Partial purification and biochemical characterization of a heteromeric protein phosphatase 2A holoenzyme from maize (Zea mays L.) leaves that dephosphorylates C4 phosphoenolpyruvate carboxylase

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 equal to 170-kDa heteromer comprised of a core PP2Ac-A heterodimer (approximately equal to 38- and approximately equal to 65-kDa subunits, respectively) complexed with a putative, approximately equal to 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 Me(2+), 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 equal to 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.