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

Despite 14-3-3 proteins being implicated in the control of the eukaryotic c ell 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 phosphope ptide, including nitrate reductase, glyceraldehyde-3-phosphate dehydrogenas e, a calcium-dependent protein kinase, sucrose-phosphate synthase (SPS) and glutamyl-tRNA synthetase. Remarkably, in cells starved of sugars or fed wi th 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. Starvat ion-induced degradation was blocked by 5-aminoimidazole-4-carboxamide ribos ide (which is converted to an AMP-mimetic) or the protease inhibitor MG132 (Cbz-leu-leu-leucinal). Extracts of sugar-starved (but not sugar-fed) Arabi dopsis cells contained an ATP-independent, MG132-sensitive, neutral proteas e that cleaved Arabidopsis SPS, and the mammalian 14-3-3-regulated transcri ption factor, FKHR. Cleavage of SPS and phosphorylated FKHR in vitro was bl ocked by binding to 14-3-3s. The finding that 14-3-3s participate in a nutr ient-sensing pathway controlling cleavage of many targets may underlie the effects of these proteins on plant development.