Phosphorylation and subsequent interaction with 14-3-3 proteins regulate plastid glutamine synthetase in Medicago truncatula.
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.
Lima
Lígia
L
Instituto de Biologia Molecular e Celular Rua do Campo Alegre, 823, 4150-180 Porto, Portugal.
Seabra
Ana
A
Melo
Paula
P
Cullimore
Julie
J
Carvalho
Helena
H
D048948:14-3-3 Proteins / Q000378:metabolism*; D000595:Amino Acid Sequence; D002118:Calcium / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D004926:Escherichia coli / Q000235:genetics; D018506:Gene Expression Regulation, Plant; D005974:Glutamate-Ammonia Ligase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D046913:Medicago truncatula / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D018515:Plant Leaves / Q000378:metabolism; D018517:Plant Roots / Q000378:metabolism; D018087:Plastids / Q000201:enzymology*; D025941:Protein Interaction Mapping; D011494:Protein Kinases / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D016415:Sequence Alignment; D012694:Serine / Q000737:chemistry
D048948:14-3-3 Proteins; D011993:Recombinant Fusion Proteins; D012694:Serine; D011494:Protein Kinases; D005974:Glutamate-Ammonia Ligase; D002118:Calcium