TY - JOUR
AU - Plaxton, William C.
AB - Abstract Phosphoenolpyruvate carboxylase (PEPC) is a tightly controlled cytosolic enzyme situated at a crucial branch point of central plant metabolism. In developing castor oil seeds (Ricinus communis) a novel, allosterically desensitized 910-kD Class-2 PEPC hetero-octameric complex, arises from a tight interaction between 107-kD plant-type PEPC and 118-kD bacterial-type (BTPC) subunits. The native Ca2+-dependent protein kinase (CDPK) responsible for in vivo inhibitory phosphorylation of Class-2 PEPC’s BTPC subunit’s at Ser-451 was highly purified from COS and identified as RcCDPK1 (XP_002526815) by mass spectrometry. Heterologously expressed RcCDPK1 catalyzed Ca2+-dependent, inhibitory phosphorylation of BTPC at Ser-451 while exhibiting: (i) a pair of Ca2+ binding sites with identical dissociation constants of 5.03 μM, (ii) a Ca2+-dependent electrophoretic mobility shift, and (iii) a marked Ca2+-independent hydrophobicity. Pull-down experiments established the Ca2+-dependent interaction of N-terminal GST-tagged RcCDPK1 with BTPC. RcCDPK1-Cherry localized to the cytosol and nucleus of tobacco bright yellow-2 cells, but colocalized with mitochondrial-surface associated BTPC-enhanced yellow fluorescent protein when both fusion proteins were coexpressed. Deletion analyses demonstrated that although its N-terminal variable domain plays an essential role in optimizing Ca2+-dependent RcCDPK1 autophosphorylation and BTPC transphosphorylation activity, it is not critical for in vitro or in vivo target recognition. Arabidopsis (Arabidopsis thaliana) CPK4 and soybean (Glycine max) CDPKβ are RcCDPK1 orthologs that effectively phosphorylated castor BTPC at Ser-451. Overall, the results highlight a potential link between cytosolic Ca2+ signaling and the posttranslational control of respiratory CO2 refixation and anaplerotic photosynthate partitioning in support of storage oil and protein biosynthesis in developing COS. Calcium plays a central role in eukaryotic signal transduction with various Ca2+-sensor proteins being critical transducers of Ca2+ signatures elicited in response to external stimuli or developmental cues. The activation of protein phosphorylation cascades is often the first and most important signaling event triggered by Ca2+ signals (DeFalco et al., 2009). Among Ca2+ sensors, including calmodulin (CaM) and CaM-like proteins (CMLs), Ca2+-dependent protein kinases (CDPKs) are unique because they function as catalytic responders that directly transduce Ca2+ signals into protein phosphorylation events that modulate physiological responses (Harper et al., 2004; DeFalco et al., 2009; Boudsocq and Sheen, 2013; Schulz et al., 2013; Simeunovic et al., 2016). This combination of signaling properties likely arose after the early fusion of a protein kinase gene with a CaM gene, and was followed by CDPK diversification into a relatively large multigene family in vascular plants, thus providing a mechanism to decode different Ca2+ signals in a temporal and spatially specific manner (Harper et al., 2004). Different CDPK isozymes exhibit distinctive tissue and subcellular locations, substrate specificities, and Ca2+ sensitivities. Thus, diverse developmental programs and stress responses are likely controlled by specific CDPKs including hormone-regulated developmental processes, seed development, pollen tube formation, and abiotic and biotic stress signaling (Harper et al., 2004; Boudsocq and Sheen, 2013; Schulz et al., 2013; Simeunovic et al., 2016). Downstream targets of CDPK action include other protein kinases, transcription factors, ion channels and pumps, cytoskeletal proteins, and metabolic enzymes such as Suc synthase, Suc P synthase, nitrate reductase, and NADPH oxidase (Bachmann et al., 1996; Douglas et al., 1998; Zhang et al., 1999; Kobayashi et al., 2007; Asai et al., 2013; Simeunovic et al., 2016; Almadanim et al., 2017). While no CDPK appears to be an integral membrane protein, most isoforms contain a myristoylation motif at their N terminus, which for several CDPKs has been shown to be important for their membrane association (Martín and Busconi, 2000; Ito et al., 2010; Asai et al., 2013). However, a major gap in our understanding of plant Ca2+ signaling and CDPK biology in general is that relatively few in vivo CDPK targets have been identified to date (Liese and Romeis, 2013; Schulz et al., 2013; Simeunovic et al., 2016). It is evident that some CDPKs, when purified as recombinant enzymes, promiscuously phosphorylate exogenous proteins in vitro at many different Ser and Thr residues, far beyond proposed CDPK phosphorylation motifs (Harper et al., 2004; Boudsocq and Sheen, 2013; Schulz et al., 2013). The aim of this study was to identify and characterize the putative CDPK that catalyzes in vivo inhibitory phosphorylation of bacterial-type phosphoenolpyruvate (PEP) carboxylase (BTPC) at Ser-451 in developing castor (Ricinus communis) oil seeds (COS; Dalziel et al., 2012; Hill et al., 2014). PEP carboxylase (PEPC; EC 4.1.1.31) is a tightly regulated enzyme that has been intensively studied owing to its pivotal role in assimilating atmospheric CO2 during C4 and Crassulacean acid metabolism (CAM) photosynthesis. Much attention has also been devoted to PEPC’s essential nonphotosynthetic functions, particularly the anaplerotic replenishment of tricarboxylic acid cycle intermediates withdrawn during biosynthesis and nitrogen assimilation (O’Leary et al., 2011b). To fulfill its diverse roles and complex regulation, plant PEPC belongs to a small multigene family encoding several plant-type PEPCs (PTPCs), along with a distantly related BTPC (O’Leary et al., 2011b). PTPC genes encode closely related 100-110-kD polypeptides containing conserved seryl-phosphorylation (activatory) and lysyl-monoubiquitination (inhibitory) sites and that typically oligomerize as tetrameric Class-1 PEPCs (Supplemental Fig. S1; Tripodi et al., 2005; Uhrig et al., 2008b; O’Leary et al., 2011b; Ruiz-Ballesta et al., 2014, 2016). By contrast, plant BTPC genes encode distantly related 116- to 118-kD polypeptides that are more similar to prokaryotic PEPCs. Purification of native PEPCs from unicellular green algae and then developing castor oil seeds (COS) led to the discovery of unusual high-Mr Class-2 PEPC heteromeric complexes composed of tightly associated PTPC and BTPC subunits, and that are largely desensitized to allosteric effectors relative to Class-1 PEPCs (Supplemental Fig. S1; Rivoal et al., 2001; Blonde and Plaxton, 2003; Gennidakis et al., 2007; Uhrig et al., 2008a; O’Leary et al., 2009). Plant BTPC polypeptides and thus Class-2 PEPC complexes have only been documented in biosynthetically active tissues (i.e. developing seeds and pollen, immature leaves), as opposed to PTPCs that are constitutively expressed in the cytosol as housekeeping Class-1 PEPCs (Igawa et al., 2010; O’Leary et al., 2011a, 2011b). Although COS Class-1 PEPC localizes diffusely throughout the cytosol, the Class-2 PEPC associates with the outer mitochondrial envelope, an interaction mediated by its BTPC subunits (Park et al., 2012). Class-2 PEPC’s unique kinetic and regulatory properties, and dynamic subcellular targeting to the mitochondrial surface, support the hypothesis that it facilitates rapid refixation of respiratory CO2 while sustaining a large anaplerotic flux to replenish tricarboxylic acid cycle C-skeletons withdrawn in support of storage oil and protein biosynthesis in developing COS. Although BTPCs lack the N-terminal seryl phosphorylation motif characteristic of nonphotosynthetic and C4/CAM photosynthetic PTPCs, BTPC is in vivo phosphorylated at multiple sites during COS development (Uhrig et al., 2008a; O’Leary et al., 2011c; Dalziel et al., 2012). This includes inhibitory phosphorylation at Ser-425 and Ser-451, which both occur within a distinctive approximately 10-kD intrinsically disordered region not found in PTPCs (Dalziel et al., 2012; O’Leary et al., 2011c). Despite the apparent important role of multisite BTPC phosphorylation in the posttranslational control of photosynthate partitioning and anaplerotic C-flux at the PEP branchpoint during COS development, nothing was known about the responsible protein kinases or related signaling pathways. Thus, Hill et al. (2014) purified the native BTPC Ser-451 kinase from developing COS by greater than 500-fold and provided kinetic evidence that it belongs to the castor CDPK family. This study used mass spectrometry (MS) to identify the BTPC Ser-451 kinase as RcCDPK1, one of 20 predicted castor CDPK isozymes (Hill et al., 2014). We also demonstrate the specificity of RcCDPK1 for phosphorylating BTPC at Ser-451, as well as its in vivo interaction with BTPC on the surface of mitochondria. RESULTS AND DISCUSSION Identification of Castor Bean BTPC Ser-451 Kinase as RcCDPK1 Peptides derived from a total tryptic digest of the final BTPC Ser-451 kinase preparation of Hill et al. (2014) were sequenced via nanoHPLC tandem MS (LC-MS/MS). Database searches identified numerous proteins (Supplemental Table S1) including a single protein kinase, RcCDPK1 (protein #36 on the list). Reliable identification was achieved by nine tryptic peptides whose respective sequences precisely matched corresponding regions of RcCDPK1’s predicted sequence (30.2% sequence coverage; protein accession: XP_002526815; Fig. 1). The predicted size of RcCDPK1 is 56 kD, a value similar to the native molecular mass of 63 kD estimated for BTPC Ser-451 kinase isolated from developing COS (Hill et al., 2014). Figure 1. Open in new tabDownload slide Alignment of tryptic peptides derived from purified castor bean BTPC Ser-451 kinase with the deduced sequence of RcCDPK1 and several orthologs from other plants. LC-MS/MS analysis of the final BTPC Ser-451 kinase preparation of Hill et al. (2014) resulted in nine peptide sequences unique to RcCDPK1 that are underlined with a solid gray line. The protein kinase catalytic domain and four EF-hand (Ca2+-binding) motifs are overlined with dashed and solid black lines, respectively. The conserved phosphomimetic Asp (D) residue that occurs within the activation loop of RcCDPK1 and its orthologs is highlighted with a bold font (i.e. Asp-190 in the case of RcCDPK1). Black shading indicates identical residues, whereas gray shading denotes residues conserved among the six proteins. Gaps, indicated by dashes, were introduced to maximize alignment. Inset: schematic diagram of RcCDPK1 functional domains (AIJ, autoinhibitory junction; CTVD, C-terminal variable domain; NTVD, N-terminal variable domain). Figure 1. Open in new tabDownload slide Alignment of tryptic peptides derived from purified castor bean BTPC Ser-451 kinase with the deduced sequence of RcCDPK1 and several orthologs from other plants. LC-MS/MS analysis of the final BTPC Ser-451 kinase preparation of Hill et al. (2014) resulted in nine peptide sequences unique to RcCDPK1 that are underlined with a solid gray line. The protein kinase catalytic domain and four EF-hand (Ca2+-binding) motifs are overlined with dashed and solid black lines, respectively. The conserved phosphomimetic Asp (D) residue that occurs within the activation loop of RcCDPK1 and its orthologs is highlighted with a bold font (i.e. Asp-190 in the case of RcCDPK1). Black shading indicates identical residues, whereas gray shading denotes residues conserved among the six proteins. Gaps, indicated by dashes, were introduced to maximize alignment. Inset: schematic diagram of RcCDPK1 functional domains (AIJ, autoinhibitory junction; CTVD, C-terminal variable domain; NTVD, N-terminal variable domain). The deduced RcCDPK1 protein corresponds to a 497-amino acid polypeptide having 78% to 84% sequence identity with the orthologs aligned in Figure 1; maximal identity (83% to 84%) was achieved with CPK4 and CPK11 from Arabidopsis (Arabidopsis thaliana, AtCPK4 and AtCPK11, respectively) and soybean (Glycine max) CDPKβ (GmCDPKβ). RcCDPK1 and its orthologs possess all five modular domains characteristic of CDPKs: an N-terminal variable domain that differs both in sequence and length, a protein kinase catalytic domain, an autoinhibitory junction, a CaM-like domain containing four EF-hand motifs implicated in Ca2+ binding, and a C-terminal variable domain (Fig. 1). RcCDPK1, AtCPK4, and AtCPK11 were determined to be soluble rather than membrane bound (Dammann et al., 2003; Boudsocq et al., 2012; Hill et al., 2014). This is consistent with the absence of a membrane-targeting myristoylation sequence (MGXXXS) at their N terminus (Fig. 1) that is typical of most CDPKs (Schulz et al., 2013). The closest paralog of RcCDPK1 is RcCDPK2 (70% sequence identity; Hill et al., 2014), the kinase that in vivo phosphorylates castor Suc synthase-1 (RcSUS1) at Ser-11 in developing COS (Fedosejevs et al., 2016). As with all known eukaryotic protein kinases, the catalytic kinase domain of plant CDPKs contains a conserved activation loop that begins and ends with Asp-Phe-Gly and Ala-Pro-Glu motifs, respectively (e.g. RcCDPK1 residues 175 to 201; Fig. 1; Taylor and Kornev, 2011; Liese and Romeis, 2013). Stimulus-dependent phosphorylation of a Thr/Ser residue within this activation loop is a prerequisite for activation of most protein kinases (Taylor and Kornev, 2011). It is notable that phosphorylation-dependent regulation within the kinase activation loop does not occur for CDPKs (Liese and Romeis, 2013), as they instead have had the target Thr/Ser substituted with a phosphomimetic Asp or Glu residue; e.g. Asp in the case of RcCDPK1 (i.e. Asp-190) and its orthologs (Fig. 1). RcCDPK1 Is Highly Expressed During Castor Bean Development Many CDPK genes are transcriptionally responsive to developmental and stress stimuli (Harper et al., 2004; Schulz et al., 2013). RcCDPK1 is also differentially expressed in castor plants, with maximal transcript levels occurring in male flowers and mature leaves, and the endosperm and cotyledons of developing COS (Fig. 2A). RcCDPK1’s expression profile in COS endosperm matched the pattern of in vivo BTPC phosphorylation at Ser-451 with both peaking at the maturation stage (stage IX) of seed development (Fig. 2B; Dalziel et al., 2012). However, extractable BTPC Ser-451 kinase activity showed a somewhat dissimilar developmental profile as it peaked at stage VII, but was followed by a marked decrease at stage IX (Hill et al., 2014). These results are consistent with the posttranslational control of RcCDPK1 during COS development. By comparison, BTPC transcript levels maximized during the middle phase of COS development and showed a pronounced drop at stage IX, before becoming undetectable in fully mature COS (Fig. 2B). BTPC’s expression profile parallels that previously obtained via semiquantitative RT-PCR, and correlates well with relative levels of the encoded 118-kD BTPC polypeptides (p118; Gennidakis et al., 2007; O’Leary et al., 2011a). Thus, maximal BTPC expression during COS development occurs in advance of the subsequent peaks in RcCDPK1 expression, BTPC Ser-451 kinase activity, and in vivo BTPC phosphorylation at Ser-451 (Fig. 2B; Dalziel et al., 2012; Hill et al., 2014). Figure 2. Open in new tabDownload slide Analysis of RcCDPK1 expression. Levels of mRNA were analyzed by qRT-PCR using gene-specific primers. Castor Actin (AY360221) was used as the internal control for normalization. A, Tissue-specific expression of RcCDPK1. Developing and germinating seed tissues were, respectively, harvested from stage VII and 5 d postimbibition COS, male flowers were harvested at maturity, whereas female flowers were harvested at 5 DPA (corresponding to proembryo or stage I COS). B, The profile of RcCDPK1 transcripts was compared with that of BTPC (RcPPC4; EF634318) in endosperm of developing COS. Stages III, V, VII, and IX correspond to heart-shaped embryo, midcotyledon, full cotyledon, and maturation stages of endosperm development, respectively; the lane labeled “Dry” designates fully mature COS. All values in (A) and (B) represent means ± se of n = 4 determinations using cDNAs prepared from two biological replicates. E, endosperm; dE, endosperm from stage-VII developing COS that had been depodded for 48 h; C, cotyledon; dC, cotyledon from stage-VII developing COS that had been depodded for 48 h; Bud, leaf bud; Exp, expanding leaf; Mat, mature leaf; H, hypocotyl; RM, root middle; RT, root tip; MF, male flower; Int, integument of female flowers; P, pericarp of female flowers; n.d., not detected. Figure 2. Open in new tabDownload slide Analysis of RcCDPK1 expression. Levels of mRNA were analyzed by qRT-PCR using gene-specific primers. Castor Actin (AY360221) was used as the internal control for normalization. A, Tissue-specific expression of RcCDPK1. Developing and germinating seed tissues were, respectively, harvested from stage VII and 5 d postimbibition COS, male flowers were harvested at maturity, whereas female flowers were harvested at 5 DPA (corresponding to proembryo or stage I COS). B, The profile of RcCDPK1 transcripts was compared with that of BTPC (RcPPC4; EF634318) in endosperm of developing COS. Stages III, V, VII, and IX correspond to heart-shaped embryo, midcotyledon, full cotyledon, and maturation stages of endosperm development, respectively; the lane labeled “Dry” designates fully mature COS. All values in (A) and (B) represent means ± se of n = 4 determinations using cDNAs prepared from two biological replicates. E, endosperm; dE, endosperm from stage-VII developing COS that had been depodded for 48 h; C, cotyledon; dC, cotyledon from stage-VII developing COS that had been depodded for 48 h; Bud, leaf bud; Exp, expanding leaf; Mat, mature leaf; H, hypocotyl; RM, root middle; RT, root tip; MF, male flower; Int, integument of female flowers; P, pericarp of female flowers; n.d., not detected. RcCDPK1 transcript abundance in the endosperm and cotyledon of developing COS was reduced by greater than 50% within 48 h after photosynthate supply was eliminated by excision of intact fruit clusters (i.e. depodding; Fig. 2A). However, this was not paralleled by a decrease in BTPC Ser-451 kinase activity nor a reduction in BTPC phosphorylation at Ser-451 in the developing endosperm (Dalziel et al., 2012; Hill et al., 2014). This provides further evidence in support of posttranscriptional RcCDPK1 control in developing COS. A similar lack of correlation of AtCPK4 and AtCPK11 transcripts with their respective protein levels and kinase activity was noted after abscisic acid treatment of Arabidopsis (Zhu et al., 2007). RcCDPK1 Cloning, Heterologous Expression, and Antibody Production Full-length RcCDPK1 as well as its N-terminal variable domain truncation mutant (i.e. ∆N-RcCDPK1, lacking the first 29 amino acid residues), were heterologously expressed as N-terminal His6-tagged fusion proteins in Escherichia coli BL21 and purified to apparent homogeneity by Ni2+-affinity chromatography (Fig. 3A). RcCDPK1 and ∆N-RcCDPK1 both demonstrated Ca2+-binding-dependent mobility shifts during SDS-PAGE (Fig. 3A), as described previously for several CDPKs including RcCDPK1 orthologs AtCPK4, AtCPK11, and GmCDPKβ (Harmon et al., 1987; Zhu et al., 2007; Boudsocq et al., 2012). Figure 3. Open in new tabDownload slide SDS-PAGE and immunoblot analysis of RcCDPK1 and ∆N-RcCDPK1. A, SDS-PAGE of 2.5 μg each of the final His6-tagged RcCDPK1 and ∆N-RcCDPK1 preparations was performed after the addition of 2 mm CaCl2 or 2 mm EGTA to the samples as indicated. The gel was stained with Coomassie Brilliant Blue R-250. B, Immunoblotting after SDS-PAGE was performed with a 1:50 dilution of affinity-purified anti-RcCDPK1 peptide antibodies. Protein loading was: His6-RcCDPK1 and His6-∆N-RcCDPK1 (50 ng each); “RcCDPK1” represents 75 ng of recombinant RcCDPK1 after incubation with enterokinase to remove its His6-tag; “COS Phenyl Sepharose Eluate” represents 20 μg of BTPC Ser-451 kinase enriched extract from stages V to VII developing COS (prepared as described in legend for Supplemental Fig. S4); whereas “COS BTPC-K” represents 2 μg of the final preparation of BTPC Ser-451 kinase purified by Hill et al. (2014). CaCl2 (0.2 mM) was added to all samples before SDS-PAGE and immunoblotting. “M” denotes various protein molecular mass standards in (A) and (B). Figure 3. Open in new tabDownload slide SDS-PAGE and immunoblot analysis of RcCDPK1 and ∆N-RcCDPK1. A, SDS-PAGE of 2.5 μg each of the final His6-tagged RcCDPK1 and ∆N-RcCDPK1 preparations was performed after the addition of 2 mm CaCl2 or 2 mm EGTA to the samples as indicated. The gel was stained with Coomassie Brilliant Blue R-250. B, Immunoblotting after SDS-PAGE was performed with a 1:50 dilution of affinity-purified anti-RcCDPK1 peptide antibodies. Protein loading was: His6-RcCDPK1 and His6-∆N-RcCDPK1 (50 ng each); “RcCDPK1” represents 75 ng of recombinant RcCDPK1 after incubation with enterokinase to remove its His6-tag; “COS Phenyl Sepharose Eluate” represents 20 μg of BTPC Ser-451 kinase enriched extract from stages V to VII developing COS (prepared as described in legend for Supplemental Fig. S4); whereas “COS BTPC-K” represents 2 μg of the final preparation of BTPC Ser-451 kinase purified by Hill et al. (2014). CaCl2 (0.2 mM) was added to all samples before SDS-PAGE and immunoblotting. “M” denotes various protein molecular mass standards in (A) and (B). For production of RcCDPK1-specific antibodies (anti-RcCDPK1), a 29-amino acid oligopeptide matching RcCDPK1’s N-terminal variable domain was synthesized with an extra N-terminal Cys residue (Supplemental Fig. S2A) to enable its conjugation to keyhole limpet hemocyanin before rabbit immunization. The affinity-purified anti-RcCDPK1 detection limit was 10 ng of the corresponding peptide on dot blots, with the signal being quenched by the blocking parent peptide (Supplemental Fig. S2B). Immunoblots of His6-RcCDPK1 cross reacted with the anti-RcCDPK1, whereas His6-∆N-RcCDPK1 was not detected, as expected (Fig. 3B). The anti-RcCDPK1 appears to be monospecific as it cross reacted with a single 56-kD immunoreactive polypeptide that comigrated with the recombinant, full-length RcCDPK1 (after cleavage of its His6-tag) on immunoblots of both a phenyl-Sepharose-enriched developing COS extract and the final BTPC Ser-451 kinase preparation of Hill et al. (2014) that was analyzed by LC-MS/MS in this study (Fig. 3B). RcCDPK1 Catalyzes Ca2+-Dependent, Inhibitory Phosphorylation of Castor BTPC at Ser-451 Kinetic studies of heterologously expressed RcCDPK1 were conducted by monitoring Pi incorporation from unlabeled ATP or [γ-32P]-ATP into the p118/BTPC subunits of recombinant Class-2 PEPC consisting of a 1:1 stoichiometric ratio of castor BTPC (RcPPC4) and an Arabidopsis PTPC isozyme (AtPPC3; O’Leary et al., 2009). RcCDPK1 readily catalyzed Ca2+-dependent Ser-451 phosphorylation of the p118 BTPC subunits of Class-2 PEPC (Figs. 4A, 5, and Fig. 6A). Furthermore, and as reported for native BTPC Ser-451 kinase (Hill et al., 2014): RcCDPK1 exhibited a relatively narrow substrate specificity, as it phosphorylated BTPC in addition to histone III-S, but was unable to phosphorylate a S451D phosphomimetic BTPC mutant, dephosphorylated COS PTPC or RcSUS1 (Fig. 5), or a pair of synthetic dephosphopeptides containing sequences flanking BTPC’s Ser-451 phosphosite (Supplemental Fig. S3). This not only illustrates the selectivity of RcCDPK1 for phosphorylating BTPC at Ser-451, but also the importance of the structural context of the interaction. Furthermore, all PTPCs and Suc synthases examined to date share an orthologous phosphorylation motif near their N-terminus, i.e. ϕ-5-X-4-basic-3-X-2-X-1-Ser-X+1-X+2-X+3-ϕ+4 (where ϕ is a hydrophobic residue, X is any amino acid, and subscripts denote residue positions relative to the seryl phosphorylation site; Winter and Huber, 2000; O’Leary et al., 2011b; Fedosejevs et al., 2014, 2016). This differs from the unique and highly conserved BTPC recognition motif identified for RcCDPK1, namely: ϕ-4-X-3-Basic-2-X-1-Ser-X+1-X+2-Basic+3-ϕ+4 (Dalziel et al., 2012; Hill et al., 2014; Supplemental Fig. S3). Our combined results therefore demonstrate the importance of not only the linear sequence flanking BTPC’s Ser-451 site, but also the overall conformation of BTPC subunits within the Class-2 PEPC complex. This provides additional evidence that RcCDPK1 is a relatively specific Ser/Thr protein kinase. Figure 4. Open in new tabDownload slide Calcium-dependent phosphorylation of castor BTPC at Ser-451. A and B, Recombinant Class-2 PEPC (10 µg) containing an equivalent ratio of castor BTPC (RcPPC4) and AtPPC3 subunits (O’Leary et al., 2009) was incubated for 20 min at 30°C in 25-μL of the standard phosphorylation assay mix containing 0.1 mm ATP, 10 mm MgCl2, and 0.2 mm CaCl2 (+) or 2 mm EGTA (−) and (A) various CDPKs (250 ng each), or (B) soluble protein extracts (25 μg each) from flowers of 28-d old Arabidopsi Col-0 or mutant atcpk11 knockout plants. C, RcCDPK1 was assayed as above in the presence of 0.2 mm CaCl2 and the indicated PEP concentrations. After SDS-PAGE, the gels were subjected to immunoblotting with anti-pSer451 or anti-BTPC as indicated. Results of (A) to (C) are representative of three independent experiments. Figure 4. Open in new tabDownload slide Calcium-dependent phosphorylation of castor BTPC at Ser-451. A and B, Recombinant Class-2 PEPC (10 µg) containing an equivalent ratio of castor BTPC (RcPPC4) and AtPPC3 subunits (O’Leary et al., 2009) was incubated for 20 min at 30°C in 25-μL of the standard phosphorylation assay mix containing 0.1 mm ATP, 10 mm MgCl2, and 0.2 mm CaCl2 (+) or 2 mm EGTA (−) and (A) various CDPKs (250 ng each), or (B) soluble protein extracts (25 μg each) from flowers of 28-d old Arabidopsi Col-0 or mutant atcpk11 knockout plants. C, RcCDPK1 was assayed as above in the presence of 0.2 mm CaCl2 and the indicated PEP concentrations. After SDS-PAGE, the gels were subjected to immunoblotting with anti-pSer451 or anti-BTPC as indicated. Results of (A) to (C) are representative of three independent experiments. Figure 5. Open in new tabDownload slide Substrate specificity of RcCDPK1. Purified recombinant Class-2 PEPC containing S451D mutant or wild-type BTPC subunits (O’Leary et al., 2009; Dalziel et al., 2012), in vitro dephosphorylated COS Class-1 PEPC or RcSUS1 (Tripodi et al., 2005; Fedosejevs et al., 2014), or histone III-S from calf thymus (10 μg) were incubated for 10 min at 30°C in 25-μL of the standard [γ-32P]ATP phosphorylation assay mix containing 0.2 mm CaCl2 and 250 ng of RcCDPK1. After SDS-PAGE (2 μg protein per lane), the gel was subjected to (A) phosphorimaging and (B) stained with Coomassie Blue R-250 (CBB-250). Results are representative of three independent experiments. Figure 5. Open in new tabDownload slide Substrate specificity of RcCDPK1. Purified recombinant Class-2 PEPC containing S451D mutant or wild-type BTPC subunits (O’Leary et al., 2009; Dalziel et al., 2012), in vitro dephosphorylated COS Class-1 PEPC or RcSUS1 (Tripodi et al., 2005; Fedosejevs et al., 2014), or histone III-S from calf thymus (10 μg) were incubated for 10 min at 30°C in 25-μL of the standard [γ-32P]ATP phosphorylation assay mix containing 0.2 mm CaCl2 and 250 ng of RcCDPK1. After SDS-PAGE (2 μg protein per lane), the gel was subjected to (A) phosphorimaging and (B) stained with Coomassie Blue R-250 (CBB-250). Results are representative of three independent experiments. Figure 6. Open in new tabDownload slide The N-terminal variable domain is required for optimal RcCDPK1 activity. A, The indicated amounts of RcCDPK1 and ∆N-RcCDPK1 were incubated with 20 μg of recombinant Class-2 PEPC for 10 min at 30°C in the standard [γ-32P]ATP phosphorylation assay mix containing 0.2 mm CaCl2. After SDS-PAGE and phosphorimaging, 32P incorporation into BTPC subunits was quantified by digestion of excised Coomassie Blue R-250 stained p118 bands from SDS gels with H2O2, and liquid scintillation counting. B, RcCDPK1 and ∆N-RcCDPK1 (2 μg each) were incubated for 30 min at 30°C in 10 μL of the standard [γ-32P]ATP phosphorylation assay mix containing 0.2 mm CaCl2 (+) or 2 mm EGTA (−), but lacking an exogenous substrate. After SDS-PAGE, autophosphorylation activity was visualized by phosphorimaging. 32P incorporation was quantified by digestion of excised protein-stained RcCDPK1 and ∆N-RcCDPK1 polypeptides from SDS gels with H2O2, and liquid scintillation counting. Results of (A) and (B) are representative of three independent experiments. Figure 6. Open in new tabDownload slide The N-terminal variable domain is required for optimal RcCDPK1 activity. A, The indicated amounts of RcCDPK1 and ∆N-RcCDPK1 were incubated with 20 μg of recombinant Class-2 PEPC for 10 min at 30°C in the standard [γ-32P]ATP phosphorylation assay mix containing 0.2 mm CaCl2. After SDS-PAGE and phosphorimaging, 32P incorporation into BTPC subunits was quantified by digestion of excised Coomassie Blue R-250 stained p118 bands from SDS gels with H2O2, and liquid scintillation counting. B, RcCDPK1 and ∆N-RcCDPK1 (2 μg each) were incubated for 30 min at 30°C in 10 μL of the standard [γ-32P]ATP phosphorylation assay mix containing 0.2 mm CaCl2 (+) or 2 mm EGTA (−), but lacking an exogenous substrate. After SDS-PAGE, autophosphorylation activity was visualized by phosphorimaging. 32P incorporation was quantified by digestion of excised protein-stained RcCDPK1 and ∆N-RcCDPK1 polypeptides from SDS gels with H2O2, and liquid scintillation counting. Results of (A) and (B) are representative of three independent experiments. Phosphorylation of the BTPC subunits of a PTPC-inactive (R644A) Class-2 PEPC mutant (O’Leary et al., 2009) by RcCDPK1 inhibited its PEPC activity by 55 ± 7% (mean ± se, n = 3 determinations) when assayed under suboptimal conditions (pH 7.3, 1 mm PEP, 10 mm l-malate). This validates our previous studies of a S451D phosphomimetic BTPC mutant, as well as the purified native BTPC Ser-451 kinase of developing COS (Dalziel et al., 2012; Hill et al., 2014). These results also corroborate our earlier report (Hill et al., 2014) that BTPC Ser-451 kinase activity is subject to additional posttranslational controls beyond fluctuations in Ca2+ levels, namely inhibition by PEP; i.e. addition of 1 to 5 mm PEP potently inhibited BTPC phosphorylation at Ser-451 by RcCDPK1 (Fig. 4C). PEP appears to directly affect the kinase as similar levels of BTPC Ser-451 kinase inhibition occurred when BTPC or histone III-S served as its substrates (Hill et al., 2014). By contrast, the Class-1 PEPC (i.e. PTPC) protein kinase of developing COS was activated approximately 40% by 1 mm PEP (Murmu and Plaxton, 2007). Reciprocal control of COS RcCDPK1 and PTPC protein kinase by PEP provides an intriguing regulatory mechanism whereby a drop in cytosolic PEP levels would diminish overall anaplerotic PEP carboxylation to oxaloacetate; i.e. by simultaneously enhancing inhibitory phosphorylation of Class-2 PEPC’s BTPC subunits at Ser-451, while attenuating phosphorylation-activation of Class-1 PEPC’s PTPC subunits at Ser-11. Reduced cytosolic PEP levels likely occurs after the elimination of photosynthate import that arises during the final stages of COS development or after depodding when vascular connection with the parent plant has been lost; both processes are accompanied by enhanced in vivo BTPC phosphorylation at Ser-451 and complete PTPC dephosphorylation at Ser-11 (Tripodi et al., 2005; Murmu and Plaxton, 2007; Dalziel et al., 2012). RcCDPK1 also exhibited prominent Ca2+-activated autophosphorylation activity (Fig. 6B). In vitro autophosphorylation at multiple sites has been widely reported for plant CDPKs and may influence their activity and substrate accessibility (Liese and Romeis, 2013). However, the occurrence and functions of in vivo CDPK phosphorylation remain obscure. Autophosphorylation is important for the regulation and/or subcellular localization of eukaryotic protein kinases (Endicott et al., 2012) including at least one plant CDPK, tobacco CDPK2 (NtCDPK2; Witte et al., 2010). Both constitutively phosphorylated residues and stress-induced in vivo phosphorylation, catalyzed by either NtCDPK2 itself or by upstream protein kinases, were reported (Witte et al., 2010). Therefore, an important objective will be to establish RcCDPK1’s (auto)phosphorylation sites, and the impact of site-specific phosphorylation on kinase function. RcCDPK1 orthologs AtCPK4 and GmCDPKβ were also expressed as His6-fusion proteins in E. coli, along with a distantly related Arabidopsis CDPK, AtCPK34. Anti-pSer-451-based kinase assays established that both orthologs, but not AtCPK34, effectively catalyzed Ca2+-dependent phosphorylation of castor BTPC at Ser-451 (Fig. 4A). It is notable that castor BTPC’s Ser-451 phosphorylation site and its adjacent basophilic CDPK recognition motif (Supplemental Fig. S3) are conserved among vascular plant BTPC orthologs, including the Arabidopsis BTPC, AtPPC4 (Hill et al., 2014). Thus, future studies need to determine if AtCPK4 and/or AtCPK11 participate in the in vivo regulatory phosphorylation of AtPPC4, particularly in developing pollen where AtCPK4, AtCPK11, and AtPPC4 are all highly expressed (Harper et al., 2004; Igawa et al., 2010). Extracts prepared from flowers of Col-0 plants but not from atcpk11 mutants showed Ca2+-dependent kinase activity against castor BTPC at Ser-451 suggesting that AtCPK11 is functionally as well as structurally homologous to RcCDPK1 (Fig. 4B). RcCDPK1 Exhibits a Pair of High-affinity Ca2+-binding Sites and Strong Intrinsic Hydrophobicity We next assessed the in vitro Ca2+-binding and hydrophobic properties of RcCDPK1 as these vary among CDPKs and can offer insights into their in vivo roles. RcCDPK1’s binding stoichiometry and affinity for Ca2+ in the presence of Mg2+ was evaluated by isothermal titration calorimetry (Fig. 7A). The data best fit a model that predicted two sets of high affinity Ca2+ binding sites with identical Kd values of 5.03 μM. RcCDPK1’s Kd(Ca2+) values are within the physiological range and compare favorably with K 0.5(Ca2+) values of 2.7 μm reported for the native BTPC Ser-451 kinase purified from developing COS (Hill et al., 2014), as well as 3.1 and 4.5 μm determined for RcCDPK1 orthologs AtCPK4 and AtCPK11, respectively (Boudsocq et al., 2012). Figure 7. Open in new tabDownload slide Biophysical studies of RcCDPK1. A, Analysis of Ca2+ binding to RcCDPK1 by isothermal titration calorimetry. A representative data curve of the RcCDPK1-Ca2+ interaction at 30°C in 25 mm HEPES-KOH (pH 7.5), 100 mm NaCl, and 10 mm MgCl2 is presented. The top panel shows the calorimetric titration of 2.0 mL of 20 μm RcCDPK1 with 500 μm CaCl2 using 29 injections of 10 μL each. The lower panel presents the corresponding integrated binding isotherm modeled to two sets of binding sites. B, 8-Anilinonaphthalene-1-sulfonic acid fluorescence emission spectra from 430 to 600 nm were collected to examine the exposed hydrophobicity of 25 μm RcCDPK1 versus petunia CaM81 in 25 mm HEPES-KOH (pH 7.5) containing 100 mm KCl and 1 mm DTT, with various additions as shown. Background fluorescence in the absence of RcCDPK1 or CaM81 is also shown. C, Far-UV circular dichroism spectroscopy was used to study the impact of Ca2+ addition on the secondary structure characteristics of RcCDPK1 in 5 mm Tris-HCl or HEPES-KOH (pH 7.5). Each spectrum is representative of at least 10 averaged scans. Figure 7. Open in new tabDownload slide Biophysical studies of RcCDPK1. A, Analysis of Ca2+ binding to RcCDPK1 by isothermal titration calorimetry. A representative data curve of the RcCDPK1-Ca2+ interaction at 30°C in 25 mm HEPES-KOH (pH 7.5), 100 mm NaCl, and 10 mm MgCl2 is presented. The top panel shows the calorimetric titration of 2.0 mL of 20 μm RcCDPK1 with 500 μm CaCl2 using 29 injections of 10 μL each. The lower panel presents the corresponding integrated binding isotherm modeled to two sets of binding sites. B, 8-Anilinonaphthalene-1-sulfonic acid fluorescence emission spectra from 430 to 600 nm were collected to examine the exposed hydrophobicity of 25 μm RcCDPK1 versus petunia CaM81 in 25 mm HEPES-KOH (pH 7.5) containing 100 mm KCl and 1 mm DTT, with various additions as shown. Background fluorescence in the absence of RcCDPK1 or CaM81 is also shown. C, Far-UV circular dichroism spectroscopy was used to study the impact of Ca2+ addition on the secondary structure characteristics of RcCDPK1 in 5 mm Tris-HCl or HEPES-KOH (pH 7.5). Each spectrum is representative of at least 10 averaged scans. Calcium-binding to EF-hand containing proteins, including CDPKs, initiates conformational changes in their conserved CaM-related domain that typically results in exposure of hydrophobic clefts believed to be critical to target recognition and interaction (DeFalco et al., 2009). These conformational changes are a hallmark of Ca2+ sensors and have been exploited for their efficient purification via Ca2+-dependent hydrophobic interaction chromatography. Although RcCDPK1 exhibited a Ca2+-dependent electrophoretic mobility shift reminiscent of a classic Ca2+ sensor (Figs. 3A and 6B), its unusual Ca2+-independent hydrophobicity was demonstrated by 8-anilinonaphthalene-1-sulfonic acid-based fluorescence spectroscopy and hydrophobic interaction chromatography using phenyl-Sepharose (Fig. 7B and Supplemental Fig. S4). 8-Anilinonaphthalene-1-sulfonic acid exhibits a blue shift and increases fluorescence when it interacts with nonpolar surfaces on proteins, whereas Ca2+-dependent binding to phenyl-Sepharose has been reported for CaM as well as several, but not all, CDPKs and CMLs. Fluorescence spectroscopy revealed that apo-RcCDPK1 [in contrast to petunia (Petunia) CaM81, a positive control] exhibits considerable hydrophobicity that was unaffected by the addition of up to 1 mm Ca2+ (Fig. 7B). Similarly, the native BTPC Ser-451 kinase from developing COS exhibited tight Ca2+-independent binding to phenyl-Sepharose and could only be eluted from this resin using a buffer containing 10% (v/v) ethylene glycol (i.e. a chaotropic solvent; Supplemental Fig. S4) or a detergent such as 0.5% (v/v) Triton X-100 (results not shown). Similar results were reported for cytosolic CDPKs that, respectively, phosphorylate Suc synthase at Ser-11 in developing COS (Fedosejevs et al., 2016) and nitrate reductase in spinach leaves (Bachmann et al., 1996; Douglas et al., 1998). Calcium-mediated conformational changes in RcCDPK1 were further investigated by probing its secondary structure using far-UV circular dichroism spectroscopy. This indicated that apo-RcCDPK1 was folded in solution, and exhibited a large positive band below 200 nm, with maxima at 191 nm and negative bands with local minima at 209 nm and 220 nm, typical of proteins having significant α-helical structure (Fig. 7C). The apo- and Ca2+-bound RcCDPK1 spectra were almost identical, indicating little change in secondary structure composition upon Ca2+ binding. Conformational changes associated with Ca2+ binding to RcCDPK1 may trigger a reorientation of helices, as occurs with CaM (Zhang et al., 1995), rather than pronounced changes in helical content. These data demonstrate that high-affinity Ca2+ binding by RcCDPK1 induces predominately tertiary but not secondary structural changes associated with increased surface hydrophobicity as exhibited by canonical Ca2+ sensors such as CaM (DeFalco et al., 2009). The strong intrinsic hydrophobicity, and lack of increased hydrophobicity upon exposure to Ca2+, distinguishes RcCDPK1 from CaM and most CMLs examined to date. This indicates that while Ca2+ is clearly important for regulating RcCDPK1 activity (Figs. 4A and 6B; Hill et al., 2014), it may do so through structural changes that lead to relief of autoinhibition rather than exposure of hydrophobic target-interacting regions. Given the relatively large number of CDPK isozymes in a given plant, such unique biochemical features may reflect the diversity needed to ensure specificity of activity, especially whenever multiple CDPKs are coexpressed. RcCDPK1 Is Targeted to the Cytosol and Nucleus of Tobacco Bright Yellow-2 Cells, But Relocalizes to the Mitochondrial Surface when Coexpressed with BTPC The subcellular location and potential interaction of fluorescent protein fusions of RcCDPK1 and ∆N-RcCDPK1 with castor BTPC were assessed in tobacco bright yellow-2 (BY-2) suspension cells, a well-established model system for protein localization and interaction studies (Brandizzi et al., 2003). Cells were transiently transformed using gene constructs encoding a variety of fluorescent protein-tagged RcCDPK1 and BTPC fusion proteins (Fig. 8A), and imaged by confocal laser-scanning microscopy (CLSM). C-terminal Cherry-tagged versions of RcCDPK1 and ƊN-RcCDPK1, similar to Cherry alone, localized to the cytosol and nucleus of individual, representative transformed BY-2 cells (Fig. 8, B to D). Likewise, a RcCDPK1-GFP fusion, as well as N-terminal Cherry-tagged versions of RcCDPK1 and ƊN-RcCDPK1, localized to the cytosol and nucleus in BY-2 cells (Supplemental Fig. S5, A to C), indicating that the position and/or type of appended fluorescent protein does not influence the subcellular localization of RcCDPK1 or ƊN-RcCDPK1. Notably, the partial nuclear targeting of RcCDPK1 may be due to its putative nuclear localization signal, -P58KRK61- (Rodriguez Milla et al., 2006), which is conserved in the orthologs aligned in Figure 1, including AtCPK4 whose GFP-tagged fusion protein was also localized to the cytosol and nucleus (of transgenic Arabidopsis plants; Dammann et al., 2003). Figure 8. Open in new tabDownload slide Relocalization of RcCDPK1 and ƊN-RcCPDK1 by castor BTPC to the mitochondria of tobacco BY-2 cells. A, Schematic illustration of fluorescent protein fusion constructs of wild-type and mutant versions of RcCDPK1 and castor BTPC that were transiently expressed in tobacco BY-2 cells. BTPC’s intrinsically disordered region is highlighted in blue. The numbers above each construct indicate amino acid residue positions. B to I, Representative CLSM micrographs of individual tobacco BY-2 cells (co)expressing (as indicated by panel labels) the following: (B) RcCDPK1-Cherry, (C) ∆N-RcCDPK1-Cherry, (D) Cherry, (E) RcCDPK1-Cherry and p118-EYFP, (F) ∆N-RcCDPK1-Cherry and p118-EYFP, (G) RcCDPK1-Cherry and p54-EYFP, (H) RcCDPK1-Cherry and p64-EYFP, and (I) RcCDPK1-Cherry and p40-EYFP. Note that the fluorescence attributable to RcCDPK1 and p118 (and mutant versions thereof) was false-colorized magenta and green, respectively. Shown in (B) to (D) are the corresponding differential interference contrast images. Corresponding merged images are presented in (E) and (F); white color represents protein colocalization. Arrowheads and boxes representing portions of the cells shown at higher magnification in the insets in (E) and (F) indicate obvious examples of colocalization of RcCDPK1-Cherry or ƊN-RcCDPK1-Cherry with p118-EYFP at (aggregated) mitochondria. Scale bar = 10 μm. Figure 8. Open in new tabDownload slide Relocalization of RcCDPK1 and ƊN-RcCPDK1 by castor BTPC to the mitochondria of tobacco BY-2 cells. A, Schematic illustration of fluorescent protein fusion constructs of wild-type and mutant versions of RcCDPK1 and castor BTPC that were transiently expressed in tobacco BY-2 cells. BTPC’s intrinsically disordered region is highlighted in blue. The numbers above each construct indicate amino acid residue positions. B to I, Representative CLSM micrographs of individual tobacco BY-2 cells (co)expressing (as indicated by panel labels) the following: (B) RcCDPK1-Cherry, (C) ∆N-RcCDPK1-Cherry, (D) Cherry, (E) RcCDPK1-Cherry and p118-EYFP, (F) ∆N-RcCDPK1-Cherry and p118-EYFP, (G) RcCDPK1-Cherry and p54-EYFP, (H) RcCDPK1-Cherry and p64-EYFP, and (I) RcCDPK1-Cherry and p40-EYFP. Note that the fluorescence attributable to RcCDPK1 and p118 (and mutant versions thereof) was false-colorized magenta and green, respectively. Shown in (B) to (D) are the corresponding differential interference contrast images. Corresponding merged images are presented in (E) and (F); white color represents protein colocalization. Arrowheads and boxes representing portions of the cells shown at higher magnification in the insets in (E) and (F) indicate obvious examples of colocalization of RcCDPK1-Cherry or ƊN-RcCDPK1-Cherry with p118-EYFP at (aggregated) mitochondria. Scale bar = 10 μm. We next studied whether subcellular targeting of RcCDPK1-Cherry was altered upon its coexpression with a castor BTPC-enhanced yellow fluorescent protein (EYFP) fusion. The in vivo association of castor BTPC with the surface of mitochondria in developing COS, as well as after its expression as a EYFP-fusion protein in tobacco BY-2 cells, has been well documented (Park et al., 2012). Likewise, transiently expressed, EYFP-tagged castor BTPC (i.e. p118-EYFP) colocalized with an endogenous mitochondrial marker enzyme (i.e. cytochrome c oxidase; Supplemental Fig. S5D). When RcCDPK1-Cherry was coexpressed with p118-EYFP it partially colocalized with BTPC at aggregated mitochondria (Fig. 8E), which are considered to have coalesced owing to BTPC expression and association (Park et al., 2012). This supports the hypothesis of an in vivo interaction between RcCDPK1 and BTPC, and is reminiscent of the reported occurrence of an unspecified CDPK isoform and multiple phosphoproteins in the outer envelope of mitochondria purified from potato tubers (Pical et al., 1993). Allied control experiments confirmed that the colocalization of RcCDPK1-Cherry with p118-EYFP at aggregated mitochondria (Fig. 8E), was not due to p118-EYFP fluorescence bleed-through during CLSM imaging (Supplemental Fig. S5E). Furthermore, no protein colocalization occurred when p118-EYFP was coexpressed with Cherry alone (Supplemental Fig. S5F), nor when RcCDPK1-Cherry was coexpressed with mito-EYFP, which consisted of the E1α subunit of the COS mitochondrial pyruvate dehydrogenase complex appended to EYFP (Supplemental Fig. S5G). BTPC’s Intrinsically Disordered Region Is an RcCDPK1 Interaction Domain When PEPC was first isolated from developing COS, 64-kD BTPC polypeptides were identified by LC-MS/MS and determined to be the binding partner of PTPC subunits in the purified Class-2 PEPC complex (Blonde and Plaxton, 2003). Subsequent research revealed that the native BTPC exists as a p118, but that upon COS extraction it is rapidly cleaved by an endogenous thiol endopeptidase into 54-kD and 64-kD polypeptides (p54 and p64; Gennidakis et al., 2007). BTPC’s proteolytic cleavage site (Lys-446/Ile-447) as well as its in vivo Ser-425 and Ser-451 phosphorylation sites all occur within an approximate 10-kD intrinsically disordered region, a distinctive feature of green algal and vascular plant BTPCs that mediates their tight interaction with coexpressed PTPC subunits to form the Class-2 PEPC heteromeric complex (O’Leary et al., 2011b; Park et al., 2012). To map the RcCDPK1-interaction domain of COS BTPC, we initially focused our attention on p54 and p64. As with full-length p118-EYFP (Fig. 8E), RcCDPK1-Cherry partially colocalized with p54-EYFP, but not with p64-EYFP, at (aggregated) mitochondria in BY-2 cells (Fig. 8, G and H). Based on these results, we next tested whether the intrinsically disordered region of BTPC is a potential RcCDPK1-interaction domain. Specifically, RcCDPK1-Cherry was coexpressed with an additional BTPC truncation mutant, namely p40-EYFP (consisting of p54, but without the disordered region; corresponding to BTPC residues 1 to 329). As shown in Figure 8I, p40-EYFP localized to the mitochondria, as expected (Park et al., 2012), but failed to relocalize coexpressed RcCDPK1-Cherry from the cytosol to mitochondria. Taken together, these results indicate that the interaction of RcCDPK1 and BTPC/p118 is mediated, at least in part, by BTPC’s intrinsically disordered region. Similarly, COS PTPC’s p107 subunits localized to the cytosol in the tobacco BY-2 cells, but relocalized to the mitochondrial surface when coexpressed with BTPC/p118 or with various deletion mutants containing portions of BTPC’s disordered region (Park et al., 2012). Intrinsically disordered regions are widespread among different proteins, provide a docking site to promote protein-protein interactions by recruiting binding partners, and are susceptible to site-specific phosphorylation (Dunker et al., 2005). Castor BTPC is no exception because its Ser-425 and Ser-451 phosphorylation sites occur within its disordered region (O’Leary et al., 2011c; Dalziel et al., 2012). Furthermore, this domain appears to be essential for mediating BTPC’s in vivo interaction with PTPC within a Class-2 PEPC complex (Park et al., 2012), as well as with RcCDPK1 (Fig. 8). RcCDPK1 Binding to BTPC Is Ca2+ Dependent N-terminal GST-tagged RcCDPK1 (GST-RcCDPK1) was heterologously expressed and purified, then adsorbed to glutathione beads and incubated with recombinant Class-1 or Class-2 PEPCs followed by affinity chromatography. GST-RcCDPK1 bound to the BTPC-containing Class-2 PEPC in a Ca2+-dependent manner (Fig. 9). Thus, conformational changes induced by Ca2+ binding not only activate RcCDPK1 (Figs. 4A and 6B; Hill et al., 2014), but also control the ability of RcCDPK1 to recruit its BTPC substrate. GST-RcCDPK1 specifically associated with the BTPC subunits of Class-2 PEPC, because no interaction was detected when it was incubated with purified Class-1 PEPC (i.e. a PTPC homotetramer) isolated from AtPPC3-overexpressing E. coli cells (Fig. 9). Figure 9. Open in new tabDownload slide In vitro interaction of GST-RcCDPK1 or GST-∆N-RcCDPK1 with BTPC subunits of recombinant Class-2 PEPC is Ca2+ dependent. GST, GST-RcCDPK1, and GST-∆N-RcCDPK1 (2 μg each) were, respectively, immobilized on glutathione beads and incubated with pure recombinant Class-1 and/or Class-2 PEPCs (5 μg each; O’Leary et al., 2009) in the presence of 0.5 mm CaCl2 (+) or 2 mm EGTA (−) as indicated. Bound proteins were eluted with a buffer containing GSH and analyzed via SDS-PAGE (2.5 μg protein/lane), (A) protein staining with Coomassie Brilliant Blue R-250, or (B) immunoblotting with anti-BTPC or anti-PTPC. Figure 9. Open in new tabDownload slide In vitro interaction of GST-RcCDPK1 or GST-∆N-RcCDPK1 with BTPC subunits of recombinant Class-2 PEPC is Ca2+ dependent. GST, GST-RcCDPK1, and GST-∆N-RcCDPK1 (2 μg each) were, respectively, immobilized on glutathione beads and incubated with pure recombinant Class-1 and/or Class-2 PEPCs (5 μg each; O’Leary et al., 2009) in the presence of 0.5 mm CaCl2 (+) or 2 mm EGTA (−) as indicated. Bound proteins were eluted with a buffer containing GSH and analyzed via SDS-PAGE (2.5 μg protein/lane), (A) protein staining with Coomassie Brilliant Blue R-250, or (B) immunoblotting with anti-BTPC or anti-PTPC. The N-terminal Variable Domain Is Important for RcCDPK1’s Trans- and Autophosphorylation Activities, But Not for its Ca2+-Dependent Binding to BTPC The N-terminal variable domain has been the focus of several CDPK studies examining its role in substrate recognition/binding, subcellular localization, and catalysis. For example, this domain of NtCDPK1 appears to mediate a Ca2+-dependent interaction with its substrate, the transcription factor REPRESSION OF SHOOT GROWTH FACTOR that controls expression of gibberellin biosynthetic genes (Ito et al., 2010). N-terminal variable domain deletion eliminated the in vitro and in vivo interaction of NtCDPK1 with REPRESSION OF SHOOT GROWTH FACTOR, while significantly inhibiting its ability to in vitro phosphorylate this substrate at Ser-114 (Ito et al., 2010). Similarly, the N-terminal variable domain of: (i) AtCPK32 is necessary but not solely sufficient for its interaction with a substrate AtABF4 (Choi et al., 2005), and (ii) a potato CDPK, StCDPK5, is essential for ensuring its proper subcellular location and consequent discrimination of its substrate, NADPH oxidase (Asai et al., 2013). We approached this problem by expressing ∆N-RcCDPK1 as His6-, Cherry-, and GST-tagged fusion proteins. N-terminal variable domain deletion triggered a marked reduction in the ability of Ca2+-activated RcCDPK1 to phosphorylate castor BTPC at Ser-451 (Figs. 4A and 6A). Similarly, parallel autophosphorylation assays revealed that relative autophosphorylation activity of ∆N-RcCDPK1 was approximately 90% lower than that of the nontruncated RcCDPK1 (Fig. 6B). Ca2+ addition enhanced RcCDPK1 autophosphorylation activity by approximately 50%, but had no obvious impact on ∆N-RcCDPK autophosphorylation. By contrast, deletion of the N-terminal variable domain did not disrupt the apparent in vivo interaction of ∆N-RcCDPK1-Cherry with p118-EYFP in tobacco BY-2 cells (Fig. 8F), nor hinder the ability of GST-∆N-RcCDPK1 to specifically interact with its BTPC substrate in a Ca2+-dependent fashion (Fig. 9). Overall, these results indicate that although the N-terminal variable domain of RcCDPK1 is not critical for target recognition, it clearly contributes to fine-tuning kinase activity for optimal Ca2+-dependent phosphorylation of BTPC at Ser-451. CONCLUSIONS In summary, we have identified BTPC as a target for a specific CDPK isozyme in developing COS, and likely across plant taxa. This suggests an intriguing potential link between cytosolic Ca2+ signaling and the control of carbon partitioning at the critical PEP branchpoint in a biosynthetically active, heterotrophic plant tissue. These results have implications not only for PEPC’s broader role in the control of cytosolic carbohydrate partitioning and anaplerosis, but also for the regulation of the enigmatic BTPC by reversible phosphorylation. It is notable that the developmental pattern of in vivo BTPC phosphorylation at Ser-451 in COS (Dalziel et al., 2012; Hill et al., 2014), as well as the basophilic phosphorylation motif flanking its Ser-451 residue (Supplemental Fig. S3), are distinct from those of COS PTPC and RcSUS1 (Tripodi et al., 2005; Fedosejevs et al., 2016). These differences imply specific protein kinase-phosphatase pairings in controlling the phosphorylation status of BTPC, PTPC versus RcSUS1 in developing COS. Indeed, a low molecular mass (approximately 30 kD), Ca2+-independent, and highly specific PTPC kinase mediates Ser-11 phosphorylation/activation of the PTPC subunits of Class-1 PEPC during COS development (Tripodi et al., 2005; Murmu and Plaxton, 2007). Furthermore, RcCDPK2 phosphorylates RcSUS1 at Ser-11 in developing COS and shows many additional and prominent differences compared to RcCDPK1 including: (i) a relatively nonspecific in vitro substrate selectivity, including abundant peptide kinase activity; (ii) a significantly lower K 0.5(Ca2+) value of approximately 200 nM; (iii) insensitivity to metabolite effectors including PEP; (iv) partial association with microsomal membranes; and (iv) a completely opposite developmental profile in COS (i.e. RcCDPK2 expression, RcSUS1 Ser-11 kinase activity, and in vivo RcSUS1 phosphorylation at Ser-11 progressively decrease during COS development; Fedosejevs et al., 2014, 2016). Several attempts at metabolic engineering of PEPC have failed due to a lack of in-depth knowledge of the enzyme’s multifaceted posttranslational controls (O’Leary et al., 2011b). Owing to the commercial interest in modifying photosynthate partitioning to agronomically important end-products such as oil or storage proteins during seed development, and the key position of PEPC in controlling this process, our castor bean PEPC research should prove informative for developing innovative strategies for engineering PEPC activity. Given PEPC’s critical position in central plant metabolism, it is perhaps not surprising that the emerging model for regulation of nonphotosynthetic PEPCs involves a complex and unique set of posttranslational control mechanisms that work hand-in-hand with the well-documented regulation of PTPC-containing Class-1 PEPCs (including C4 and CAM photosynthetic PEPCs) by allosteric effectors and reversible phosphorylation (Supplemental Fig. S1; O’Leary et al., 2011b). These control mechanisms include BTPC’s remarkable role as a catalytic and regulatory subunit of the allosterically desensitized Class-2 PEPC heteromeric complex (O’Leary et al., 2009), and RcCDPK1’s Ca2+-dependent, inhibitory phosphorylation of the BTPC subunits at Ser-451. A major challenge will be to link diverse posttranslational PEPC controls with the in vivo regulation of PEP partitioning to specific metabolic pathways. This now includes consideration of how transient alterations in cytosolic Ca2+ signatures and PEP levels may serve to modulate RcCDPK1 phosphorylation of BTPC at Ser-451 and thereby contribute to the control of anaplerotic PEP flux and respiratory CO2 recycling by Class-2 PEPCs. Given the central importance of protein phosphorylation as a posttranslational regulatory mechanism, the diversity of CDPKs in plants, and the lack of in vivo CDPK targets that have been pinpointed to date, our RcCDPK1 and RcCDPK2 studies represent a significant advance that helps to set the stage for future research aimed at exploring the interplay between Ca2+ signaling and the integration and control of plant carbon metabolism. MATERIALS AND METHODS Plant Material, Protein Extraction, PEPC Activity Assays, and Protein Concentration Determination Castor (Ricinus communis L.; cv Baker 296) plants were cultivated in a greenhouse as described in O’Leary et al. (2011a). Tissues were rapidly harvested, frozen in liquid N2, and stored at −80°C until used. Tobacco (Nicotiana tabacum L.) BY-2 suspension-cultured cells were maintained as described by Park et al. (2012). A homozygous Arabidopsis (Arabidopsis thaliana) mutant line (atcpk11, SALK_054495) was kindly provided by Prof. Jeffrey Harper (University of Nevada). Col-0 and atcpk11 seeds were sown in a standard soil mixture (Sunshine Aggregate Plus Mix 1; SunGro) and stratified at 4°C for 3 d. Plants were cultivated in growth chambers at 23°C (16/8 h photoperiod at 100 μmol m−2 s−1 PAR) and fertilized biweekly by subirrigation with 0.25× Hoagland’s media. Flowers (1 g) from 28-d-old plants were ground to a powder under liquid N2 and homogenized (1:2; w/v) in 50 mm HEPES-KOH (pH 7.3) containing 1 mm EDTA, 1 mm EGTA, 10 mm MgCl2, 20% (v/v) glycerol, 5 mm thiourea, 1 mm DTT, 1% (w/v) polyvinyl(polypyrrolidone), 1 mm 2,2′-dipyridyl disulfide, 1 mm PMSF, 25 mm NaF, 1 mm Na3VO4, and 1 mm Na2MoO4. After centrifugation, the supernatant fluid was subjected to nonradioactive BTPC Ser-451 kinase assays as described below and in the legend for Figure 4A. PEPC activity was assayed at 25°C by following NADH oxidation at 340 nm using a Molecular Devices microplate spectrophotometer as described in O’Leary et al. (2009) and Hill et al. (2014). Protein concentrations were routinely determined by Coomassie Blue G-250 dye binding using bovine γ-globulin (Pierce) as the protein standard. LC-MS/MS and Protein Identification Proteins in the final BTPC Ser-451 kinase preparation of Hill et al. (2014) were reduced with 10 mm DTT, alkylated with 55 mm iodoacetamide, dialyzed against 10 mm ammonium bicarbonate, and dried using a CentriVap centrifugal concentrator (Labconco). Protein digestion was performed using sequencing-grade trypsin (Promega) in 25 mm ammonium bicarbonate at 1:100 ratio of trypsin to protein substrates. Tryptic peptides were dried and reconstituted with 4 μL of 0.1% formic acid, and identified using an on-line nanoAcquity UPLC (Waters) coupled with an Orbitrap Fusion Tribrid Mass Spectrometer (Thermo Fisher Scientific). The peptides were trapped by a 2G-V/MT Trap symmetry C18 column (5 μm particles, 180 µm i.d. × 20 mm length) for 3 min at a flow rate of 5 μL/min, and separated on a BEH130 C18 analytical column (1.7 μm particles, 100 μm i.d. × 100 mm length) at 300 nL/min for 1 h. The mobile phase was set up to a linear gradient from 5% to 30% solvent B (0.1% formic acid in acetonitrile) over 40 min for peptide elution, followed by flushing with 85% solvent B for 15 min and reequilibrating the column with solvent A (0.1% formic acid) for 10 min. MS survey scan was acquired with a high resolution of 60,000 at the mass region of m/z 350 to 1800, and MS/MS measurements were performed by collision-induced dissociation at data-dependent acquisition mode for scanning the top 20 most intense ions at multipally charged states of 2+ to 7+, respectively. Dynamic exclusion was set to 60 s. MS/MS data were searched against the R. communis protein sequence in the NCBI database using Mascot Server (version 2.5.1; Matrix Science). The search parameters were restricted to tryptic peptides at a maximum of two missed cleavages. Cys carbamidomethylation was designated as a fixed modification, and N-terminal protein acetylation, deamidation of Asn and Gln, oxidation of Met, and phosphorylation of Ser, Thr, and Tyr were considered as variable modifications. Mass tolerances were set up to 10 ppm for Orbitrap MS ions and 0.8 D for ion-trap MS/MS fragment ions. Peptide assignments were filtered by an ion score cutoff at 20, and the identified MS/MS spectra were verified manually. qRT-PCR Total RNA was extracted using an RNeasy kit (QIAGEN) following the manufacturer’s protocols. The DNase-treated RNA was used for cDNA synthesis with a QuantiTect reverse transcription kit (QIAGEN). Quantitative PCR was performed using a 7500 Real-Time PCR System (Applied Biosystems) and GoTaq qPCR Master Mix (Promega). The reaction procedures were as follows: denature at 95°C for 5 min, followed by 40 cycles of 95°C for 15 s, 60°C for 15 s, and 72°C for 34 s. The castor Actin (AY360221) gene (RcActin) was used as an internal control. Primers (Supplemental Table S3) were designed using the software DNAman (Version 5.0; Lynnon Biosoft), and results were analyzed with the software Applied Biosystems 7500 (Version 2.0.1). Relative gene expression was calculated using the relative 2–ƊƊCt method (Livak and Schmittgen, 2001). All the experiments were repeated at least three times using cDNAs prepared from two biological replicates. RcCDPK1 Cloning and Heterologous Expression Standard PCR amplification was used to introduce restriction sites for all subcloning procedures, and all constructs were confirmed by DNA sequencing. Cloning of a cDNA encoding RcCDPK1 was initiated by designing gene-specific primers (Supplemental Table S2). A full-length cDNA library from stage V developing COS endosperm (Gennidakis et al., 2007) was used as the template to amplify a PCR product that was amplified and cloned into pGEM-T vector (Promega) for sequencing. Sequence assembling was performed using the software DNAman (Version 5.0). Alignment of RcCDPK1’s deduced amino acid sequence with several of its orthologs was performed using the software Vector NTI (Life Technologies) with default parameters. For heterologous expression of RcCDPK1, its full-length cDNA was subcloned into a pET30a(+) expression vector (Novagen) carrying an N-terminal His6-tag. A second construct encoding the His6-tagged N-terminally truncated RcCDPK1 (residues 30 to 497; ∆N-RcCDPK1) was also produced. For recombinant protein production, both constructs were separately transformed into Escherichia coli [BL21-CondonPlus (DE3)-RIL); Stratagene], and cultured overnight at 37°C in 500 mL of LB broth containing 50 μg/mL kanamycin to an A 600 of approximately 0.6. Protein production was induced using 0.4 mm isopropyl-β-d-thiogalactoside for 3 h at 37°C. Cells (approximately 5 gFW) were harvested by centrifugation, resuspended in 40-mL of ice-cold buffer A (50 mm NaH2PO4, pH 8.0, containing 300 mm NaCl), and lysed at 4°C by passage through a French Pressure Cell at 18,000 p.s.i. After centrifugation, the supernatant fluid was loaded at 1 mL/min onto a column (1.6 × 10 cm) of PrepEase His-tagged High Yield Purification Ni2+-affinity resin (Affymetrix) equilibrated in buffer A. The column was washed with buffer A until the A 280 approached baseline and then eluted with buffer A containing 250 mm imidazole. Pooled peak fractions were concentrated to 1 mL with an Amicon Ultra-15 Centrifugal Filter unit (30-kD cutoff; EMD Millipore), divided into 25-μL aliquots, frozen in liquid N2 and stored in −80°C. For cleaving the His6-tag, purified RcCDPK1 was incubated with enterokinase (New England Biolabs) according to the manufacturer’s recommendations. For the generation of GST-RcCDPK1 and GST-∆N-RcCDPK1, the respective constructs were ligated into a pGEX-4T-3 vector (GE Healthcare). The proteins were heterologously expressed in E. coli as described above and purified using PrepEase Protein Purification Glutathione Agarose 4B (Affymetrix) according to the manufacturer’s instructions. Purified GST-RcCDPK1 and GST-∆N-RcCDPK1 were concentrated and stored as described above. RcCDPK1 Antibody Production and Immunoblotting Rabbit antiserum against RcCDPK1 was produced using a synthetic peptide (Synpeptide; www.synpeptide.com) designed to match amino acids 1 to 29 (i.e. the N-terminal variable domain; Supplemental Fig. S2A), with an additional Cys residue introduced at the N-terminus. Purified peptide was coupled to maleimide-activated keyhole limpet hemocyanin (Life Technologies), dialyzed against Pi buffered saline (pH 7.4), filter-sterilized, and emulsified with Titermax Gold (CytRx). After collection of preimmune serum, desalted conjugate (750 μg) was injected subcutaneously into a rabbit, and a booster injection (250 μg) was administered at 28 d. Two weeks after the final injection, blood was collected in Vacutainer tubes (Becton Dickinson) by cardiac puncture. Clotted cells were removed by centrifugation at 1000 g, and the immune serum frozen in liquid N2 and stored at −80°C in 0.04% (w/v) NaN3. For immunoblotting, anti-RcCDPK1 was affinity-purified against 500 μg of nitrocellulose-bound recombinant RcCDPK1, as described in Dalziel et al. (2012). Production of anti-PTPC, anti-BTPC and anti-pSer451, SDS-PAGE, immunoblotting, and chromogenic detection of immunoreactive polypeptides were carried out as described in Gennidakis et al. (2007), O’Leary et al. (2009), and Dalziel et al. (2012). All immunoblots were replicated at least three times with representative results shown in the figures. In Vitro Kinase Assays Kinase activity was assayed by monitoring P incorporation from nonradioactive or [γ-32P]-labeled ATP into the p118 BTPC subunits of purified, heterologously expressed Class-2 PEPC as described in Hill et al. (2014). Recombinant CDPKs (250 ng) were routinely incubated with 10 μg of the Class-2 PEPC substrate (corresponding to 5 μg p118/BTPC subunits) in a 25 μL reaction mix containing 50 mm HEPES-KOH (pH 7.3), 10 mm MgCl2, 1 mm DTT, 0.1 mm Na3VO4, 0.1 mm Na2MoO4, 10% (v/v) glycerol, and 0.2 mm CaCl2. Reactions were initiated by the addition of 0.2 mm ATP or [γ-32P]ATP (1000 cpm/pmol), incubated at 30°C for up to 20 min, and terminated by addition of SDS-PAGE sample buffer followed by heating at 100°C for 3 min. For nonradioactive kinase assays, P incorporation from ATP into p118 was determined by subjecting 10-μL aliquots of kinase assays to SDS-PAGE and immunoblotting with anti-pSer451 and anti-BTPC. For radiometric assays, the level of 32P incorporation from [γ-32P]ATP into p118 was visualized by subjecting 10-μL aliquots of kinase assays to SDS-PAGE and developing Coomassie Blue R-250 stained gels overnight in a phosphorimager cassette (Molecular Dynamics) followed by scanning of the cassette using a Typhoon 6000 (GE Healthcare). 32P incorporation was quantified by liquid scintillation counting after H2O2 digestion of excised SDS gel slices containing Coomassie Blue R-250-stained p118 as described in Hill et al. (2014). Autophosphorylation Assays RcCDPK1 and ∆N-RcCDPK1 (2 µg each) were separately incubated at 30°C for 30 min with 0.2 mm [γ-32P]ATP (1000 cpm/pmol) in the following autophosphorylation assay buffer: 25 mm HEPES-KOH, pH 7.3, containing 10 mm MgCl2 and 0.2 mm CaCl2 or 2 mm EGTA in a final volume of 10 μL. Reactions were terminated by addition of SDS-PAGE sample buffer and heating at 100°C for 3 min. The samples were subjected to SDS-PAGE and phosphorimaging as described above. Biophysical Studies Isothermal scanning calorimetry was performed at 30°C on a VP-ITC Microcalorimeter (MicroCal). RcCDPK1 (10 mg in 1.5 mL) was further purified by gel filtration at 0.3 mL/min on a Superdex 200 HiLoad 16/60 column (GE Healthcare) equilibrated with 25 mm HEPES-KOH (pH 7.5) containing 100 mm NaCl, 2 mm EDTA, and 2 mm EGTA. Pooled peak fractions were concentrated to 1 mL using an Amicon Ultra-15 concentrator (30-kD cutoff; EMD Millipore) and dialyzed against 25 mm HEPES-KOH (pH 7.5) containing 100 mm NaCl and 10 mm MgCl2. RcCDPK1 (20 µM) and 10-μL injections (29 injections at 360 s intervals) of 500 μm CaCl2 were used in each experiment (with appropriate buffer blanks). Origin 7.0 software (MicroCal) was used to obtain values for stoichiometry (N) and dissociation constants (Kd), and binding-type input parameters were adjusted to obtain the best fitting model for each experiment. Fluorescence spectroscopy was performed using 25 μM RcCDPK1 and 250 μm 8-anilinonaphthalene-1-sulfonic acid in 10 mm Tris-HCl (pH 7.5) containing 100 mm KCl and 1 mm DTT (with various additions described in Fig. 7B). RcCDPK1’s fluorescence emission spectra were recorded at 23°C using black Fluotrac-200 96-well microplates on a SPECTRAmax GEMINI XS Spectrofluorometer (Molecular Dynamics). Petunia CaM81 (15 μM; Bender et al., 2014) was monitored alongside RcCDPK1 as a positive control for Ca2+-dependent hydrophobic exposure. Far-UV circular dichroism spectra from 182 nm to 260 nm were acquired at 23°C on a Chirascan CD Spectrometer (Applied Photophysics) using a cylindrical quartz cuvette with a pathlength of 0.1 mm. Samples of 15 μm RcCDPK1 were used for data acquisition as described in Figure 7C. Spectra from a minimum of 10 replicate scans were averaged and corrected for background. Transient Transformation and Imaging of Tobacco BY-2 Suspension Cells Full-length and a 5′-truncated version (corresponding to ƊN-RcCDPK1) of the RcCDPK1 cDNA sequence (with or without a stop codon) were amplified via PCR with the appropriate primers (Supplemental Table S2) from pET30a-RcCDPK1 (see above). Resulting DNA fragments were gel-purified and subcloned into pRTL2-Cherry using XmaI and NheI to yield RcCDPK1-Cherry and ƊN-RcCDPK1-Cherry. To generate Cherry-RcCDPK1 and Cherry-ƊN-RcCDPK1, purified fragments were subcloned into pRTL2-Cherry (a transient expression vector containing the 35S cauliflower mosaic virus promoter and the open reading frame of the red fluorescent protein Cherry; Gidda et al., 2011) using BamHI. To construct RcCDPK1-GFP, pGreen35S C-GFP and an RcCDPK1 PCR fragment were digested with BamHI and EcoRV, gel purified, and then ligated. Mito-EYFP was constructed by amplifying the full-length open reading frame encoding the mitochondrial pyruvate dehydrogenase complex E1-α subunit from a developing COS cDNA library (Gennidakis et al., 2007) using the appropriate oligonucleotide primers (Supplemental Table S2). Amplified products were digested with NcoI and EcoRI and ligated into the corresponding sites in pSAT6-EYFP-C1 (Tzfira et al., 2005). Construction of the BTPC-EYFP plasmid was previously reported (Park et al., 2012). All constructs were confirmed by DNA sequencing. Transient (co)transformations of tobacco BY-2 suspension-cultured cells was performed with 2 to 5 μg of plasmid DNA using a biolistic particle delivery system (Bio-Rad) as described in Park et al. (2012). Bombarded cells were incubated for approximately 6 h to allow for gene expression and protein sorting, fixed in 4% (w/v) formaldehyde, and then either processed for immunostaining or imaged directly using a model no. SP2 CLSM (Leica) at the Molecular and Cellular Imaging Facility (University of Guelph) as previously described in Park et al. (2012). Primary and secondary antibodies used were rabbit anti-Arabidopsis cytochrome c oxidase subunit II affinity-peptide purified IgGs (Cedarlane Labs) and goat anti-rabbit rhodamine red-X conjugated IgGs (Jackson Immunoresearch Laboratories), respectively. Fluorophore emissions were collected sequentially in all fluorescent protein fusion coexpression experiments; single-labeling experiments showed no detectable crossover (i.e. bleed through) at the settings used for data collection. False colorizations of images (i.e. EYFP to green and Cherry to magenta) and merges were generated using the software ImageJ (http://imagej.nih.gov/ij/), and figure compositions were generated using Adobe Photoshop CS6 and/or Illustrator CS6 (Adobe Systems). All micrographs shown are representative images obtained from at least three independent experiments. GST Pull-Down Assays Purified GST-RcCDPK1 or GST-∆N-RcCDPK1 (2 μg each) was incubated with bait proteins (3 μg each) and glutathione agarose beads (40 μL; Affymetrix) in a binding buffer [25 mm HEPES-KOH (pH 7.0), 100 mm NaCl, 0.05% (v/v) β-mercaptoethanol, 0.1% (v/v) Triton X-100, and 0.5 mm CaCl2 or 2 mm EGTA] for 2 h at 4°C with end-over-end mixing. The beads were washed three times with binding buffer, and bound proteins eluted using 40 μL of 50 mm Tris-HCl (pH 8.0) containing 20 mm reduced glutathione, and analyzed by SDS-PAGE and immunoblotting as described above. Accession Numbers Sequence data from this article can be found in the GenBank/EMBL databases under the following accession numbers: NM_117025 (AtCPK4), NM_103271 (AtCPK11), NM_001248700 (GmCDPKβ), NM_001072396 (OsCPK24), XM_002526769 (RcCDPK1), NM_001112282 (ZmCPK11). Supplemental Data The following supplemental materials are available. Supplemental Figure S1. Model illustrating the biochemical complexity of castor bean PEPC. Supplemental Figure S2. Dot-blot assessment of affinity-purified antibodies raised against a synthetic peptide matching RcCDPK1’s N-terminal variable domain. Supplemental Figure S3. RcCDPK1 or ∆N-RcCDPK1 cannot phosphorylate a pair of synthetic peptides containing residues flanking BTPC’s Ser-451 phosphorylation site. Supplemental Figure S4. Phenyl-Sepharose elution profile for native BTPC Ser-451 kinase activity from developing COS. Supplemental Figure S5. Various fluorescent protein-tagged versions of RcCDPK1 and ∆N-RcCDPK1 localize to the cytosol and nucleus in tobacco BY-2 cells. Supplemental Table S1. Proteins reliably identified by nanoHPLC-MS/MS analysis of a tryptic digest of the final preparation of partially purified native BTPC Ser-451 kinase from developing COS (Hill et al., 2014). Supplemental Table S2. Primers used for cloning and PCR. ACKNOWLEDGMENTS We are grateful to Prof. Steven Huber (University of Illinois) for helpful discussions as well as the gift of heterologous expression plasmids for AtCPK4, AtCPK34, and GmCDPKβ. We are also indebted to Prof. Joonho Park (Seoul National University of Science and Technology) for preparation of the Mito-EYFP construct, Mr. Kim Munro (Queen’s Protein Function Discovery Facility) for assisting with isothermal titration calorimetry and circular dichroism spectroscopy, Ms. Deni Ogunrinde (Department of Biomedical and Molecular Sciences, Queen’s University) for assisting with fluorescence spectroscopy, Prof. Jeffrey Harper (University of Nevada) for the gift of atcpk11 knockout seeds, and Prof. Greg Moorhead (University of Calgary) for helpful discussions and encouragement. 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Plant Cell 19 : 3019 – 3036 Google Scholar Crossref Search ADS PubMed WorldCat Author notes 1 This research was supported by Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery, and Research Tool and Infrastructure grants (to W.A.S., R.T.M., and W.C.P.), as well as the Queen’s and Guelph Research Chair programs (to W.C.P. and R.T.M). 2 Present address: Division of Plant Biology, The Samuel Roberts Noble Foundation, Ardmore, Oklahoma 73401. * Address correspondence to plaxton@queensu.ca. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: William Plaxton (plaxton@queensu.ca). S.Y., W.A.S., R.T.M., and W.C.P. designed and supervised this study; S.Y., A.T.H., M.P., 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. prepared the article; all authors read, contributed to, and approved the article. [OPEN] Articles can be viewed without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.17.00288 © 2017 American Society of Plant Biologists. All Rights Reserved. © The Author(s) 2017. Published by Oxford University Press on behalf of American Society of Plant Biologists. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.
TI - Regulatory Phosphorylation of Bacterial-Type PEP Carboxylase by the Ca2+-Dependent Protein Kinase RcCDPK1 in Developing Castor Oil Seeds  
JF - Plant Physiology
DO - 10.1104/pp.17.00288
DA - 2017-06-05
UR - https://www.deepdyve.com/lp/oxford-university-press/regulatory-phosphorylation-of-bacterial-type-pep-carboxylase-by-the-ZanVIfRhoR
SP - 1012
EP - 1027
VL - 174
IS - 2
DP - DeepDyve
ER -