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 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 (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.

Transport and Use of Bicarbonate in Plants: Current Knowledge and Challenges Ahead

“…There is a tight interaction between PTPC and BTPC, which forms the class 2 PEP. This is an enzyme complex that, in contrast to class 1 PEPC, is mostly insensitive to allosteric inhibition by high malate concentrations [ 169 , 170 ]. While class 1 PEPCs are constitutively expressed in the cytosol, the BPEPC is associated with the outer mitochondrial surface.…”

Transcript profiling indicates a widespread role for bacterial-type phosphoenolpyruvate carboxylase in malate-accumulating sink tissues

“…2 ). RcCDPK1 was recently identified as a calcium dependent protein kinase isozyme that catalyzes in vivo regulatory inhibitory BTPC phosphorylation at Ser-451 in developing COS ( Hill et al , 2014 ; Ying et al , 2017 ). BTPC characterization from additional species will therefore help to corroborate and extend the role of calcium-dependent BTPC phosphorylation.…”

“…Tryptic peptide sequencing by LC MS/MS identified RcPPC3 and RcPPC4, namely castor PTPC and BTPC, respectively, as the subunits of COS Class-2 PEPC ( Blonde and Plaxton 2003 , Gennidakis et al , 2007 ). BTPC and PTPC transcripts and polypeptides are coordinately expressed during COS development; their developmental profiles correlate with Class-2 PEPC formation as well as with the stages of rapid endosperm growth and oil accumulation ( Blonde and Plaxton, 2003 ; Gennidakis et al , 2007 ; O’Leary et al , 2011 a ; Ying et al , 2017 ). Although both subunit types exhibit PEPC activity within the Class-2 PEPC complex, the BTPC subunits demonstrated remarkable insensitivity towards PTPC allosteric inhibitors such as L-malate ( O’Leary et al , 2009 ; O’Leary et al , 2011 b ).…”

Phosphorylation-dependent sub-functionalization of the calcium-dependent protein kinase CPK28

“…Autophosphorylation has been correlated with the activation (15,53,(73)(74)(75) activation is reversible with contact sites between the CaM-like domain and kinase domain stabilizing both active and inactive forms (31). Many of the residues that stabilize these conformations are conserved between plants and protists (32) suggesting similar contact sites may exist in plants.…”