Post-translational regulation of cytosolic glutamine synthetase by reversible phosphorylation and 14-3-3 protein interaction


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Regulation of the cytosolic isozyme of glutamine synthetase (GS1; EC was studied in leaves of Brassica napus L. Expression and immunodetection studies showed that GS1 was the only active GS isozyme in senescing leaves. By use of [γ-32P]ATP followed by immunodetection, it was shown that GS1 is a phospho-protein. GS1 is regulated post-translationally by reversible phosphorylation catalysed by protein kinases and microcystin-sensitive serine/threonine protein phosphatases. Dephosphorylated GS1 is much more susceptible to degradation than the phosphorylated form. The phosphorylation status of GS1 changes during light/dark transitions and depends in vitro on the ATP/AMP ratio. Phosphorylated GS1 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 GS1. 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.