Phosphoproteomic identification of targets of the Arabidopsis sucrose nonfermenting-like kinase SnRK2.8 reveals a connection to metabolic processes

Abstract

SnRK2.8 is a member of the sucrose nonfermenting-related kinase family that is down-regulated when plants are deprived of nutrients and growth is reduced. When this kinase is over expressed in Arabidopsis, the plants grow larger. To understand how this kinase modulates growth, we identified some of the proteins that are phosphorylated by this kinase. A new phosphoproteomic method was used in which total protein from plants overexpressing the kinase was compared with total protein from plants in which the kinase was inactivated. Protein profiles were compared on two-dimensional gels following staining by a dye that recognizes phosphorylated amino acids. Candidate target proteins were confirmed with in vitro phosphorylation assays, using the kinase and target proteins that were purified from Escherichia coli. Seven target proteins were confirmed as being phosphorylated by SnRK2.8. Certain targets, such as 14-3-3 proteins, regulate as yet unidentified proteins, whereas other targets, such as glyoxalase I and ribose 5-phosphate isomerase, detoxify byproducts from glycolysis and catalyze one of the final steps in carbon fixation, respectively. Also, adenosine kinase and 60S ribosomal protein were confirmed as targets of SnRK2.8. Using mass spectrometry, we identified phosphorylated residues in the SnRK2.8, the 14-3-3 , and the 14-3-3 . These data show that the expression of SnRK2.8 is correlated with plant growth, which may in part be due to the phosphorylation of enzymes involved in metabolic processes.14-3-3 ͉ nutrient deprivation ͉ plant ͉ potassium T he mammalian AMPK (AMP-activated protein kinase), the yeast SNF (sucrose nonfermenting) 1 protein kinase, and the plant SnRK (SNF1-related protein kinase) are highly conserved and play broad roles in growth and metabolic responses to cellular stress (1). Mammalian cells sense glucose levels through the mammalian target of rapamycin kinase, which is part of an AMPK complex. AMPK is implicated in the development and treatment of metabolic disorders, including obesity and type 2 diabetes (2), and mutations in AMPK causes cardiac abnormalities (3). In yeast, SNF1 is required for regulation of glucose-responsive genes necessary for pseudohyphal growth in response to nutrient limitations (4) and for controlling the onset of meiosis in yeast (5). SNF1 provides a mechanism for crosstalk between metabolic pathways and cell cycle signaling processes (6). Mammal AMPK and yeast SNF1 act as energy-level sensors that function to regulate metabolism during low-energy conditions.

SNF1-related Protein Kinases 2 Are Negatively Regulated by a Plant-specific Calcium Sensor

“…Similarly to SnRK2.2, SnRK2.3, and SnRK2.6, this enzyme is involved in the control of stress-responsive gene expression. It has also been shown that SnRK2.8 is a positive * This work was supported by Ministry of Science and Higher Education regulator of plant growth, most probably by phosphorylation and regulation of the activity of several enzymes involved in metabolism (13). The results of Diédhiou et al (14) have revealed that overexpression of SAPK4 (a rice SnRK2) increases rice tolerance to salinity and oxidative stress.…”

Sucrose-induced Receptor Kinase SIRK1 Regulates a Plasma Membrane Aquaporin in Arabidopsis

“…The identification of SnRK2.5 suggests a link to further cytosolic signaling cascades. Members of the SnRK family were previously associated with signaling in relation to metabolic stresses (98,99), and in particular the SnRK2 kinases were suggested to function in intracellular osmotic responses (26). There are indications that phosphorylation is required for the activation of SnRK2.5, but the precise sites of activation are not yet identified (25).…”

Quantitative Proteomics in Plants: Choices in Abundance

“…Experiments isolating compartments of the endomembrane system were performed successfully using either ICAT (Dunkley et al, 2004) or iTRAQ (Dunkley et al, 2006). Similarly, studies investigating changes in protein phosphorylation under various treatment conditions or developmental stages in plants have been performed using 2-D gels (Shin et al, 2007), in vivo metabolic labeling (Benschop et al, 2007), iTRAQ (Nü hse et al, 2007), and label-free methods (Niittyla et al, 2007). Therefore, multiple methods are capable of providing important new insights into plant biology.…”