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

Summary Regulation of the cytosolic isozyme of glutamine synthetase (GS1; EC 6.3.1.2) 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 t...

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Published in:The Plant journal : for cell and molecular biology Vol. 24; no. 2; pp. 171 - 181
Main Authors: Finnemann, Jørgen, Schjoerring, Jan K.
Format: Journal Article
Language:English
Published: Oxford, UK Blackwell Science Ltd 01-10-2000
Blackwell Science
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Summary:Summary Regulation of the cytosolic isozyme of glutamine synthetase (GS1; EC 6.3.1.2) 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.
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ISSN:0960-7412
1365-313X
DOI:10.1046/j.1365-313x.2000.00863.x