Article

Threonine at position 306 of the KAT1 potassium channel is essential for channel activity and is a target site for ABA-activated SnRK2/OST1/SnRK2.6 protein kinase.

Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku University, Aobayama 6-6-07, Sendai 980-8579, Japan.
Biochemical Journal (Impact Factor: 4.65). 09/2009; 424(3):439-48. DOI: 10.1042/BJ20091221
Source: PubMed

ABSTRACT The Arabidopsis thaliana K+ channel KAT1 has been suggested to have a key role in mediating the aperture of stomata pores on the surface of plant leaves. Although the activity of KAT1 is thought to be regulated by phosphorylation, the endogenous pathway and the primary target site for this modification remained unknown. In the present study, we have demonstrated that the C-terminal region of KAT1 acts as a phosphorylation target for the Arabidopsis calcium-independent ABA (abscisic acid)-activated protein kinase SnRK2.6 (Snf1-related protein kinase 2.6). This was confirmed by LC-MS/MS (liquid chromatography tandem MS) analysis, which showed that Thr306 and Thr308 of KAT1 were modified by phosphorylation. The role of these specific residues was examined by single point mutations and measurement of KAT1 channel activities in Xenopus oocyte and yeast systems. Modification of Thr308 had minimal effect on KAT1 activity. On the other hand, modification of Thr306 reduced the K+ transport uptake activity of KAT1 in both systems, indicating that Thr306 is responsible for the functional regulation of KAT1. These results suggest that negative regulation of KAT1 activity, required for stomatal closure, probably occurs by phosphorylation of KAT1 Thr306 by the stress-activated endogenous SnRK2.6 protein kinase.

0 Bookmarks
 · 
83 Views
  • [Show abstract] [Hide abstract]
    ABSTRACT: Cellular membranes act as signaling platforms and control solute transport. Membrane receptors, transporters, and enzymes communicate with intracellular processes through protein-protein interactions. Using a split-ubiquitin yeast two-hybrid screen that covers a test-space of 6.4 × 10(6) pairs, we identified 12,102 membrane/signaling protein interactions from Arabidopsis. Besides confirmation of expected interactions such as heterotrimeric G protein subunit interactions and aquaporin oligomerization, >99% of the interactions were previously unknown. Interactions were confirmed at a rate of 32% in orthogonal in planta split-green flourescent protein interaction assays, which was statistically indistinguishable from the confirmation rate for known interactions collected from literature (38%). Regulatory associations in membrane protein trafficking, turnover, and phosphorylation include regulation of potassium channel activity through abscisic acid signaling, transporter activity by a WNK kinase, and a brassinolide receptor kinase by trafficking-related proteins. These examples underscore the utility of the membrane/signaling protein interaction network for gene discovery and hypothesis generation in plants and other organisms.
    Science 05/2014; 344(6185):711-6. · 31.20 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Elucidating how plants sense and respond to water loss is important for identifying genetic and chemical interventions that may help sustain crop yields in water limiting environments. Currently, the molecular mechanisms involved in the initial perception and response to dehydration are not well understood. Modern mass spectrometric methods for quantifying changes in the phosphoproteome provide an opportunity to identify key phosphorylation events involved in this process. Here we have used both untargeted and targeted isotope-assisted mass spectrometric methods of phosphopeptide quantitation to characterize proteins in Arabidopsis thaliana whose degree of phosphorylation is rapidly altered by hyperosmotic treatment. Thus, protein phosphorylation events responsive to 5 minutes of 0.3 M mannitol treatment were first identified using 15N metabolic labeling and untargeted mass spectrometry with a high-resolution ion trap instrument. The results from these 'discovery' experiments were then validated using targeted mass spectrometry with a triple quadrupole. Targeted experiments were conducted with plants grown under nine different environmental conditions to determine whether the phosphorylation changes were specific for osmosignaling or involved cross talk with other signaling pathways. The results indicate that regulatory proteins such as members of the Mitogen-activated protein kinase kinase kinase (MAP3K) and MAP4K gene family are specifically phosphorylated in response to osmotic stress. Proteins involved in 5'mRNA decapping and phosphatidylinositol 3,5-bisphosphate synthesis were also identified as targets of dehydration-induced phosphoregulation. The results of these experiments demonstrate the utility of targeted phosphoproteomic analysis in understanding protein regulation networks and provide new insight into cellular processes involved in osmotic stress response.
    Plant physiology 05/2014; · 6.56 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Some intracellular organelles found in eukaryotes such as plants have arisen through the endocytotic engulfment of prokaryotic cells. This accounts for the presence of plant membrane intrinsic proteins that have homologs in prokaryotic cells. Other organelles, such as those of the endomembrane system, are thought to have evolved through infolding of the plasma membrane. Acquisition of intracellular components (organelles) in the cells supplied additional functions for survival in various natural environments. The organelles are surrounded by biological membranes, which contain membrane-embedded K(+) transport systems allowing K(+) to move across the membrane. K(+) transport systems in plant organelles act coordinately with the plasma membrane intrinsic K(+) transport systems to maintain cytosolic K(+) concentrations. Since it is sometimes difficult to perform direct studies of organellar membrane proteins in plant cells, heterologous expression in yeast and Escherichia coli has been used to elucidate the function of plant vacuole K(+) channels and other membrane transporters. The vacuole is the largest organelle in plant cells; it has an important task in the K(+) homeostasis of the cytoplasm. The initial electrophysiological measurements of K(+) transport have categorized three classes of plant vacuolar cation channels, and since then molecular cloning approaches have led to the isolation of genes for a number of K(+) transport systems. Plants contain chloroplasts, derived from photoautotrophic cyanobacteria. A novel K(+) transport system has been isolated from cyanobacteria, which may add to our understanding of K(+) flux across the thylakoid membrane and the inner membrane of the chloroplast. This chapter will provide an overview of recent findings regarding plant organellar K(+) transport proteins.
    Journal of plant physiology 05/2014; 171(9):743-7. · 2.50 Impact Factor

Full-text (2 Sources)

View
12 Downloads
Available from
May 28, 2014