14-3-3-regulated Ca(2+)-dependent protein kinase CPK3 is required for sphingolipid-induced cell death in Arabidopsis.

In eukaryotic cells, sphingoid long chain bases (LCBs) such as sphingosine or phytosphingosine (PHS) behave as second messengers involved in various processes including programmed cell death (PCD). In plants, induction of PCD by LCBs has now been described, but the signalling pathway is still enigmatic. Using Arabidopsis, we identify new key steps in this pathway. We demonstrate that PHS induces activation of the calcium-dependent kinase CPK3, which phosphorylates its binding partners, the 14-3-3 proteins. This phosphorylation leads to the disruption of the complex and to CPK3 degradation. Using cpk3 knockout lines, we demonstrate that CPK3 is a positive regulator of LCB-mediated PCD. These findings establish 14-3-3-regulated CPK3 as a key component of the LCB pathway leading to PCD in plants.

Affinity purification of diverse plant and human 14-3-3-binding partners.

Many proteins that bind to a 14-3-3 column in competition with a 14-3-3-binding phosphopeptide have been purified from plant and mammalian cells and tissues. New 14-3-3 targets include enzymes of biosynthetic metabolism, vesicle trafficking, cell signalling and chromatin function. These findings indicate central regulatory roles for 14-3-3s in partitioning carbon among the pathways of sugar, amino acid, nucleotide and protein biosynthesis in plants. Our results also suggest that the current perception that 14-3-3s bind predominantly to signalling proteins in mammalian cells is incorrect, and has probably arisen because of the intensity of research on mammalian signalling and for technical reasons.

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 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.

Phosphorylation-dependent interactions between enzymes of plant metabolism and 14-3-3 proteins.

Far-Western overlays of soluble extracts of cauliflower revealed many proteins that bound to digoxygenin (DIG)-labelled 14-3-3 proteins. Binding to DIG-14-3-3s was prevented by prior dephosphorylation of the extract proteins or by competition with 14-3-3-binding phosphopeptides, indicating that the 14-3-3 proteins bind to phosphorylated sites. The proteins that bound to the DIG-14-3-3s were also immunoprecipitated from extracts with anti-14-3-3 antibodies, demonstrating that they were bound to endogenous plant 14-3-3 proteins. 14-3-3-binding proteins were purified from cauliflower extracts, in sufficient quantity for amino acid sequence analysis, by affinity chromatography on immobilised 14-3-3 proteins and specific elution with a 14-3-3-binding phosphopeptide. Purified 14-3-3-binding proteins included sucrose-phosphate synthase, trehalose-6-phosphate synthase, glutamine synthetases, a protein (LIM17) that has been implicated in early floral development, an approximately 20 kDa protein whose mRNA is induced by NaCl, and a calcium-dependent protein kinase that was capable of phosphorylating and rendering nitrate reductase (NR) sensitive to inhibition by 14-3-3 proteins. In contrast to the phosphorylated NR-14-3-3 complex which is activated by dissociation with 14-3-3-binding phosphopeptides, the total sugar-phosphate synthase activity in plant extracts was inhibited by up to 40% by a 14-3-3-binding phosphopeptide and the phosphopeptide-inhibited activity was reactivated by adding excess 14-3-3 proteins. Thus, 14-3-3 proteins are implicated in regulating several aspects of primary N and C metabolism. The procedures described here will be valuable for determining how the phosphorylation and 14-3-3-binding status of defined target proteins change in response to extracellular stimuli.

Induction of Stomatal Closure by Vanadate or a Light/Dark Transition Involves Ca2+-Calmodulin-Dependent Protein Phosphorylations.

Previous studies indicate that a continual source of adenosine 5[prime]-triphosphate is required for both opening and closing of stomata. However, vanadate (Na3VO4 at 500 [mu]M) as well as a light/dark transition induced stomatal closing in epidermal peels of Commelina communis L., showing that the stoppage or even the decrease of the activity of the plasma membrane H+-adenosine 5[prime]-triphosphatase is sufficient to induce stomatal closure. Furthermore, stomatal closing in response to Na3VO4 or a light/dark transition was suppressed by inhibitors of metabolism (10 [mu]M carbonyl cyanide m-chlorophenylhydrazone) and of protein kinases (20 [mu]M 1-[5-iodonaphthalene-1-sulfonyl]-1H-hexa-hydro-1,4-diaz-epine), calmodulin antagonists (20 [mu]M N-[6-aminohexyl]-5-chloro-1-naphthalenesulfonamide), and the anion channel blocker 5-nitro-2,3-phenylpropyllamino benzoic acid (50 [mu]M). These data suggest that the slow, outward rectifying anion channel, whose opening would be related to the membrane potential, and at least one step requiring a protein phosphorylation by a Ca2+-calmodulin-dependent protein kinase of the myosin light chain kinase type might be implicated in the induction of stomatal closing by vanadate or a light/dark transition.