title: Regulatory phosphorylation of Sorghum leaf phosphoenolpyruvate carboxylase. Identification of the protein-serine kinase and some elements of the signal-transduction cascade.
abstract: The phosphoenolpyruvate (PPrv) carboxylase isozyme involved in C4 photosynthesis undergoes a day/night reversible phosphorylation process in leaves of the C4 plant, Sorghum. Ser8 of the target enzyme oscillates between a high (light) and a low (dark) phosphorylation status. Both in vivo and in vitro, phosphorylation of dark-form carboxylase was accompanied by an increase in the apparent Ki of the feedback inhibitor L-malate and an increase in Vmax. Feeding detached leaves various photosynthetic inhibitors, i.e. 3-(3,4-dichlorophenyl)-1,1-dimethylurea, gramicidin and DL-glyceraldehyde, prevented PPrv carboxylase phosphorylation in the light, thus suggesting that the cascade involves the photosynthetic apparatus as the light signal receptor, and presumably has the electron transfer chain and the Calvin-Benson cycle as components in the signal-transduction chain. Two protein-serine kinases capable of phosphorylating PPrv carboxylase in vitro have been partially purified from light-adapted leaves. One was isolated on a calmodulin-Sepharose column; it was calcium-dependent but did not require calmodulin for activity. The other was purified on a blue-dextran-agarose column and the only Me2+ required for activity was Mg2+. In reconstituted phosphorylation assays, only the latter caused the expected decrease in malate sensitivity of PPrv carboxylase suggesting that this protein is the genuine PPrv-carboxylase-kinase. Desalted extracts from light-adapted leaves possessed a considerably greater phosphorylation capacity with immunopurified dephosphorylated PPrv carboxylase as substrate than did dark extracts. This light stimulation was insensitive to type 2A protein phosphatase inhibitors, okadaic acid and microcystin-LR, which suggests that the kinase is a controlled step in the cascade which leads to phosphorylation of PPrv carboxylase. The higher phosphorylation capacity of light-adapted leaf tissue was nullified by pretreatment with the cytosolic protein synthesis inhibitor, cycloheximide. Thus, protein turnover is involved as part of the mechanism controlling the activity of the kinase purified on blue-dextran-agarose. However, no information is available with respect to the specific nature of the link between the above-mentioned light transducing steps and the protein kinase that achieves the physiological response. Finally, the in vivo phosphorylation site (Ser8) in the N-terminal region of the C4 type Sorghum PPrv carboxylase is also present in a non-photosynthetic form of the Sorghum enzyme (Ser7), as deduced by cDNA sequence analysis.
authors.affiliation: Laboratoire de Physiologie Végétale Moléculaire, Université Paris-Sud, Centre d'Orsay, France.
authors.forename: N
authors.initials: N
authors.lastname: Bakrim
authors.forename: C
authors.initials: C
authors.lastname: Echevarria
authors.forename: C
authors.initials: C
authors.lastname: Cretin
authors.forename: M
authors.initials: M
authors.lastname: Arrio-Dupont
authors.forename: J N
authors.initials: JN
authors.lastname: Pierre
authors.forename: J
authors.initials: J
authors.lastname: Vidal
authors.forename: R
authors.initials: R
authors.lastname: Chollet
authors.forename: P
authors.initials: P
authors.lastname: Gadal
mesh_terms: D000595:Amino Acid Sequence; D001345:Autoradiography; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D008027:Light; D008293:Malates / Q000378:metabolism; D008969:Molecular Sequence Data; D010730:Phosphoenolpyruvate Carboxylase / Q000302:isolation & purification / Q000378:metabolism*; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors; D010766:Phosphorylation; D010788:Photosynthesis; D010944:Plants / Q000201:enzymology*; D011494:Protein Kinases / Q000302:isolation & purification / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D012689:Sequence Homology, Nucleic Acid; D015398:Signal Transduction
chemical_list: D008293:Malates; D011500:Protein Synthesis Inhibitors; C030298:malic acid; D011494:Protein Kinases; D017346:Protein Serine-Threonine Kinases; D010749:Phosphoprotein Phosphatases; D010730:Phosphoenolpyruvate Carboxylase