing 812

COS (prepared as described in legend for Supplemental Fig. S4); whereas ‘COS BTPC-K’ 813

represents 2 µg of the final preparation of BTPC Ser-451 kinase purified by Hill et al. 814

(2014). CaCl2 (0.2 mM) was added to all samples prior to SDS-PAGE and immunoblotting. 815

‘M’ denotes various protein molecular mass standards in panels A and B. 816

817

Figure 4. Calcium-dependent phosphorylation of castor BTPC at Ser-451. A and B, 818

Recombinant Class-2 PEPC (10 µg) containing an equivalent ratio of castor BTPC 819

(RcPPC4) and Arabidopsis PTPC (AtPPC3) subunits (O’Leary et al., 2009) was incubated 820

for 20 min at 30 oC in 25-μL of the standard phosphorylation assay mix containing 0.1 mM 821

ATP, 10 mM MgCl2, and 0.2 mM CaCl2 (+) or 2 mM EGTA (-) and (A) various CDPKs (250 822

ng each), or (B) soluble protein extracts (25 µg each) from flowers of 28-d old Arabidopsis 823

Col-0 or mutant atcpk11 knockout plants. (C) RcCDPK1 was assayed as above in the 824

presence of 0.2 mM CaCl2 and the indicated PEP concentrations. Following SDS-PAGE, 825

the gels were subjected to immunoblotting with anti-pSer451 or anti-BTPC as indicated. 826

Results of panels A-C are representative of three independent experiments. 827

828

Figure 5. Substrate specificity of RcCDPK1. Purified recombinant Class-2 PEPC 829

containing S451D mutant or wild-type BTPC subunits (O’Leary et al., 2009; Dalziel et al., 830

2012), in vitro dephosphorylated COS Class-1 PEPC or RcSUS1 (Tripodi et al., 2005; 831

Fedosejevs et al., 2014), or histone III-S from calf thymus (10 μg) were incubated for 10 832

min at 30 °C in 25-μL of the standard [γ-32P]ATP phosphorylation assay mix containing 0.2 833



32

32

mM CaCl2 and 250 ng of RcCDPK1. Following SDS-PAGE (2 μg protein per lane), the gel 834

was subjected to (A) phosphorimaging and (B) stained with Coomassie Blue R-250 (CBB-835

250). Results are representative of three independent experiments. 836

837

Figure 6. The N-terminal variable domain is required for optimal RcCDPK1 activity. 838

A, The indicated amounts of RcCDPK1 and ∆N-RcCDPK1 were incubated with 20 μg of 839

recombinant Class-2 PEPC for 10 min at 30 °C in the standard [γ-32P]ATP phosphorylation 840

assay mix containing 0.2 mM CaCl2. Following SDS-PAGE and phosphorimaging, 32P 841

incorporation into BTPC subunits was quantified by digestion of excised Coomassie blue 842

R-250 stained p118 bands from SDS gels with H2O2, and liquid scintillation counting. 843

B, RcCDPK1 and ∆N-RcCDPK1 (2 µg each) were incubated for 30 min at 30 °C in 10 µL of 844

the standard [γ-32P]ATP phosphorylation assay mix containing 0.2 mM CaCl2 (+) or 2 mM 845

EGTA (-), but lacking an exogenous substrate. Following SDS-PAGE, autophosphorylation 846

activity was visualized by phosphorimaging. 32P incorporation was quantified by digestion 847

of excised protein-stained RcCDPK1 and ∆N-RcCDPK1 polypeptides from SDS gels with 848

H2O2, and liquid scintillation counting. Results of panels A and B are representative of three 849

independent experiments. 850

851

Figure 7. Biophysical studies of RcCDPK1. A, Analysis of Ca2+ binding to RcCDPK1 by 852

isothermal titration calorimetry. A representative data curve of the RcCDPK1-Ca2+ 853

interaction at 30 oC in 25 mM HEPES-KOH (pH 7.5), 100 mM NaCl, and 10 mM MgCl2 is 854

presented. The top panel shows the calorimetric titration of 2.0 mL of 20 μM RcCDPK1 with 855

500 μM CaCl2 using 29 injections of 10-μL each. The lower panel presents the 856

corresponding integrated binding isotherm modelled to two sets of binding sites. B, 8-857

Anilinonaphthalene-1-sulfonic acid fluorescence emission spectra from 430 - 600 nm were 858

collected to examine the exposed hydrophobicity of 25 μM RcCDPK1 versus petunia 859

CaM81 in 25 mM HEPES-KOH (pH 7.5) containing 100 mM KCl and 1 mM DTT, with 860

various additions as shown. Background fluorescence in the absence of RcCDPK1 or 861

CaM81 is also shown. C, Far-UV circular dichroism spectroscopy was used to study the 862

impact of Ca2+ addition on the secondary structure characteristics of RcCDPK1 in 5 mM 863



33

33

Tris-HCl or HEPES-KOH (pH 7.5). Each spectrum is representative of at least 10 averaged 864

scans. 865

866

Figure 8. Relocalization of RcCDPK1 and ΔN-RcCPDK1 by castor BTPC to the 867

mitochondria of tobacco BY-2 cells. A, Schematic illustration of fluorescent protein fusion 868

constructs of wild-type and mutant versions of RcCDPK1 and castor BTPC that were 869

transiently expressed in tobacco BY-2 cells. BTPC’s intrinsically disordered region is 870

highlighted in blue. The numbers above each construct indicate amino acid residue 871

positions. B-I, Representative CLSM micrographs of individual tobacco BY-2 cells 872

(co)expressing (as indicated by panel labels) the following: (B) RcCDPK1-Cherry, (C) ∆N-873

RcCDPK1-Cherry, (D) Cherry, (E) RcCDPK1-Cherry and p118-EYFP, (F) ∆N-RcCDPK1-874

Cherry and p118-EYFP, (G) RcCDPK1-Cherry and p54-EYFP, (H) RcCDPK1-Cherry and 875

p64-EYFP, and (I) RcCDPK1-Cherry and p40-EYFP. Note that the fluorescence 876

attributable to RcCDPK1 and p118 (and mutant versions thereof) was false colourized 877

magenta and green, respectively. Shown in (B-D) are the corresponding differential 878

interference contrast (DIC) images. Corresponding merged images are presented in (E) 879

and (F); white colour represents protein co-localization. Arrowheads and boxes 880

representing portions of the cells shown at higher magnification in the insets in (E) and (F) 881

indicate obvious examples of co-localization of RcCDPK1-Cherry or ΔN-RcCDPK1-Cherry 882

with p118-EYFP at (aggregated) mitochondria. Scale bar = 10 μm. 883

884

Figure 9. In vitro interaction of GST-RcCDPK1 or GST-∆N-RcCDPK1 with BTPC subunits 885

of recombinant Class-2 PEPC is Ca2+-dependent. GST, GST-RcCDPK1, and GST-∆N-886

RcCDPK1 (2 μg each) were respectively immobilized on glutathione beads and incubated 887

with pure recombinant Class-1 and/or Class-2 PEPCs (5 μg each) (O’Leary et al., 2009) in 888

the presence of 0.5 mM CaCl2 (+) or 2 mM EGTA (-) as indicated. Bound proteins were 889

eluted with a buffer containing GSH and analyzed via SDS-PAGE (2.5 µg protein/lane) and: 890

(A) protein staining with Coomassie Brilliant Blue R-250, or (B) immunoblotting with anti-891

PTPC or anti-BTPC. 892

893



34

34

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abscisic acid signal transduction in Arabidopsis. Plant Cell 19: 3019–3036 1048

1049



AtCPK11 (1) ------------------METKPNPRRPS-----NTVLPYQTPRLRDHYLLGKKLGQGQFGTTYLCTEKSTSANYACKSIPKRKLVCREDYEDVWREIQI AtCPK4 (1) -------------------MEKPNPRRPS-----NSVLPYETPRLRDHYLLGKKLGQGQFGTTYLCTEKSSSANYACKSIPKRKLVCREDYEDVWREIQI

OsCPK24 (1) MQPDPSGSGGDGNANAKAKLAPPPVTAAG--GRPVSVLPHKTANVRDHYRIGKKLGQGQFGTTYLCVDKASGGEFACKSIPKRKLLCREDYEDVWREIQI ZmCPK11 (1) MQPDPSG-----NANAKTKLPQLVTAPAPSSGRPASVLPYKTANVRDHYRIGKKLGQGQFGTTYQCVGKADGAEYACKSIPKRKLLCREDYEDVYREIQI GmCDPKβ (1) -------------------MQKHGFASK------RNVLPYQTARLRDHYVLGKKLGQGQFGTTYLCTHKVTGKLYACKSIPKRKLMCQEDYDDVWREIQI RcCDPK1 (1) -------------------MKKQSAGGSSTTKPAHTVLPYQTSRLRDHYLIGKKLGQGQFGTTYLCTNKATNAQYACKSIPKRKLLCKEDYEDVWREIQI

AtCPK11 (78) MHHLSEHPNVVRIKGTYEDSVFVHIVMEVCEGGELFDRIVSKGHFSEREAVKLIKTILGVVEACHSLGVMHRDLKPENFLFDSPKDDAKLKATDFGLSVF AtCPK4 (77) MHHLSEHPNVVRIKGTYEDSVFVHIVMEVCEGGELFDRIVSKGCFSEREAAKLIKTILGVVEACHSLGVMHRDLKPENFLFDSPSDDAKLKATDFGLSVF

OsCPK24 (99) MHHLSEHPNVVRIRGAYEDALFVHIVMELCAGGELFDRIVAKGHYTERAAAQLIRTIVAVVEGCHSLGVMHRDLKPENFLFASAAEDAPLKATDFGLSMF ZmCPK11 (96) MHHLSEHPNVVRIRGAYEDALFVHIVMELCAGGELFDRIVAKGHYSERAAAKLIKTIVGVVEGCHSLGVMHRDLKPENFLFASTAEEAPLKATDFGLSMF GmCDPKβ (76) MHHLSEHPNVVQIQGTYEDSVFVHLVMELCAGGELFDRIIQKGHYSEREA LIKTIVGVVEACHSLGVMHRDLKPENFLFDTPGEDAQMKATDFGLSVI RcCDPK1 (82) MHHLSEHPNVVQIKGTYEDSMFVHLVMELCAGGELFDRIVAKGQYSEKEAAKLIKTIVGVVEACHSLGVMHRDLKPENFLFDTPGDDAKLKATDFGLSVF

AtCPK11 (178) YKPGQYLYDVVGSPYYVAPEVLKKCYGPEIDVWSAGVILYILLSGVPPFWAETESGIFRQILQGKLDFKSDPWPTISEAAKDLIYKMLERSPKKRISAHE AtCPK4 (177) YKPGQYLYDVVGSPYYVAPEVLKKCYGPEIDVWSAGVILYILLSGVPPFWAETESGIFRQILQGKIDFKSDPWPTISEGAKDLIYKMLDRSPKKRISAHE

OsCPK24 (199) YKPGDKFSDVVGSPYYVAPEVLQKCYGPESDVWSAGVILYILLCGVPPFWAETEAGIFRQILRGKLDFESEPWPSISDSAKDLVRNMLCRDPTKRLTAHE ZmCPK11 (196) YKPGDKFSDVVGSPYYVAPEVLQKCYGPEADVWSAGVILYILLCGVPPFWAETEAGIFRQILRGKLDFESEPWPSISDSAKDLVCNMLTRDPKKRFSAHE GmCDPKβ (176) LQARQAFHDVVGSPYYVAPEVLCKQYGPEVDVWSAGVILYILLSGVPPFWAETEAGIFRQILNGDLDFVSEPWPSISENAKELVKQMLDRDPKKRISAHE RcCDPK1 (182) YKPGQYFSDVVGSPYYVAPEVLLKRYGPEVDVWSAGVILYILLSGVPPFWAETESGIFRHILQGKIDFESEPWPKISDSAKDLIKKMLERDPRQRISAHE

AtCPK11 (278) ALCHPWIVDEQAAPDKPLDPAVLSRLKQFSQMNKIKKMALRVIAERLSEEEIGGLKELFKMIDTDNSGTITFEELKAGLKRVGSELMESEIKSLMDAADI AtCPK4 (277) ALCHPWIVDEHAAPDKPLDPAVLSRLKQFSQMNKIKKMALRVIAERLSEEEIGGLKELFKMIDTDNSGTITFEELKAGLKRVGSELMESEIKSLMDAADI

OsCPK24 (299) VLCHPWIVDDAVAPDKPIDSAVLSRLKHFSAMNKLKKMALRVIAESLSEEEIGGLKELFKMIDTDDSGTITFDELKEGLKRVGSELTEHEIQALMEAADI ZmCPK11 (296) VLCHAWIVDDAVAPDKPIDSAVLSRLKHFSAMNKLKKMALRVIAESLSEEEIGGLKELFKMIDTDSSGTITFDELKDGLKRVGSELTENEIQALMEAADI GmCDPKβ (276) VLCNPWVVDD-IAPDKPLDSAVLTRLKHFSAMNKLKKMALRVIAERLSEEEIGGLKELFKMIDTDNSGTITFEELKEGLKSVGSNLMESEIKSLMEAADI RcCDPK1 (282) VLCHPWIVDDTVAPDKPLDSAVLSRLKKFSAMHKLKKMALRVIAERLSEEEIGGLKELFKMLDTDSSGTITFEELKEGLLRVGSELMECEIKALMEAADI

AtCPK11 (378) DNSGTIDYGEFLAATLHMNKMEREENLVAAFSYFDKDGSGYITIDELQSACTEFGLCDTPLDDMIKEIDLDNDGKIDFSEFTAMMRKGDG-VGRSRTMMK AtCPK4 (377) DNSGTIDYGEFLAATLHINKMEREENLVVAFSYFDKDGSGYITIDELQQACTEFGLCDTPLDDMIKEIDLDNDGKIDFSEFTAMMKKGDG-VGRSRTMRN

OsCPK24 (399) DNSGTIDYGEFIAATLHMNKLEREENLVSAFSFFDKDGSGFITIDELSQACREFGLDDLHLEDMIKDVDQNNDGQIDYSEFTAMMRKGNAGGAGRRTMRN ZmCPK11 (396) DNSGTIDYGEFIAATLHMNKLEREENLVSAFSFFDKDGSGFITIDELSQACREFGLDDLHLEDMIKDVDQNNDGQIDYSEFTAMMRKGNAGATGRRTMRN GmCDPKβ (375) DNNGSIDYGEFLAATLHLNKMEREENLVAAFAYFDKDGSGYITIDELQQACKDFSLGDVHLDEMIKEIDQDNDGRIDYAEFAAMMKKGDPNMGRSRTMKG RcCDPK1 (382) DNSGTIDYGEFLAATLHLNKMEREENLLAAFSYFDKDGSGYITVDELQQACKDFGLDDVHLDEMIKEIDEDNDGRIDYAEFTSMMRKGDEDIGRSRTMRS

AtCPK11 (477) NLNFNIADAFGVDGE----KSDD--- AtCPK4 (476) NLNFNIAEAFGVEDTSSTAKSDDSPK OsCPK24 (499) SLQLNLGEILNPSNS----------- ZmCPK11 (496) SLHLNLGELLNPSKT----------- GmCDPKβ (475) NLNFNIADAFGMKDSS---------- RcCDPK1 (482) HLNFNLADALGVKDLN----------

Mr (kD) Identity (%)55.9 8456.4 83 56.6 7956.6 7855.2 84 56.0 100

NH2 COOH1 30 288 331 472 495

Kinase Domain AIJ

NTVD

Ca2+-binding (EF hand)

Domain CTVD

RcCDPK1

∆

EF Hand

EF Hand

EF Hand EF Hand

AUTO-INHIBITORY JUNCTION CALMODULIN-LIKE DOMAIN

KINASE DOMAIN

N-TERMINAL VARIABLE DOMAIN

C-TERMINAL VARIABLE DOMAIN

FIGURE 1



0

1

2

3

4

5

6

7

8

9

Rela

tive

Tran

scrip

t Lev

el

E dE C dC BudExp Mat E C H RM RT MF Int PDeveloping

SeedLeaf Germinating

SeedlingFlower

A

B

0

1

2

3

Ⅲ Ⅴ Ⅶ Ⅸ

RcCDPK1 BTPC

Castor Bean Developmental StageDry

n.d. n.d.Rela

tive

Tran

scrip

t Lev

el

FIGURE 2



250150

100

75

25

37

50

A BHis6-RcCDPK1 His6-∆N-RcCDPK1+Ca2+ M +Ca2+-Ca2+ -Ca2+

60

M

25015010075

25

37

5060

20

His 6-RcCDPK1

His 6-∆N

-RcCDPK1

RcCDPK1

COS BTPC-K

(final p

rep)

COS Phenyl

Sepharose Eluate

kD kD

FIGURE 3



Ca2+: + − + − + − + − + −RcCDPK1 AtCPK4 GmCDPK-βAtCPK34ΔN-RcCDPK1

Anti-pSer451p118

Anti-BTPCp118

[PEP] (mM) 0 1.0 2.5 5.0

p118

p118

Anti-pSer451

Anti-BTPC

A

B

Anti-pSer451

Anti-BTPC

Col-0 atcpk11

p118

p118

Ca2+: + − +

C

FIGURE 4



150

100

50

75

37

25

Class-2

PEPC (W

T)

Class-2

PEPC (S

451D

)Hist

one Ⅲ

-SCOS C

lass-1

PEPC

RcSUS1

SDS/PAGECBB-250

SDS/PAGEPhosphorimager

B

A

p118 (BTPC)p107 (PTPC)

p118 (BTPC) 150

100

50

75

37

25

p93 (RcSUS1)

kD

FIGURE 5



p118

RcCDPK1 ΔN-RcCDPK1

Incorporationof 32P (cpm): 720 1188 2392 4771 6950 - - - 122 333

Amount of kinase (ng): 62.5 125 250 500 1000 62.5 125 250 500 1000

A

Ca2+: + − + − RcCDPK1 ΔN-RcCDPK1

60kD

Incorporationof 32P (cpm): 1170 658 175 213

B

FIGURE 6



0

500

1000

1500

2000

2500

3000

3500

Fluo

resc

ence

(IAU

)

CaM81

1 µM CaCl2

10 µM CaCl2

0

100

200

300

400

500

600

700

Fluo

resc

ence

(IAU

)

RcCDPK11 mM EDTA + 1mM EGTA

1 mM MgCl2

1 mM CaCl2

10 mM MgCl2 + 1 mM CaCl2

CaCl2added to EGTA wells

Wavelength (nm)430 455 480 505 530 555 580

50 µM CaCl2

ANS alone

1 mM CaCl2

B

-15

-10

-5

0

5

10

15

180 190 200 210 220 230 240 250 260

Mill

ideg

rees

(deg

10-3

cm

2 dm

ol-1)

Wavelength (nm)

C

Tris, 1 mM CaCl2

Tris, 2.5 mM CaCl2

Tris, 0.5 mM EGTA

Hepes, 1 mM CaCl2

AFIGURE 7



A

EYFP

EYFPp40

p541 325 446

1EYFPp64

447 467 1052

329

RcCDPK11 495

mCherry

∆N-RcCDPK130 495

mCherry

p1181 1052325 467

EYFP

B C DRcCDPK1-Cherry DIC DN-RcCDPK1-Cherry DIC Cherry DIC

ERcCDPK1-Cherry p118-EYFP merge

FDN-RcCDPK1-Cherry p118-EYFP merge

GRcCDPK1-Cherry p54-EYFP

HRcCDPK1-Cherry p64-EYFP

IRcCDPK1-Cherry p40-EYFP

FIGURE 8



Anti-PTPC

GST

GST-RcC

DPK1 GST-R

cCDPK1

GST-ΔN-R

cCDPK1

GST-RcC

DPK1

GST-ΔN-R

cCDPK1

+ + − + − +

Anti-BTPC p118 (BTPC)

p107 (PTPC)

A

B

Class-2 PEPC

Class-1PEPC

kD

p118 (BTPC)p107 (PTPC)

250-150-100-75-

50-

37-

25-

20-

MCa2+:

FIGURE 9



Parsed CitationsAlmadanim MC, Alexandre BM, Rosa MTG, Sapeta H, Leitão AE, Ramalho JC, Lam TT, Negrão S, Abreu IA, Oliveira MM (2017) Ricecalcium-dependent protein kinase OsCPK17 targets plasma membrane intrinsic protein and sucrose phosphate synthase and isrequired for a proper cold stress response. Plant Cell Environ. doi: 10.1111/pce.12916

Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Asai S, Ichikawa T, Nomura H, Kobayashi M, Kamiyoshihara Y, Mori H, Kadota Y, Zipfel C, Jones JDG, Yoshioka H (2013) Thevariable domain of a plant calcium-dependent protein kinase (CDPK) confers subcellular localization and substrate recognition forNADPH oxidase. J Biol Chem 288: 14332-14340


Bachmann M, Shiraishi N, Campbell WH, Yoo BC, Harmon AC, Huber SC (1996) Identification of Ser-543 as the major regulatoryphosphorylation site in spinach leaf nitrate reductase. Plant Cell 8: 505-517


Bender KW, Dobney S, Ogunrinde A, Chiasson D, Mullen RT, Teresinski HJ, Singh P, Munro K, Smith SP, Snedden WA (2014) Thecalmodulin-like protein CML43 functions as a salicylic-acid-inducible root-specific Ca2+ sensor in Arabidopsis. Biochem J 457: 127-136


Blonde JD, Plaxton WC (2003) Structural and kinetic properties of high and low molecular mass phosphoenolpyruvate carboxylaseisoforms from the endosperm of developing castor oilseeds. J Biol Chem 278: 11867-11873


Boudsocq M, Droillard M-J, Regad L, Lauriere C (2012) Characterization of Arabidopsis calcium-dependent protein kinases:activated or not by calcium? Biochem J 447: 291-299


Boudsocq M, Sheen J (2013) CDPKs in immune and stress signaling. Trends Plant Sci 18: 30-40Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Brandizzi F, Irons S, Kearns A, Hawes C (2003) BY-2 cells: culture and transformation for live cell imaging. Curr Protoc Cell Biol 1-7Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Choi HI, Park HJ, Park JH, Kim S, Im MY, Seo HH, Kim YW, Hwang I, Kim SY (2005) Arabidopsis calcium-dependent protein kinaseAtCPK32 interacts with ABF4, a transcriptional regulator of abscisic acid-responsive gene expression, and modulates its activity.Plant Physiol 139: 1750-1761


Dalziel KJ, O'Leary B, Brikis C, Rao SK, She Y-M, Cyr T, Plaxton WC (2012) The bacterial-type phosphoenolpyruvate carboxylaseisozyme from developing castor oil seeds is subject to in vivo regulatory phosphorylation at serine-451. FEBS Lett 586: 1049-1054


Dammann C, Ichida A, Hong BM, Romanowsky SM, Hrabak EM, Harmon AC, Pickard BG, Harper JF (2003) Subcellular targeting ofnine calcium-dependent protein kinase isoforms from Arabidopsis. Plant Physiol 132: 1840-1848


DeFalco TA, Bender KW, Snedden WA (2010) Breaking the code: Ca2+ sensors in plant signalling. Biochem J 425: 27-40Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Douglas P, Moorhead G, Hong Y, Morrice N, MacKintosh C (1998) Purification of a nitrate reductase kinase from Spinaceaoleracea leaves, and its identification as a calmodulin-domain protein kinase. Planta 206: 435-442

Pubmed: Author and TitleCrossRef: Author and Title


http://www.ncbi.nlm.nih.gov/pubmed?term=Almadanim MC%2C Alexandre BM%2C Rosa MTG%2C Sapeta H%2C Leit�o AE%2C Ramalho JC%2C Lam TT%2C Negr�o S%2C Abreu IA%2C Oliveira MM %282017%29 Rice calcium%2Ddependent protein kinase OsCPK17 targets plasma membrane intrinsic protein and sucrose phosphate synthase and is required for a proper cold stress response%2E Plant Cell Environ%2E doi%3A 10%2E1111%2Fpce%2E12916&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Almadanim MC, Alexandre BM, Rosa MTG, Sapeta H, Leit�o AE, Ramalho JC, Lam TT, Negr�o S, Abreu IA, Oliveira MM (2017) Rice calcium-dependent protein kinase OsCPK17 targets plasma membrane intrinsic protein and sucrose phosphate synthase and is required for a proper cold stress response. Plant Cell Environ. doi: 10.1111/pce.12916

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Almadanim&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Rice calcium-dependent protein kinase OsCPK17 targets plasma membrane intrinsic protein and sucrose phosphate synthase and is required for a proper cold stress response.&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Rice calcium-dependent protein kinase OsCPK17 targets plasma membrane intrinsic protein and sucrose phosphate synthase and is required for a proper cold stress response.&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Almadanim&as_ylo=2017&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Asai S%2C Ichikawa T%2C Nomura H%2C Kobayashi M%2C Kamiyoshihara Y%2C Mori H%2C Kadota Y%2C Zipfel C%2C Jones JDG%2C Yoshioka H %282013%29 The variable domain of a plant calcium%2Ddependent protein kinase %28CDPK%29 confers subcellular localization and substrate recognition for NADPH oxidase%2E J Biol Chem 288%3A 14332%2D14340&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Asai S, Ichikawa T, Nomura H, Kobayashi M, Kamiyoshihara Y, Mori H, Kadota Y, Zipfel C, Jones JDG, Yoshioka H (2013) The variable domain of a plant calcium-dependent protein kinase (CDPK) confers subcellular localization and substrate recognition for NADPH oxidase. J Biol Chem 288: 14332-14340

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Asai&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=The variable domain of a plant calcium-dependent protein kinase (CDPK) confers subcellular localization and substrate recognition for NADPH oxidase&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=The variable domain of a plant calcium-dependent protein kinase (CDPK) confers subcellular localization and substrate recognition for NADPH oxidase&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Asai&as_ylo=2013&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Bachmann M%2C Shiraishi N%2C Campbell WH%2C Yoo BC%2C Harmon AC%2C Huber SC %281996%29 Identification of Ser%2D543 as the major regulatory phosphorylation site in spinach leaf nitrate reductase%2E Plant Cell 8%3A 505%2D517&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Bachmann M, Shiraishi N, Campbell WH, Yoo BC, Harmon AC, Huber SC (1996) Identification of Ser-543 as the major regulatory phosphorylation site in spinach leaf nitrate reductase. Plant Cell 8: 505-517

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Bachmann&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Identification of Ser-543 as the major regulatory phosphorylation site in spinach leaf nitrate reductase&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Identification of Ser-543 as the major regulatory phosphorylation site in spinach leaf nitrate reductase&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Bachmann&as_ylo=1996&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Bender KW%2C Dobney S%2C Ogunrinde A%2C Chiasson D%2C Mullen RT%2C Teresinski HJ%2C Singh P%2C Munro K%2C Smith SP%2C Snedden WA %282014%29 The calmodulin%2Dlike protein CML43 functions as a salicylic%2Dacid%2Dinducible root%2Dspecific Ca2%2B sensor in Arabidopsis%2E Biochem J 457%3A 127%2D136&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Bender KW, Dobney S, Ogunrinde A, Chiasson D, Mullen RT, Teresinski HJ, Singh P, Munro K, Smith SP, Snedden WA (2014) The calmodulin-like protein CML43 functions as a salicylic-acid-inducible root-specific Ca2+ sensor in Arabidopsis. Biochem J 457: 127-136

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Bender&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=The calmodulin-like protein CML43 functions as a salicylic-acid-inducible root-specific Ca2+ sensor in Arabidopsis&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=The calmodulin-like protein CML43 functions as a salicylic-acid-inducible root-specific Ca2+ sensor in Arabidopsis&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Bender&as_ylo=2014&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Blonde JD%2C Plaxton WC %282003%29 Structural and kinetic properties of high and low molecular mass phosphoenolpyruvate carboxylase isoforms from the endosperm of developing castor oilseeds%2E J Biol Chem 278%3A 11867%2D11873&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Blonde JD, Plaxton WC (2003) Structural and kinetic properties of high and low molecular mass phosphoenolpyruvate carboxylase isoforms from the endosperm of developing castor oilseeds. J Biol Chem 278: 11867-11873

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Blonde&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Structural and kinetic properties of high and low molecular mass phosphoenolpyruvate carboxylase isoforms from the endosperm of developing castor oilseeds&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Structural and kinetic properties of high and low molecular mass phosphoenolpyruvate carboxylase isoforms from the endosperm of developing castor oilseeds&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Blonde&as_ylo=2003&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Boudsocq M%2C Droillard M%2DJ%2C Regad L%2C Lauriere C %282012%29 Characterization of Arabidopsis calcium%2Ddependent protein kinases%3A activated or not by calcium%3F Biochem J 447%3A 291%2D299&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Boudsocq M, Droillard M-J, Regad L, Lauriere C (2012) Characterization of Arabidopsis calcium-dependent protein kinases: activated or not by calcium? Biochem J 447: 291-299

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Boudsocq&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Characterization of Arabidopsis calcium-dependent protein kinases: activated or not by calcium&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Characterization of Arabidopsis calcium-dependent protein kinases: activated or not by calcium&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Boudsocq&as_ylo=2012&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Boudsocq M%2C Sheen J %282013%29 CDPKs in immune and stress signaling%2E Trends Plant Sci 18%3A 30%2D40&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Boudsocq M, Sheen J (2013) CDPKs in immune and stress signaling. Trends Plant Sci 18: 30-40

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Boudsocq&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=CDPKs in immune and stress signaling&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=CDPKs in immune and stress signaling&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Boudsocq&as_ylo=2013&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Brandizzi F%2C Irons S%2C Kearns A%2C Hawes C %282003%29 BY%2D2 cells%3A culture and transformation for live cell imaging%2E Curr Protoc Cell Biol 1%2D7&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Brandizzi F, Irons S, Kearns A, Hawes C (2003) BY-2 cells: culture and transformation for live cell imaging. Curr Protoc Cell Biol 1-7

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Brandizzi&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Brandizzi&as_ylo=2003&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Choi HI%2C Park HJ%2C Park JH%2C Kim S%2C Im MY%2C Seo HH%2C Kim YW%2C Hwang I%2C Kim SY %282005%29 Arabidopsis calcium%2Ddependent protein kinase AtCPK32 interacts with ABF4%2C a transcriptional regulator of abscisic acid%2Dresponsive gene expression%2C and modulates its activity%2E Plant Physiol 139%3A 1750%2D1761&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Choi HI, Park HJ, Park JH, Kim S, Im MY, Seo HH, Kim YW, Hwang I, Kim SY (2005) Arabidopsis calcium-dependent protein kinase AtCPK32 interacts with ABF4, a transcriptional regulator of abscisic acid-responsive gene expression, and modulates its activity. Plant Physiol 139: 1750-1761

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Dunker AK, Cortese MS, Romero P, Iakoucheva LM, Uversky VN (2005) Flexible nets - the roles of intrinsic disorder in proteininteraction networks. FEBS J 272: 5129-5148


Endicott JA, Noble MEM, Johnson LN (2012) The structural basis for control of eukaryotic protein kinases. Annu Rev Biochem 81:587-613


Fedosejevs ET, Gerdis SA, Ying S, Pyc M, Anderson EM, Snedden WA, Mullen RT, She Y-M, Plaxton WC (2016) The calcium-dependent protein kinase RcCDPK2 phosphorylates sucrose synthase at Ser11 in developing castor oil seeds. Biochem J 473:3667-3682


Fedosejevs ET, Ying S, Park J, Anderson EM, Mullen RT, She Y-M, Plaxton WC (2014) Biochemical and molecular characterizationof RcSUS1, a cytosolic sucrose synthase phosphorylated in vivo at serine 11 in developing castor oil seeds. J Biol Chem 289:33412-33424


Gennidakis S, Rao S, Greenham K, Uhrig RG, O'Leary B, Snedden WA, Lu C, Plaxton WC (2007) Bacterial- and plant-typephosphoenolpyruvate carboxylase polypeptides interact in the hetero-oligomeric Class-2 PEPC complex of developing castor oilseeds. Plant J 52: 839-849


Harmon A, Putnam-Evans C, Cormier M (1987) A calcium-dependent but calmodulin-independent protein kinase from soybean.Plant Physiol 83: 830-837


Harper JE, Breton G, Harmon A (2004) Decoding Ca2+ signals through plant protein kinases. Annu Rev Plant Biol 55: 263-288Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Hill AT, Ying S, Plaxton WC (2014) Phosphorylation of bacterial-type phosphoenolpyruvate carboxylase by a Ca2+-dependentprotein kinase suggests a link between Ca2+ signalling and anaplerotic pathway control in developing castor oil seeds. Biochem J458: 109-118


Igawa T, Fujiwara M, Tanaka I, Fukao Y, Yanagawa Y (2010) Characterization of bacterial-type phosphoenolpyruvate carboxylaseexpressed in male gametophyte of higher plants. BMC Plant Biol 10: 200-200


Ito T, Nakata M, Fukazawa J, Ishida S, Takahashi Y (2010) Alteration of substrate specificity: the variable N-terminal domain oftobacco Ca2+-dependent protein kinase is important for substrate recognition. Plant Cell 22: 1592-1604


Kobayashi M, Ohura I, Kawakita K, Yokota N, Fujiwara M, Shimamoto K, Doke N, Yoshioka H (2007) Calcium-dependent proteinkinases regulate the production of reactive oxygen species by potato NADPH oxidase. Plant Cell 19: 1065-1080


Liese A, Romeis T (2013) Biochemical regulation of in vivo function of plant calcium-dependent protein kinases (CDPK). BiochimBiophys Acta 1833: 1582-1589


Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(T)(-DeltaDelta C) method. Methods 25: 402-408

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http://search.crossref.org/?page=1&rows=2&q=Endicott JA, Noble MEM, Johnson LN (2012) The structural basis for control of eukaryotic protein kinases. Annu Rev Biochem 81: 587-613

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Martin ML, Busconi L (2000) Membrane localization of a rice calcium-dependent protein kinase (CDPK) is mediated bymyristoylation and palmitoylation. Plant J 24: 429-435


Milla MAR, Uno Y, Chang IF, Townsend J, Maher EA, Quilici D, Cushman JC (2006) A novel yeast two-hybrid approach to identifyCDPK substrates: Characterization of the interaction between AtCPK11 and AtDi19, a nuclear zinc finger protein. FEBS Lett 580:904-911


O'Leary B, Fedosejevs ET, Hill AT, Bettridge J, Park J, Rao SK, Leach CA, Plaxton WC (2011a) Tissue-specific expression andpost-translational modifications of plant- and bacterial-type phosphoenolpyruvate carboxylase isozymes of the castor oil plant,Ricinus communis L. J Exp Bot 62: 5485-5495


O'Leary B, Park J, Plaxton WC (2011b) The remarkable diversity of plant PEPC (phosphoenolpyruvate carboxylase): recentinsights into the physiological functions and post-translational controls of non-photosynthetic PEPCs. Biochem J 436: 15-34


O'Leary B, Rao SK, Kim J, Plaxton WC (2009) Bacterial-type phosphoenolpyruvate carboxylase (PEPC) functions as a catalytic andregulatory subunit of the novel Class-2 PEPC complex of vascular plants. J Biol Chem 284: 24797-24805


O'Leary B, Rao SK, Plaxton WC (2011c) Phosphorylation of bacterial-type phosphoenolpyruvate carboxylase at Ser(425) provides afurther tier of enzyme control in developing castor oil seeds. Biochem J 433: 65-74


Park J, Khuu N, Howard ASM, Mullen RT, Plaxton WC (2012) Bacterial- and plant-type phosphoenolpyruvate carboxylase isozymesfrom developing castor oil seeds interact in vivo and associate with the surface of mitochondria. Plant J 71: 251-262


Pical C, Fredlund K, Petit P, Sommarin M, Moller I (1993) The outer-membrane of plant mitochondria contains a calcium-dependentprotein-kinase and multiple phosphoproteins. FEBS Letts 336: 347-351


Rivoal J, Trzos S, Gage DA, Plaxton WC, Turpin DH (2001) Two unrelated phosphoenolpyruvate carboxylase polypeptidesphysically interact in the high molecular mass isoforms of this enzyme in the unicellular green alga Selenastrum minutum. J BiolChem 276: 12588-12597


Ruiz-Ballesta I, Baena G, Gandullo J, Wang L, She Y-M, Plaxton WC, Echevarría C (2016) New insights into the post-translationalmodification of multiple phosphoenolpyruvate carboxylase isoenzymes by phosphorylation and monoubiquitination duringsorghum seed development and germination. J Exp Bot 67: 3523-3536


Ruiz-Ballesta I, Feria A-B, Ni H, She Y-M, Plaxton WC, Echevarra C (2014) In vivo monoubiquitination of anapleroticphosphoenolpyruvate carboxylase occurs at Lys624 in germinating sorghum seeds. J Exp Bot 65: 443-451


Schulz P, Herde M, Romeis T (2013) Calcium-dependent protein kinases: hubs in plant stress signaling and development. PlantPhysiol 163: 523-530


Simeunovic A, Mair A, Wurzinger B, Teige M (2016) Know where your clients are: subcellular localization and targets of calcium- www.plantphysiol.orgon May 28, 2020 - Published by Downloaded from

Copyright © 2017 American Society of Plant Biologists. All rights reserved.

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http://scholar.google.com/scholar?as_q=Analysis of relative gene expression data using real-time quantitative PCR and the 2(T)(-Delta Delta C) method&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Livak&as_ylo=2001&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Martin ML%2C Busconi L %282000%29 Membrane localization of a rice calcium%2Ddependent protein kinase %28CDPK%29 is mediated by myristoylation and palmitoylation%2E Plant J 24%3A 429%2D435&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Martin ML, Busconi L (2000) Membrane localization of a rice calcium-dependent protein kinase (CDPK) is mediated by myristoylation and palmitoylation. Plant J 24: 429-435

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Martin&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Membrane localization of a rice calcium-dependent protein kinase (CDPK) is mediated by myristoylation and palmitoylation&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Membrane localization of a rice calcium-dependent protein kinase (CDPK) is mediated by myristoylation and palmitoylation&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Martin&as_ylo=2000&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Milla MAR%2C Uno Y%2C Chang IF%2C Townsend J%2C Maher EA%2C Quilici D%2C Cushman JC %282006%29 A novel yeast two%2Dhybrid approach to identify CDPK substrates%3A Characterization of the interaction between AtCPK11 and AtDi19%2C a nuclear zinc finger protein%2E FEBS Lett 580%3A 904%2D911&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Milla MAR, Uno Y, Chang IF, Townsend J, Maher EA, Quilici D, Cushman JC (2006) A novel yeast two-hybrid approach to identify CDPK substrates: Characterization of the interaction between AtCPK11 and AtDi19, a nuclear zinc finger protein. FEBS Lett 580: 904-911

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Milla&hl=en&c2coff=1


http://scholar.google.com/scholar?as_q=&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Milla&as_ylo=2006&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=O%27Leary B%2C Fedosejevs ET%2C Hill AT%2C Bettridge J%2C Park J%2C Rao SK%2C Leach CA%2C Plaxton WC %282011a%29 Tissue%2Dspecific expression and post%2Dtranslational modifications of plant%2D and bacterial%2Dtype phosphoenolpyruvate carboxylase isozymes of the castor oil plant%2C Ricinus communis L%2E J Exp Bot 62%3A 5485%2D5495&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=O'Leary B, Fedosejevs ET, Hill AT, Bettridge J, Park J, Rao SK, Leach CA, Plaxton WC (2011a) Tissue-specific expression and post-translational modifications of plant- and bacterial-type phosphoenolpyruvate carboxylase isozymes of the castor oil plant, Ricinus communis L. J Exp Bot 62: 5485-5495

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=O'Leary&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Tissue-specific expression and post-translational modifications of plant- and bacterial-type phosphoenolpyruvate carboxylase isozymes of the castor oil plant, Ricinus communis L&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Tissue-specific expression and post-translational modifications of plant- and bacterial-type phosphoenolpyruvate carboxylase isozymes of the castor oil plant, Ricinus communis L&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=O'Leary&as_ylo=2011&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=O%27Leary B%2C Park J%2C Plaxton WC %282011b%29 The remarkable diversity of plant PEPC %28phosphoenolpyruvate carboxylase%29%3A recent insights into the physiological functions and post%2Dtranslational controls of non%2Dphotosynthetic PEPCs%2E Biochem J 436%3A 15%2D34&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=O'Leary B, Park J, Plaxton WC (2011b) The remarkable diversity of plant PEPC (phosphoenolpyruvate carboxylase): recent insights into the physiological functions and post-translational controls of non-photosynthetic PEPCs. Biochem J 436: 15-34


http://scholar.google.com/scholar?as_q=The remarkable diversity of plant PEPC (phosphoenolpyruvate carboxylase): recent insights into the physiological functions and post-translational controls of non-photosynthetic PEPCs&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=The remarkable diversity of plant PEPC (phosphoenolpyruvate carboxylase): recent insights into the physiological functions and post-translational controls of non-photosynthetic PEPCs&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=O'Leary&as_ylo=2011&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=O%27Leary B%2C Rao SK%2C Kim J%2C Plaxton WC %282009%29 Bacterial%2Dtype phosphoenolpyruvate carboxylase %28PEPC%29 functions as a catalytic and regulatory subunit of the novel Class%2D2 PEPC complex of vascular plants%2E J Biol Chem 284%3A 24797%2D24805&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=O'Leary B, Rao SK, Kim J, Plaxton WC (2009) Bacterial-type phosphoenolpyruvate carboxylase (PEPC) functions as a catalytic and regulatory subunit of the novel Class-2 PEPC complex of vascular plants. J Biol Chem 284: 24797-24805


http://scholar.google.com/scholar?as_q=Bacterial-type phosphoenolpyruvate carboxylase (PEPC) functions as a catalytic and regulatory subunit of the novel Class-2 PEPC complex of vascular plants&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

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http://www.ncbi.nlm.nih.gov/pubmed?term=O%27Leary B%2C Rao SK%2C Plaxton WC %282011c%29 Phosphorylation of bacterial%2Dtype phosphoenolpyruvate carboxylase at Ser%28425%29 provides a further tier of enzyme control in developing castor oil seeds%2E Biochem J 433%3A 65%2D74&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=O'Leary B, Rao SK, Plaxton WC (2011c) Phosphorylation of bacterial-type phosphoenolpyruvate carboxylase at Ser(425) provides a further tier of enzyme control in developing castor oil seeds. Biochem J 433: 65-74


http://scholar.google.com/scholar?as_q=Phosphorylation of bacterial-type phosphoenolpyruvate carboxylase at Ser(425) provides a further tier of enzyme control in developing castor oil seeds&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Phosphorylation of bacterial-type phosphoenolpyruvate carboxylase at Ser(425) provides a further tier of enzyme control in developing castor oil seeds&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=O'Leary&as_ylo=2011&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Park J%2C Khuu N%2C Howard ASM%2C Mullen RT%2C Plaxton WC %282012%29 Bacterial%2D and plant%2Dtype phosphoenolpyruvate carboxylase isozymes from developing castor oil seeds interact in vivo and associate with the surface of mitochondria%2E Plant J 71%3A 251%2D262&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Park J, Khuu N, Howard ASM, Mullen RT, Plaxton WC (2012) Bacterial- and plant-type phosphoenolpyruvate carboxylase isozymes from developing castor oil seeds interact in vivo and associate with the surface of mitochondria. Plant J 71: 251-262

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Park&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Bacterial- and plant-type phosphoenolpyruvate carboxylase isozymes from developing castor oil seeds interact in vivo and associate with the surface of mitochondria&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Bacterial- and plant-type phosphoenolpyruvate carboxylase isozymes from developing castor oil seeds interact in vivo and associate with the surface of mitochondria&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Park&as_ylo=2012&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Pical C%2C Fredlund K%2C Petit P%2C Sommarin M%2C Moller I %281993%29 The outer%2Dmembrane of plant mitochondria contains a calcium%2Ddependent protein%2Dkinase and multiple phosphoproteins%2E FEBS Letts 336%3A 347%2D351&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Pical C, Fredlund K, Petit P, Sommarin M, Moller I (1993) The outer-membrane of plant mitochondria contains a calcium-dependent protein-kinase and multiple phosphoproteins. FEBS Letts 336: 347-351

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Pical&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=The outer-membrane of plant mitochondria contains a calcium-dependent protein-kinase and multiple phosphoproteins&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=The outer-membrane of plant mitochondria contains a calcium-dependent protein-kinase and multiple phosphoproteins&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Pical&as_ylo=1993&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Rivoal J%2C Trzos S%2C Gage DA%2C Plaxton WC%2C Turpin DH %282001%29 Two unrelated phosphoenolpyruvate carboxylase polypeptides physically interact in the high molecular mass isoforms of this enzyme in the unicellular green alga Selenastrum minutum%2E J Biol Chem 276%3A 12588%2D12597&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Rivoal J, Trzos S, Gage DA, Plaxton WC, Turpin DH (2001) Two unrelated phosphoenolpyruvate carboxylase polypeptides physically interact in the high molecular mass isoforms of this enzyme in the unicellular green alga Selenastrum minutum. J Biol Chem 276: 12588-12597

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http://scholar.google.com/scholar?as_q=Two unrelated phosphoenolpyruvate carboxylase polypeptides physically interact in the high molecular mass isoforms of this enzyme in the unicellular green alga Selenastrum minutum&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Two unrelated phosphoenolpyruvate carboxylase polypeptides physically interact in the high molecular mass isoforms of this enzyme in the unicellular green alga Selenastrum minutum&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Rivoal&as_ylo=2001&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Ruiz%2DBallesta I%2C Baena G%2C Gandullo J%2C Wang L%2C She Y%2DM%2C Plaxton WC%2C Echevarr�a C %282016%29 New insights into the post%2Dtranslational modification of multiple phosphoenolpyruvate carboxylase isoenzymes by phosphorylation and monoubiquitination during sorghum seed development and germination%2E J Exp Bot 67%3A 3523%2D3536&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Ruiz-Ballesta I, Baena G, Gandullo J, Wang L, She Y-M, Plaxton WC, Echevarr�a C (2016) New insights into the post-translational modification of multiple phosphoenolpyruvate carboxylase isoenzymes by phosphorylation and monoubiquitination during sorghum seed development and germination. J Exp Bot 67: 3523-3536

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http://scholar.google.com/scholar?as_q=New insights into the post-translational modification of multiple phosphoenolpyruvate carboxylase isoenzymes by phosphorylation and monoubiquitination during sorghum seed development and germination&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=New insights into the post-translational modification of multiple phosphoenolpyruvate carboxylase isoenzymes by phosphorylation and monoubiquitination during sorghum seed development and germination&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Ruiz-Ballesta&as_ylo=2016&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Ruiz%2DBallesta I%2C Feria A%2DB%2C Ni H%2C She Y%2DM%2C Plaxton WC%2C Echevarra C %282014%29 In vivo monoubiquitination of anaplerotic phosphoenolpyruvate carboxylase occurs at Lys624 in germinating sorghum seeds%2E J Exp Bot 65%3A 443%2D451&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Ruiz-Ballesta I, Feria A-B, Ni H, She Y-M, Plaxton WC, Echevarra C (2014) In vivo monoubiquitination of anaplerotic phosphoenolpyruvate carboxylase occurs at Lys624 in germinating sorghum seeds. J Exp Bot 65: 443-451

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http://scholar.google.com/scholar?as_q=In vivo monoubiquitination of anaplerotic phosphoenolpyruvate carboxylase occurs at Lys624 in germinating sorghum seeds&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Ruiz-Ballesta&as_ylo=2014&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Schulz P%2C Herde M%2C Romeis T %282013%29 Calcium%2Ddependent protein kinases%3A hubs in plant stress signaling and development%2E Plant Physiol 163%3A 523%2D530&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Schulz P, Herde M, Romeis T (2013) Calcium-dependent protein kinases: hubs in plant stress signaling and development. Plant Physiol 163: 523-530

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http://www.ncbi.nlm.nih.gov/pubmed?term=Simeunovic A%2C Mair A%2C Wurzinger B%2C Teige M %282016%29 Know where your clients are%3A subcellular localization and targets of calcium%2Ddependent protein kinases%2E J Exp Bot 67%3A 3855%2D3872&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Simeunovic A, Mair A, Wurzinger B, Teige M (2016) Know where your clients are: subcellular localization and targets of calcium-dependent protein kinases. J Exp Bot 67: 3855-3872

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http://scholar.google.com/scholar?as_q=Know where your clients are: subcellular localization and targets of calcium-dependent protein kinases&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Know where your clients are: subcellular localization and targets of calcium-dependent protein kinases&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Simeunovic&as_ylo=2016&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Taylor SS%2C Kornev AP %282011%29 Protein kinases%3A evolution of dynamic regulatory proteins%2E Trends Biochem Sci 36%3A 65%2D77&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Taylor SS, Kornev AP (2011) Protein kinases: evolution of dynamic regulatory proteins. Trends Biochem Sci 36: 65-77

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Taylor&hl=en&c2coff=1

http://scholar.google.com/scholar?as_q=Protein kinases: evolution of dynamic regulatory proteins&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

http://scholar.google.com/scholar?as_q=Protein kinases: evolution of dynamic regulatory proteins&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Taylor&as_ylo=2011&as_allsubj=all&hl=en&c2coff=1

http://www.ncbi.nlm.nih.gov/pubmed?term=Tripodi KE%2C Turner WL%2C Gennidakis S%2C Plaxton WC %282005%29 In vivo regulatory phosphorylation of novel phosphoenolpyruvate carboxylase isoforms in endosperm of developing castor oil seeds%2E Plant Physiol 139%3A 969%2D978&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Tripodi KE, Turner WL, Gennidakis S, Plaxton WC (2005) In vivo regulatory phosphorylation of novel phosphoenolpyruvate carboxylase isoforms in endosperm of developing castor oil seeds. Plant Physiol 139: 969-978

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regulatory phosphorylation of bacterial-type pep ......2017/03/31 · 35 r.t.m., and w.c.p.), as...

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