Partial purification and biochemical characterization of a heteromeric protein phosphatase 2A holoenzyme from maize ( Zea mays L.) leaves that dephosphorylates C 4 phospho enol pyruvate carboxylase

Abstract

The activity and allosteric properties of plant phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31) are controlled posttranslationally by specific reversible phosphorylation of a strictly conserved serine residue near the N-terminus. This up/down-regulation of PEPC is catalyzed by a dedicated and highly regulated serine/threonine (Ser/Thr) kinase (PEPC-kinase) and an opposing type-2A Ser/Thr phosphatase (PP2A). In marked contrast to PEPC-kinase, the PP2A holoenzyme from photosynthetic tissue has been virtually unstudied to date. In the present investigation, we have partially purified and characterized the native form of this PP2A from illuminated leaves of maize (Zea mays L.), a C4 plant, using maize [32P]PEPC as substrate. Various conventional chromatographic matrices, together with thiophosphorylated C4 PEPC-peptide and microcystin-LR affinity-supports, were exploited for the enrichment of this PP2A from soluble leaf extracts. Biochemical and immunological results indicate that the C4-leaf holoenzyme is analogous to other eukaryotic PP2As in being a approximately 170-kDa heteromer comprised of a core PP2Ac-A heterodimer (approximately 38- and approximately 65-kDa subunits, respectively) complexed with a putative, approximately 74-kDa B-type regulatory/targeting subunit. This heterotrimer lacks any strict substrate specificity in that it dephosphorylates C4 PEPC, mammalian phosphorylase a, and casein in vitro. This activity is independent of free Me2+, insensitive to levamisole and the Inhibitor-2 protein that targets PP1, activated by several polycations such as protamine and poly-L-lysine, and highly sensitive to inhibition by microcystin-LR and okadaic acid (IC50 approximately 30 pM), all of which are diagnostic features of yeast and mammalian PP2As. In addition, this C4-leaf PP2A holoenzyme (i) is inhibited in vitro by physiological concentrations of certain C4 PEPC-related metabolites (L-malate, PEP, glucose 6-phosphate, but not the activator glycine) when either 32P-labeled maize PEPC or rabbit muscle phosphorylase a is used as substrate, suggesting a direct effect on this Ser/Thr phosphatase; and (ii) displays, at best, only modest light/dark effects in vivo on its apparent molecular mass, component core subunits and activity against C4 PEPC, in marked contrast to the opposing activity of PEPC-kinase in C4 and Crassulacean acid metabolism leaves. This report represents one of the few studies of a heteromeric PP2A holoenzyme from photosynthetic tissue that dephosphorylates a known target enzyme in plants, such as PEPC, sucrose-phosphate synthase or nitrate reductase.

Phosphorylation of Phosphoenolpyruvate Carboxylase Is Not Essential for High Photosynthetic Rates in the C4 Species Flaveria bidentis

“…3). One might not expect to see close coupling between mRNA levels and the PEPC phosphorylation state because the PEPC phosphorylation level is determined by the balance of the PEPC-PK activity and the protein phosphatase activity (Dong et al, 2001). Therefore, reduction of the PEPC-PK mRNA level strongly supports our thesis that low PEPC phosphorylation in these transgenic plants is due to reduced levels of PEPC-PK.…”

“…Phosphorylation of C 4 -PEPC at its conserved Ser residue near the N terminus is thought to be controlled primarily by the light-dependent activity of a protein kinase specific to PEPC (PEPC-PK) because the activity of the protein phosphatase 2A involved in PEPC dephosphorylation appears to be constant under various conditions (Vidal and Chollet, 1997;Dong et al, 2001;Nimmo, 2003;Izui et al, 2004). Recently, cDNA clones for PEPC-PK have been cloned and characterized in vitro for a number of species (Izui et al, 2004;Shenton et al, 2006); in particular, Tsuchida et al (2001) have cloned and characterized the expression of PEPC-PK from Flaveria trinervia.…”

Update: Post-translational protein modifications in plant metabolism

“…Interestingly, this lowabundance PEPC kinase is degraded by the proteasome after polyubiquitination, but the E3 ligase is not yet known (Agetsuma et al, 2005). An unidentified phosphatase (Protein phosphatase 2A family) dephosphorylates PEPC in darkness, thereby down-regulating PEPC activity (Dong et al, 2001;Izui et al, 2004). The regulation of this phosphatase is still unknown.…”

Multilevel Control of Arabidopsis 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase by Protein Phosphatase 2A

“…A considerable number of biochemical, pharmacological, and genetic studies have shown that PP2A is involved in the control of constitutive plant processes, such as cell cycle, metabolism, ion channel fluxes, growth and development, pollination and seed germination, and in the defense against many biotic or abiotic challenges (Smith and Walker, 1996;Luan, 2003;DeLong, 2006). PP2A has been proposed to modulate several metabolic processes in vascular plants, including sucrose synthesis, quinate metabolism, nitrogen assimilation, and nocturnal CO 2 fixation (Siegl et al, 1990;MacKintosh et al, 1991;MacKintosh, 1992;Dong et al, 2001), but its involvement in isoprenoid biosynthesis had not been established. The broad distribution and functional diversity likely correspond to a multiplicity of PP2A holoenzymes (each formed by one A, one B-type, and one C subunit) reflected at the genomic level.…”