Phosphorylation of bacterial-type phosphoenolpyruvate carboxylase at Ser425 provides a further tier of enzyme control in developing castor oil seeds

Abstract

PEPC [PEP (phosphoenolpyruvate) carboxylase] is a tightly controlled anaplerotic enzyme situated at a pivotal branch point of plant carbohydrate metabolism. Two distinct oligomeric PEPC classes were discovered in developing COS (castor oil seeds). Class-1 PEPC is a typical homotetramer of 107 kDa PTPC (plant-type PEPC) subunits, whereas the novel 910-kDa Class-2 PEPC hetero-octamer arises from a tight interaction between Class-1 PEPC and 118 kDa BTPC (bacterial-type PEPC) subunits. Mass spectrometric analysis of immunopurified COS BTPC indicated that it is subject to in vivo proline-directed phosphorylation at Ser425. We show that immunoblots probed with phosphorylation site-specific antibodies demonstrated that Ser425 phosphorylation is promoted during COS development, becoming maximal at stage IX (maturation phase) or in response to depodding. Kinetic analyses of a recombinant, chimaeric Class-2 PEPC containing phosphomimetic BTPC mutant subunits (S425D) indicated that Ser425 phosphorylation results in significant BTPC inhibition by: (i) increasing its Km(PEP) 3-fold, (ii) reducing its I50 (L-malate and L-aspartate) values by 4.5- and 2.5-fold respectively, while (iii) decreasing its activity within the physiological pH range. The developmental pattern and kinetic influence of Ser425 BTPC phosphorylation is very distinct from the in vivo phosphorylation/activation of COS Class-1 PEPC's PTPC subunits at Ser11. Collectively, the results establish that BTPC's phospho-Ser425 content depends upon COS developmental and physiological status and that Ser425 phosphorylation attenuates the catalytic activity of BTPC subunits within a Class-2 PEPC complex. To the best of our knowledge, this study provides the first evidence for protein phosphorylation as a mechanism for the in vivo control of vascular plant BTPC activity.

Biochemical and Molecular Characterization of RcSUS1, a Cytosolic Sucrose Synthase Phosphorylated in Vivo at Serine 11 in Developing Castor Oil Seeds

“…The overall pattern of in vivo seryl-phosphorylation of RcSUS1 in COS, as well as the phosphorylation motif flanking its Ser-11 residue (Fig. 5A), are distinct from those of COS plant-and bacterial-type PEP carboxylases (36,37,39,55). These differences implicate novel kinase-phosphatase pairings in controlling the phosphorylation status of RcSUS1, and plant-and bacterial-type PEP carboxylases in developing COS.…”

“…7, G-I). The results: (i) implicate a direct inverse relationship between RcSUS1 expression and Ser-11 phosphorylation and the supply of sucrose from source leaves to the nonphotosynthetic COS (ii) are reminiscent of the depodding-induced dephosphorylation of COS plant-type PEP carboxylase (RcPPC3) (36,54), but (iii) contrast with the depodding-enhanced multisite in vivo phosphorylation of bacterial-type PEP carboxylase (RcPPC4) at Ser-425 and Ser-451 (37,55). The ability of plant cells to sense sugars plays a crucial role in C-partitioning and allocation between source and sink tissues.…”

Phosphoenolpyruvate Carboxylase in Arabidopsis Leaves Plays a Crucial Role in Carbon and Nitrogen Metabolism

“…In addition, monoubiquitination may also regulate plant-type PEPC activity (Uhrig et al, 2008). Recent research in castor oil seeds suggests that bacterium-type PEPC is a catalytic and regulatory subunit of class 2 PEPCs, as class 1 and class 2 PEPCs show significant differences in their sensitivity to allosteric inhibitors (O'Leary et al, 2009(O'Leary et al, , 2011b.…”

Regulatory Phosphorylation of Bacterial-Type PEP Carboxylase by the Ca2+-Dependent Protein Kinase RcCDPK1 in Developing Castor Oil Seeds

“…This includes inhibitory phosphorylation at Ser-425 and Ser-451, which both occur within a distinctive approximately 10-kD intrinsically disordered region not found in PTPCs (Dalziel et al, 2012;O'Leary et al, 2011c). Despite the apparent important role of multisite BTPC phosphorylation in the posttranslational control of photosynthate partitioning and anaplerotic C-flux at the PEP branchpoint during COS development, nothing was known about the responsible protein kinases or related signaling pathways.…”

“…Although BTPCs lack the N-terminal seryl phosphorylation motif characteristic of nonphotosynthetic and C 4 /CAM photosynthetic PTPCs, BTPC is in vivo phosphorylated at multiple sites during COS development (Uhrig et al, 2008a;O'Leary et al, 2011c;Dalziel et al, 2012). This includes inhibitory phosphorylation at Ser-425 and Ser-451, which both occur within a distinctive approximately 10-kD intrinsically disordered region not found in PTPCs (Dalziel et al, 2012;O'Leary et al, 2011c).…”