Guard cells control the aperture of stomatal pores to balance photosynthetic carbon dioxide uptake with evaporative water loss. Stomatal closure is triggered by several stimuli that initiate complex signaling networks to govern the activity of ion channels. Activation of SLOW ANION CHANNEL1 (SLAC1) is central to the process of stomatal closure and requires the leucine-rich repeat receptor-like kinase (LRR-RLK) GUARD CELL HYDROGEN PEROXIDE-RESISTANT1 (GHR1), among other signaling components. Here, based on functional analysis of nine Arabidopsis thaliana ghr1 mutant alleles identified in two independent forward-genetic ozone-sensitivity screens, we found that GHR1 is required for stomatal responses to apoplastic reactive oxygen species, abscisic acid, high CO 2 concentrations, and diurnal light/dark transitions. Furthermore, we show that the amino acid residues of GHR1 involved in ATP binding are not required for stomatal closure in Arabidopsis or the activation of SLAC1 anion currents in Xenopus laevis oocytes and present supporting in silico and in vitro evidence suggesting that GHR1 is an inactive pseudokinase. Biochemical analyses suggested that GHR1-mediated activation of SLAC1 occurs via interacting proteins and that CALCIUM-DEPENDENT PROTEIN KINASE3 interacts with GHR1. We propose that GHR1 acts in stomatal closure as a scaffolding component.

Death receptors are cell surface receptors that belong to the tumor necrosis factor receptor superfamily, the most well known members of which are tumor necrosis factor receptor 1, the CD95/Fas receptor, and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) 1 receptors DR4/TRAIL-R1 and DR5/TRAIL-R2 (for a review, see Ref. 1). Upon ligand-mediated oligomerization of CD95/Fas or TRAIL receptors, the Fas-associated death domain protein (FADD) attaches to the death receptor via homophilic death domain interactions. The death effector domain (DED) of FADD is in turn linked to the apoptotic machinery due to its affinity for the initiator caspases, procaspase-8 and procaspase-10. The close proximity of procaspase-8 molecules results in dimerization of the procaspases, the assembly of which forms an enzymatically active site (2, 3). This enzymatic activity induces serial cleavages converting the zymogen into p10 and p18 fragments, forming the proteolytically active caspase-8 heterotetramer. In responsive cells, activated caspase-8 is then able to activate effector caspases thereby initiating apoptosis (for a review, see Ref. 4). In addition to the initiator caspases, the DED in FADD also interacts with the apoptosis modulator cellular FLICE-inhibitory protein (c-FLIP; Ref. 5), which exists as two splice variants, the long splice form of FLIP (FLIP L ) and the short splice form of FLIP (FLIP S ). Together with the activated death receptor, FADD, and caspase-8 and -10, the FLIP proteins form the core of the death-inducing signaling complex (DISC; Ref. 6), which also contains a number of other proteins, many of which seem to be involved in modulating death receptor signals (for a review, see Ref. 1). The DISC assembly with pro-and antiapoptotic proteins allows for control of death receptor signaling through a number of different routes. Consequently in addition to apoptosis, stimulation of the receptors may also lead to cell survival and proliferation. Modulation of the apoptotic pathway at the level of the activated receptor is required in situations when the extrinsic, but not the intrinsic, apoptosis pathway needs to be inhibited or when localized initiator caspase activity is required for specialized signaling functions. For example, caspase-8 activity is necessary for normal T cell development (7,8) and is probably acquired through complex regulation of anti-and proapoptotic proteins in the DISC. In addition, some malignant cells are able to convert death receptor stimulation into proliferative signals (9, 10). Therefore, active regulation of the DISC proteins, both transcriptional and post-translational, determines the outcome of death receptor stimulation.c-FLIP L and c-FLIP S have been characterized as specific inhibitors of death receptor-mediated apoptosis (for a review, see Ref. 11). c-FLIP L is homologous to caspase-8, consisting of two tandemly repeated DEDs and a catalytically inactive caspase-like domain. Although c-FLIP S shares most of its se-* This work was supported by the Academy of Finlan...

Adenosine triphosphate (ATP) plays an essential role in energy transfer within the cell. In the form of NAD, adenine participates in multiple redox reactions. Phosphorylation and ATP-hydrolysis reactions have key roles in signal transduction and regulation of many proteins, especially enzymes. In each cell, proteins with many different functions use adenine and its derivatives as ligands; adenine, of course, is present in DNA and RNA. We show that an adenine binding motif, which differs according to the backbone chain direction of a loop that binds adenine (and in one variant by the participation of an aspartate side-chain), is common to many proteins; it was found from an analysis of all adenylate-containing protein structures from the Protein Data Bank. Indeed, 224 protein-ligand complexes (86 different proteins) from a total of 645 protein structure files bind ATP, CoA, NAD, NADP, FAD, or other adenine-containing ligands, and use the same structural elements to recognize adenine, regardless of whether the ligand is a coenzyme, cofactor, substrate, or an allosteric effector. The common adenine-binding motif shown in this study is simple to construct. It uses only (1) backbone polar interactions that are not dependent on the protein sequence or particular properties of amino acid side-chains, and (2) nonspecific hydrophobic interactions. This is probably why so many different proteins with different functions use this motif to bind an adenylate-containing ligand. The adenylate-binding motif reported is present in "ancient proteins" common to all living organisms, suggesting that adenine-containing ligands and the common motif for binding them were exploited very early in evolution. The geometry of adenine binding by this motif mimics almost exactly the geometry of adenine base-pairing seen in DNA and RNA.

The integrins form a large family of cell adhesion receptors. All multicellular animals express integrins, indicating that the family evolved relatively early in the history of metazoans, and homologous sequences of the component domains of integrin alpha and beta subunits are seen in prokaryotes. Some integrins, however, seem to be much younger. For example, the alphaI domain containing integrins, including collagen receptors and leukocyte integrins, have been found in chordates only. Here, we will discuss what conclusions can be drawn about integrin function by studying the evolutionary conservation of integrins. We will also look at how studying integrins in organisms such as the fruit fly and mouse has helped our understanding of integrin evolution-function relationships. As an illustration of this, we will summarize the current understanding of integrin involvement in skeletal muscle formation.

Land plants produce diverse flavonoids for growth, survival, and reproduction. Chalcone synthase is the first committed enzyme of the flavonoid biosynthetic pathway and catalyzes the production of 2′,4,4′,6′-tetrahydroxychalcone (THC). However, it also produces other polyketides, including p-coumaroyltriacetic acid lactone (CTAL), because of the derailment of the chalcone-producing pathway. This promiscuity of CHS catalysis adversely affects the efficiency of flavonoid biosynthesis, although it is also believed to have led to the evolution of stilbene synthase and p-coumaroyltriacetic acid synthase. In this study, we establish that chalcone isomerase-like proteins (CHILs), which are encoded by genes that are ubiquitous in land plant genomes, bind to CHS to enhance THC production and decrease CTAL formation, thereby rectifying the promiscuous CHS catalysis. This CHIL function has been confirmed in diverse land plant species, and represents a conserved strategy facilitating the efficient influx of substrates from the phenylpropanoid pathway to the flavonoid pathway.