title: Phosphorylation of bacterial-type phosphoenolpyruvate carboxylase by a Ca2+-dependent protein kinase suggests a link between Ca2+ signalling and anaplerotic pathway control in developing castor oil seeds.
abstract: The aim of the present study was to characterize the native protein kinase [BTPC (bacterial-type phosphoenolpyruvate carboxylase)-K (BTPC Ser451 kinase)] that in vivo phosphorylates Ser451 of the BTPC subunits of an unusual Class-2 PEP (phosphoenolpyruvate) carboxylase hetero-octameric complex of developing COS (castor oil seeds). COS BTPC-K was highly purified by PEG fractionation and hydrophobic size-exclusion anion-exchange and affinity chromatographies. BTPC-K phosphorylated BTPC strictly at Ser451 (Km=1.0 μM; pH optimum=7.3), a conserved target residue occurring within an intrinsically disordered region, as well as the protein histone III-S (Km=1.7 μM), but not a COS plant-type PEP carboxylase or sucrose synthase or α-casein. Its activity was Ca2+- (K0.5=2.7 μM) and ATP- (Km=6.6 μM) dependent, and markedly inhibited by trifluoperazine, 3-phosphoglycerate and PEP, but insensitive to calmodulin or 14-3-3 proteins. BTPC-K exhibited a native molecular mass of ~63 kDa and was soluble rather than membrane-bound. Inactivation and reactivation occurred upon BTPC-K's incubation with GSSG and then DTT respectively. Ser451 phosphorylation by BTPC-K inhibited BTPC activity by ~50% when assayed under suboptimal conditions (pH 7.3, 1 mM PEP and 10 mM L-malate). Our collective results indicate a possible link between cytosolic Ca2+ signalling and anaplerotic flux control in developing COS.
authors.affiliation: *Department of Biology, Queen's University, Kingston, ON, Canada, K7L 3N6.
authors.forename: Allyson T
authors.initials: AT
authors.lastname: Hill
authors.affiliation: *Department of Biology, Queen's University, Kingston, ON, Canada, K7L 3N6.
authors.forename: Sheng
authors.initials: S
authors.lastname: Ying
authors.forename: William C
authors.initials: WC
authors.lastname: Plaxton
mesh_terms: D001419:Bacteria / Q000201:enzymology*; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling; D002368:Castor Oil / Q000378:metabolism*; D010730:Phosphoenolpyruvate Carboxylase / Q000378:metabolism*; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D012639:Seeds / Q000378:metabolism*
chemical_list: D002368:Castor Oil; D011494:Protein Kinases; D010730:Phosphoenolpyruvate Carboxylase; D002118:Calcium