Post-translational regulation of cytosolic glutamine synthetase by reversible phosphorylation and 14-3-3 protein interaction.
Regulation of the cytosolic isozyme of glutamine synthetase (GS(1); EC 6.3.1.2) was studied in leaves of Brassica napus L. Expression and immunodetection studies showed that GS(1) was the only active GS isozyme in senescing leaves. By use of [gamma-(32)P]ATP followed by immunodetection, it was shown that GS(1) is a phospho-protein. GS(1) is regulated post-translationally by reversible phosphorylation catalysed by protein kinases and microcystin-sensitive serine/threonine protein phosphatases. Dephosphorylated GS(1) is much more susceptible to degradation than the phosphorylated form. The phosphorylation status of GS(1) changes during light/dark transitions and depends in vitro on the ATP/AMP ratio. Phosphorylated GS(1) interacts with 14-3-3 proteins as verified by two different methods: a His-tag 14-3-3 protein column affinity method combined with immunodetection, and a far-Western method with overlay of 14-3-3-GFP. The degree of interaction with 14-3-3-proteins could be modified in vitro by decreasing or increasing the phosphorylation status of GS(1). Thus, the results demonstrate that 14-3-3 protein is an activator molecule of cytosolic GS and provide the first evidence of a protein involved in the activation of plant cytosolic GS. The role of post-translational regulation of cytosolic GS and interactions between phosphorylated cytosolic GS and 14-3-3 proteins in senescing leaves is discussed in relation to nitrogen remobilization.
Finnemann
J
J
Plant Nutrition Laboratory, Department of Agricultural Sciences, The Royal Veterinary and Agricultural University, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Copenhagen, Denmark. jfi@kvl.dk
Schjoerring
J K
JK
D048948:14-3-3 Proteins; D001692:Biological Transport; D001937:Brassica / Q000235:genetics*; D003600:Cytosol / Q000201:enzymology*; D003624:Darkness; D015971:Gene Expression Regulation, Enzymologic; D005974:Glutamate-Ammonia Ligase / Q000235:genetics*; D008027:Light; D008954:Models, Biological; D008969:Molecular Sequence Data; D009584:Nitrogen / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D018515:Plant Leaves / Q000201:enzymology; D010940:Plant Proteins / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D021122:Protein Subunits; D014446:Tyrosine 3-Monooxygenase / Q000378:metabolism*
D048948:14-3-3 Proteins; D010750:Phosphoproteins; D010940:Plant Proteins; D021122:Protein Subunits; D014446:Tyrosine 3-Monooxygenase; D005974:Glutamate-Ammonia Ligase; D009584:Nitrogen