PMID:16136328

Citation	Lima, L, Seabra, A, Melo, P, Cullimore, J and Carvalho, H (2006) Phosphorylation and subsequent interaction with 14-3-3 proteins regulate plastid glutamine synthetase in Medicago truncatula. Planta 223:558-67
Abstract	In this report we demonstrate that plastid glutamine synthetase of Medicago truncatula (MtGS2) is regulated by phosphorylation and 14-3-3 interaction. To investigate regulatory aspects of GS2 phosphorylation, we have produced non-phosphorylated GS2 proteins by expressing the plant cDNA in E. coli and performed in vitro phosphorylation assays. The recombinant isoenzyme was phosphorylated by calcium dependent kinase(s) present in leaves, roots and nodules. Using an (His)6-tagged 14-3-3 protein column affinity purification method, we demonstrate that phosphorylated GS2 interacts with 14-3-3 proteins and that this interaction leads to selective proteolysis of the plastid located isoform, resulting in inactivation of the isoenzyme. By site directed mutagenesis we were able to identify a GS2 phosphorylation site (Ser97) crucial for the interaction with 14-3-3s. Phosphorylation of this target residue can be functionally mimicked by replacing Ser97 by Asp, indicating that the introduction of a negative charge contributes to the interaction with 14-3-3 proteins and subsequent specific proteolysis. Furthermore, we document that plant extracts contain protease activity that cleaves the GS2 protein only when it is bound to 14-3-3 proteins following either phosphorylation or mimicking of phosphorylation by Ser97Asp.
Links	PubMed Online version:10.1007/s00425-005-0097-8
Keywords	14-3-3 Proteins/metabolism; Amino Acid Sequence; Calcium/metabolism; Electrophoresis, Gel, Two-Dimensional; Escherichia coli/genetics; Gene Expression Regulation, Plant; Glutamate-Ammonia Ligase/chemistry; Glutamate-Ammonia Ligase/genetics; Glutamate-Ammonia Ligase/metabolism; Medicago truncatula/enzymology; Medicago truncatula/genetics; Molecular Sequence Data; Mutagenesis, Site-Directed; Phosphorylation; Plant Leaves/metabolism; Plant Roots/metabolism; Plastids/enzymology; Protein Interaction Mapping; Protein Kinases/metabolism; Recombinant Fusion Proteins/metabolism; Sequence Alignment; Serine/chemistry
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Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
MEDTR:Q84UC1		GO:0004356: glutamate-ammonia ligase activity	ECO:0000314:		F		Figure 6a shows that the authors were directly measuring the glutamine synthesis by the reaction L-glutamate + ATP + NH(4)(+) = L-glutamine + ADP + 2 H(+) + phosphate., using the transferase assay (see methods).	complete CACAO 4828
MEDTR:Q84UC1	enables	GO:0004356: glutamate-ammonia ligase activity	ECO:0000314: direct assay evidence used in manual assertion		F		Seeded From UniProt	complete

References

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