title: Regulatory Phosphorylation of Bacterial-Type PEP Carboxylase by the Ca2+-Dependent Protein Kinase RcCDPK1 in Developing Castor Oil Seeds.
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 CO2refixation and anaplerotic photosynthate partitioning in support of storage oil and protein biosynthesis in developing COS.
authors.affiliation: Department of Biology, Queen's University, Kingston, Ontario, Canada K7L 3N6 (S.Y., A.T.H., W.A.S., W.C.P.).|Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, Canada N1G 2W1 (M.P., A.M.A., R.T.M.).|Centre for Biologics Evaluation, Biologics and Genetic Therapies Directorate, Health Canada, Ottawa, Ontario, Canada K1A 0K9 (Y.-M.S.); and.|Department of Biomedical and Molecular Sciences, Queen's University, Kingston, Ontario, Canada K7L 3N6 (W.C.P.).
authors.forename: Sheng
authors.identifier: http://orcid.org/0000-0002-9964-2825
authors.initials: S
authors.lastname: Ying
authors.affiliation: Department of Biology, Queen's University, Kingston, Ontario, Canada K7L 3N6 (S.Y., A.T.H., W.A.S., W.C.P.).|Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, Canada N1G 2W1 (M.P., A.M.A., R.T.M.).|Centre for Biologics Evaluation, Biologics and Genetic Therapies Directorate, Health Canada, Ottawa, Ontario, Canada K1A 0K9 (Y.-M.S.); and.|Department of Biomedical and Molecular Sciences, Queen's University, Kingston, Ontario, Canada K7L 3N6 (W.C.P.).
authors.forename: Allyson T
authors.initials: AT
authors.lastname: Hill
authors.affiliation: Department of Biology, Queen's University, Kingston, Ontario, Canada K7L 3N6 (S.Y., A.T.H., W.A.S., W.C.P.).|Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, Canada N1G 2W1 (M.P., A.M.A., R.T.M.).|Centre for Biologics Evaluation, Biologics and Genetic Therapies Directorate, Health Canada, Ottawa, Ontario, Canada K1A 0K9 (Y.-M.S.); and.|Department of Biomedical and Molecular Sciences, Queen's University, Kingston, Ontario, Canada K7L 3N6 (W.C.P.).
authors.forename: Michal
authors.identifier: http://orcid.org/0000-0001-8702-1661
authors.initials: M
authors.lastname: Pyc
authors.affiliation: Department of Biology, Queen's University, Kingston, Ontario, Canada K7L 3N6 (S.Y., A.T.H., W.A.S., W.C.P.).|Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, Canada N1G 2W1 (M.P., A.M.A., R.T.M.).|Centre for Biologics Evaluation, Biologics and Genetic Therapies Directorate, Health Canada, Ottawa, Ontario, Canada K1A 0K9 (Y.-M.S.); and.|Department of Biomedical and Molecular Sciences, Queen's University, Kingston, Ontario, Canada K7L 3N6 (W.C.P.).
authors.forename: Erin M
authors.initials: EM
authors.lastname: Anderson
authors.affiliation: Department of Biology, Queen's University, Kingston, Ontario, Canada K7L 3N6 (S.Y., A.T.H., W.A.S., W.C.P.).|Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, Canada N1G 2W1 (M.P., A.M.A., R.T.M.).|Centre for Biologics Evaluation, Biologics and Genetic Therapies Directorate, Health Canada, Ottawa, Ontario, Canada K1A 0K9 (Y.-M.S.); and.|Department of Biomedical and Molecular Sciences, Queen's University, Kingston, Ontario, Canada K7L 3N6 (W.C.P.).
authors.forename: Wayne A
authors.identifier: http://orcid.org/0000-0001-9564-3057
authors.initials: WA
authors.lastname: Snedden
authors.affiliation: Department of Biology, Queen's University, Kingston, Ontario, Canada K7L 3N6 (S.Y., A.T.H., W.A.S., W.C.P.).|Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, Canada N1G 2W1 (M.P., A.M.A., R.T.M.).|Centre for Biologics Evaluation, Biologics and Genetic Therapies Directorate, Health Canada, Ottawa, Ontario, Canada K1A 0K9 (Y.-M.S.); and.|Department of Biomedical and Molecular Sciences, Queen's University, Kingston, Ontario, Canada K7L 3N6 (W.C.P.).
authors.forename: Robert T
authors.identifier: http://orcid.org/0000-0002-6915-7407
authors.initials: RT
authors.lastname: Mullen
authors.affiliation: Department of Biology, Queen's University, Kingston, Ontario, Canada K7L 3N6 (S.Y., A.T.H., W.A.S., W.C.P.).|Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, Canada N1G 2W1 (M.P., A.M.A., R.T.M.).|Centre for Biologics Evaluation, Biologics and Genetic Therapies Directorate, Health Canada, Ottawa, Ontario, Canada K1A 0K9 (Y.-M.S.); and.|Department of Biomedical and Molecular Sciences, Queen's University, Kingston, Ontario, Canada K7L 3N6 (W.C.P.).
authors.forename: Yi-Min
authors.initials: YM
authors.lastname: She
authors.affiliation: Department of Biology, Queen's University, Kingston, Ontario, Canada K7L 3N6 (S.Y., A.T.H., W.A.S., W.C.P.); plaxton@queensu.ca.|Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, Canada N1G 2W1 (M.P., A.M.A., R.T.M.); plaxton@queensu.ca.|Centre for Biologics Evaluation, Biologics and Genetic Therapies Directorate, Health Canada, Ottawa, Ontario, Canada K1A 0K9 (Y.-M.S.); and plaxton@queensu.ca.|Department of Biomedical and Molecular Sciences, Queen's University, Kingston, Ontario, Canada K7L 3N6 (W.C.P.) plaxton@queensu.ca.
authors.forename: William C
authors.identifier: http://orcid.org/0000-0002-8447-7249
authors.initials: WC
authors.lastname: Plaxton
mesh_terms: D000595:Amino Acid Sequence; D000917:Antibody Formation; D001665:Binding Sites; D055162:Biocatalysis; D055592:Biophysical Phenomena; D002118:Calcium / Q000378:metabolism; D002368:Castor Oil / Q000378:metabolism*; D003001:Cloning, Molecular; D018506:Gene Expression Regulation, Plant; D057927:Hydrophobic and Hydrophilic Interactions; D064267:Intrinsically Disordered Proteins / Q000378:metabolism; D008928:Mitochondria / Q000378:metabolism; D010730:Phosphoenolpyruvate Carboxylase / Q000378:metabolism*; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism; D000072417:Protein Domains; D054730:Protein Interaction Domains and Motifs; D011494:Protein Kinases / Q000737:chemistry / Q000378:metabolism*; D012278:Ricinus / Q000196:embryology / Q000201:enzymology* / Q000235:genetics; D012639:Seeds / Q000378:metabolism*; D016415:Sequence Alignment; D013379:Substrate Specificity; D017871:Calcium-Calmodulin-Dependent Protein Kinases
chemical_list: D002118:Calcium; D002368:Castor Oil; D064267:Intrinsically Disordered Proteins; D010730:Phosphoenolpyruvate Carboxylase; D010768:Phosphoserine; D011494:Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases