title: The calcium-dependent protein kinase RcCDPK2 phosphorylates sucrose synthase at Ser11 in developing castor oil seeds.
abstract: Imported sucrose is cleaved by sucrose synthase (SUS) as a critical initial reaction in the biosynthesis of storage end-products by developing seeds. Although SUS is phosphorylated at a conserved seryl residue by an apparent CDPK (Ca2+-dependent protein kinase) in diverse plant tissues, the functions and mechanistic details of this process remain obscure. Thus, the native CDPK that phosphorylates RcSUS1 (Ricinus communis SUS1) at Ser11in developing COS (castor oil seeds) was highly purified and identified as RcCDPK2 by MS/MS. Purified RcSUS1-K (-kinase) and heterologously expressed RcCDPK2 catalyzed Ca2+-dependent Ser11phosphorylation of RcSUS1 and its corresponding dephosphopeptide, while exhibiting a high affinity for free Ca2+ions [K0.5(Ca2+) < 0.4 µM]. RcSUS1-K activity, RcCDPK2 expression, and RcSUS1 Ser11phosphorylation peaked during early COS development and then declined in parallel. The elimination of sucrose import via fruit excision triggered RcSUS1 dephosphorylation but did not alter RcSUS1-K activity, suggesting a link between sucrose signaling and posttranslational RcCDPK2 control. Both RcCDPK2-mCherry and RcSUS1-EYFP co-localized throughout the cytosol when transiently co-expressed in tobacco suspension cells, although RcCDPK2-mCherry was also partially localized to the nucleus. Subcellular fractionation revealed that ∼20% of RcSUS1-K activity associates with microsomal membranes in developing COS, as does RcSUS1. In contrast with RcCDPK1, which catalyzes inhibitory phosphorylation of COS bacterial-type phosphoenolpyruvate carboxylase at Ser451, RcCDPK2 exhibited broad substrate specificity, a wide pH-activity profile centered at pH 8.5, and insensitivity to metabolite effectors or thiol redox status. Our combined results indicate a possible link between cytosolic Ca2+-signaling and the control of photosynthate partitioning during COS development.
authors: Department of Biology, Queen's University, Kingston, ON, Canada K7L 3N6.
authors: Eric T
authors: ET
authors: Fedosejevs
authors: Department of Biology, Queen's University, Kingston, ON, Canada K7L 3N6.
authors: Suzanne A
authors: SA
authors: Gerdis
authors: Department of Biology, Queen's University, Kingston, ON, Canada K7L 3N6.
authors: Sheng
authors: S
authors: Ying
authors: Department of Molecular and Cellular Biology, University of Guelph, Guelph, ON, Canada N1G 2W1.
authors: Michal
authors: M
authors: Pyc
authors: Department of Molecular and Cellular Biology, University of Guelph, Guelph, ON, Canada N1G 2W1.
authors: Erin M
authors: EM
authors: Anderson
authors: Department of Biology, Queen's University, Kingston, ON, Canada K7L 3N6.
authors: Wayne A
authors: WA
authors: Snedden
authors: Department of Molecular and Cellular Biology, University of Guelph, Guelph, ON, Canada N1G 2W1.
authors: Robert T
authors: RT
authors: Mullen
authors: Shanghai Center for Plant Stress Biology, Chinese Academy of Sciences, 3888 Chenhua Road, Shanghai 201602, China.
authors: Yi-Min
authors: YM
authors: She
authors: Department of Biology, Queen's University, Kingston, ON, Canada K7L 3N6 Department of Biomedical and Molecular Sciences, Queen's University, Kingston, ON, Canada K7L 3N6.
authors: William C
authors: WC
authors: Plaxton
mesh_terms: D002368:Castor Oil / Q000378:metabolism*; D005964:Glucosyltransferases / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008861:Microsomes / Q000378:metabolism; D010730:Phosphoenolpyruvate Carboxylase / Q000378:metabolism; D010766:Phosphorylation; D010940:Plant Proteins / Q000378:metabolism*; D011494:Protein Kinases / Q000378:metabolism*; D012639:Seeds / Q000201:enzymology* / Q000378:metabolism*; D017871:Calcium-Calmodulin-Dependent Protein Kinases
chemical_list: D002368:Castor Oil; D005964:Glucosyltransferases; D010730:Phosphoenolpyruvate Carboxylase; D010940:Plant Proteins; D011494:Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; C022699:sucrose synthase
keywords: Calcium-dependent protein kinase; Ricinus communis (castor oil plant); oil seed (metabolism); protein phosphorylation; sucrose synthase