14-3-3s regulate global cleavage of their diverse binding partners in sugar-starved Arabidopsis cells

Abstract

Despite 14‐3‐3 proteins being implicated in the control of the eukaryotic cell cycle, metabolism, cell signalling and survival, little is known about the global regulation or functions of the phosphorylation‐dependent binding of 14‐3‐3s to diverse target proteins. We identified Arabidopsis cytosolic proteins that bound 14‐3‐3s in competition with a 14‐3‐3‐binding phosphopeptide, including nitrate reductase, glyceraldehyde‐ 3‐phosphate dehydrogenase, a calcium‐dependent protein kinase, sucrose‐phosphate synthase (SPS) and glutamyl‐tRNA synthetase. Remarkably, in cells starved of sugars or fed with non‐metabolizable glucose analogues, all 14‐3‐3 binding was lost and the target proteins were selectively cleaved into proteolytic fragments. 14‐3‐3 binding reappeared after several hours of re‐feeding with sugars. Starvation‐induced degradation was blocked by 5‐amino imidazole‐4‐carboxamide riboside (which is converted to an AMP‐mimetic) or the protease inhibitor MG132 (Cbz‐leu‐leu‐leucinal). Extracts of sugar‐starved (but not sugar‐fed) Arabidopsis cells contained an ATP‐independent, MG132‐sensitive, neutral protease that cleaved Arabidopsis SPS, and the mammalian 14‐3‐3‐regulated transcription factor, FKHR. Cleavage of SPS and phosphorylated FKHR in vitro was blocked by binding to 14‐3‐3s. The finding that 14‐3‐3s participate in a nutrient‐sensing pathway controlling cleavage of many targets may underlie the effects of these proteins on plant development.

Phosphatidic Acid Binds to Cytosolic Glyceraldehyde-3-phosphate Dehydrogenase and Promotes Its Cleavage in Arabidopsis

“…The PA-induced GAPC2 cleavage did not occur in vitro when tested using either purified protein or E. coli lysate expressing GAPC2, suggesting that the GAPC2 cleavage requires other factors(s) in plants in addition to PA. GAPC was reported to bind to a 14-3-3 protein under sugar-sufficient conditions but lose its binding and undergo a selective partial cleavage in Arabidopsis cells starved of sugars (47). The proteolytic fragment of GAPC was ϳ30 kDa which was close to the size of PA-induced proteolytic fragment of GAPC observed here, raising the possibility of whether a similar proteolytic activity is used for both the sugar starvation-induced and PA-mediated proteolytic cleavages of GAPC.…”

Light Regulation of Nitrate uptake, Assimilation and Metabolism

“…Experiments with spinach did indeed support this as phosphorylated NR was shown to be more rapidly degraded than non-phosphorylated NR (Kaiser and Huber 2001). This did not however, appear to be the case in Arabidopsis cell cultures (Cotelle et al 2000) or in Nicotiana species (Lillo et al 2003). The findings for Nicotiana species are in agreement with light having a positive effect on the translation process.…”

Trehalose 6-phosphate is indispensable for carbohydrate utilization and growth in Arabidopsis thaliana

“…Interestingly, a cauliflower TPS interacts with 14-3-3 proteins (45), now known as a class II TPS (J. Harthill and C. Mackintosh, personal communication). 14-3-3 binding depends on the nutritional status of cells and determines the stability of the target protein in response to carbon supply (46). Furthermore, Arabidopsis TPSs possess putative SnRK1 phosphorylation sites, and SnRK1 is thought to be subject to regulation by G6P (47).…”