Diverse functions of 14-3-3 proteins are directly coupled to their ability to interact with targeted peptide substrates. RSX(pS/pT)XP and RX⌽X(pS/pT)XP are two canonical consensus binding motifs for 14-3-3 proteins representing the two common binding modes, modes I and II, between 14-3-3 and internal peptides. Using a genetic selection, we have screened a random peptide library and identified a group of C-terminal motifs, termed SWTY, capable of overriding an endoplasmic reticulum localization signal and redirecting membrane proteins to cell surface. Here we report that the C-terminal SWTY motif, although different from mode I and II consensus, binds tightly to 14-3-3 proteins with a dissociation constant (K D ) of 0.17 M, comparable with that of internal canonical binding peptides. We show that all residues but proline in -SWTX-COOH are compatible for the interaction and surface expression. Because SWTY-like sequences have been found in native proteins, these results support a broad significance of 14-3-3 interaction with protein C termini. The C-terminal binding consensus, mode III, represents an expansion of the repertoire of 14-3-3-targeted sequences.The 14-3-3 proteins were initially identified from brain for their abundance and acidic properties (1). They have been found in all eukaryotes and recognized for their diverse functional roles in biological processes including metabolism, cell signaling, intracellular trafficking, stress responses, cell cycle progression, and malignant transformation (see reviews in Refs. 2-4). Multiple isoforms of 14-3-3 are commonly found in a given species. Saccharomyces cerevisiae has two 14-3-3 isoforms, encoded by BMH1 and BMH2. Deletion of either does not cause growth defects, but the double deletion of both is lethal in most genetic backgrounds (5-7). Essential to 14-3-3 function, in most cases, is the ability to bind a short peptide substrate only upon phosphorylation. As the first protein module identified with preferential affinity to phosphorylated Ser/Thr substrates, 14-3-3 binding provides an inducible mechanism of translating enzymatic activity to protein-protein interaction, a molecular feature with broad significance in regulation.Structural studies and examination of the interactive sequences in human proto-oncogene Raf-1 and from random phosphopeptide libraries have established two binding consensuses, RSX(pS/pT)XP and RX⌽X(pS/pT)XP, which are also known as mode I and II recognition (8, 9). The two binding modes have provided molecular insights into the recognition of 14-3-3 binding to a large number of interacting proteins. The vast majority of binding sites identified so far lies internally and matches either mode I or II binding consensus. In addition to phosphorylation-mediated induction of 14-3-3 binding, there is evidence for additional mechanisms of interaction. For example, a tight association between 14-3-3 and multiple regions of plant plasma membrane H ϩ -ATPase is induced by additional binding of a fungal toxin, fusicoccin, which leads to the forma...

A nucleic acid thermal cycler is considered to be portable if it is under ten pounds, easily carried by one individual, and battery powered. Nucleic acid amplification includes both polymerase chain reaction (e.g. PCR, RT-PCR) and isothermal amplification (e.g. RPA, HDA, LAMP, NASBA, RCA, ICAN, SMART, SDA). There are valuable applications for portable nucleic acid thermocyclers in fields that include clinical diagnostics, biothreat detection, and veterinary testing. A system that is portable allows for the distributed detection of targets at the point of care and a reduction of the time from sample to answer. The designer of a portable nucleic acid thermocycler must carefully consider both thermal control and the detection of amplification. In addition to thermal control and detection, the designer may consider the integration of a sample preparation subsystem with the nucleic acid thermocycler. There are a variety of technologies that can achieve accurate thermal control and the detection of nucleic acid amplification. Important evaluation criteria for each technology include maturity, power requirements, cost, sensitivity, speed, and manufacturability. Ultimately the needs of a particular market will lead to user requirements that drive the decision between available technologies.

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g Cellular processes are tightly controlled through well-coordinated signaling networks that respond to conflicting cues, such as reactive oxygen species (ROS), endoplasmic reticulum (ER) stress signals, and survival factors to ensure proper cell function. We report here a direct interaction between inhibitor of B kinase (IKK) and apoptosis signal-regulating kinase 1 (ASK1), unveiling a critical node at the junction of survival, inflammation, and stress signaling networks. IKK can be activated by growth factor stimulation or tumor necrosis factor alpha engagement. IKK forms a complex with and phosphorylates ASK1 at a sensor site, Ser967, leading to the recruitment of 14-3-3, counteracts stress signal-triggered ASK1 activation, and suppresses ASK1-mediated functions. An inhibitory role of IKK in JNK signaling has been previously reported to depend on NF-B-mediated gene expression. Our data suggest that IKK has a dual role: a transcription-dependent and a transcription-independent action in controlling the ASK1-JNK axis, coupling IKK to ROS and ER stress response. Direct phosphorylation of ASK1 by IKK also defines a novel IKK phosphorylation motif. Because of the intimate involvement of ASK1 in diverse diseases, the IKK/ASK1 interface offers a promising target for therapeutic development.