Regulatory Phosphorylation of Bacterial-type PEP Carboxylase by the Ca2+-Dependent Protein Kinase RcCDPK1 in Developing Castor Oilseeds (original) (raw)

Phosphoenolpyruvate carboxylase (PEPC) is a tightly controlled cytosolic enzyme situated at a crucial branch point of central plant metabolism. In developing castor oil seeds (COS) (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 a: (i) pair of Ca2+ binding sites with identical dissociation constants of 5.03 µM, (ii) Ca2+-dependent electrophoretic mobility shift , and (iii) marked Ca2+-independent hydrophobicity. Pull-down experiments established the Ca2+-dependent inte...