½AU : QA1 Regulatory Phosphorylation of Bacterial-Type PEP Carboxylase by the Ricinus Kinase CDPK1 ½AU : 1 1[OPEN] Sheng Ying 2 , Allyson T. Hill, Michal Pyc, Erin M. Anderson, Wayne A. Snedden, Robert T. Mullen, Yi-Min She, and William C. Plaxton* 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 Vaccine Evaluation, Biologics and Genetic Therapies Directorate, Health Canada, 251 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.) ORCID IDs: 0000-0002-9964-2825 (S.Y.); 0000-0001-8702-1661 (M.P.); 0000-0001-9564-3057 (W.A.S.); 0000-0002-6915-7407 (R.T.M.); 0000-0002-8447-7249 (W.C.P.). ½AU : 4 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 to our knowledge 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 Ca 2+ -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 Ca 2+ -dependent, inhibitory phosphorylation of BTPC at Ser-451 while exhibiting: (i ) a pair of Ca 2+ binding sites with identical dissociation constants of 5.03 mM, (ii ) a Ca 2+ -dependent electrophoretic mobility shift, and (iii ) a marked Ca 2+ -independent hydrophobicity. Pull-down experiments established the Ca 2+ -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 Ca 2+ -dependent RcCDPK1 autophosphorylation and BTPC transphosphorylation activity, it is not critical for in vitro or in vivo target recognition. Arabidopsis ½AU : 5 (Arabidopsis thaliana) CPK4 and soybean (Glycine max) CDPKb are RcCDPK1 orthologs that effectively phosphorylated castor BTPC at Ser-451. Overall, the results highlight a potential link between cytosolic Ca 2+ signaling and the posttranslational control of respiratory CO 2 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 Ca 2+ -sensor proteins being critical transducers of Ca 2+ signatures elicited in re- sponse to external stimuli or developmental cues. The activation of protein phosphorylation cascades is often the first and most important signaling event triggered by Ca 2+ signals (DeFalco et al., 2009). Among Ca 2+ sensors, including calmodulin (CaM) and CaM-like proteins (CMLs), Ca 2+ -dependent protein kinases (CDPKs) are unique because they function as catalytic responders that directly transduce Ca 2+ signals into protein phosphorylation events that modulate physio- logical 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 Ca 2+ signals in a temporal and spatially specific manner (Harper et al., 2004). Different CDPK isozymes exhibit distinctive tissue and subcel- lular locations, substrate specificities, and Ca 2+ sensi- tivities. Thus, diverse developmental programs and stress responses are likely controlled by specific CDPKs including hormone-regulated developmental pro- cesses, seed development, pollen tube formation, and abiotic and biotic stress signaling (Harper et al., 2004; 1 This research was supported by Natural Sciences and Engineer- ing 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 ½AU : 2 : Division of Plant Biology, The Samuel Robert Noble Foundation, Ardmore, Oklahoma 73401. * Address correspondence to plaxton@queensu.ca. The author ½AU : 3 responsible for distribution of materials integral to the findings presented in this article in accordance with the policy de- scribed 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 experi- ments; 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 Plant Physiology Ò , May 2017, Vol. 174, pp. 1–16, www.plantphysiol.org Ó 2017 American Society of Plant Biologists. All Rights Reserved. 1