Mitogen-activated protein (MAP) kinase phosphatase 1 (MKP-1/CL100) is an inducible nuclear dual specificity protein phosphatase that can dephosphorylate and inactivate both mitogen-and stress-activated protein kinases in vitro and in vivo. However, the molecular mechanism responsible for the substrate selectivity of MKP-1 is unknown. In addition, it has been suggested that the signal transducers and activators of transcription 1 (STAT1) transcription factor is a physiological non-MAP kinase substrate for MKP-1. We have used the yeast two-hybrid assay to demonstrate that MKP-1 is able to interact selectively with the extracellular signal-regulated kinase 1/2 (ERK1/2), p38␣, and c-Jun We conclude that the substrate selectivity of MKP-1 is determined by specific protein-protein interactions coupled with catalytic activation of the phosphatase and that these interactions are restricted to members of the MAP kinase family of enzymes.The mitogen-activated protein (MAP) 1 kinases are key components of cellular signal transduction pathways, which become activated in response to a wide variety of external stimuli. They can be subdivided into at least three classes based on sequence homology and differential activation by agonists (1-3); these include the growth factor-activated MAP kinases, extracellular signal-regulated kinase 1 (ERK1) and ERK2, and the stress-activated MAP kinases c-Jun amino-terminal kinase (JNK, SAPK1) and p38 (SAPK2) MAP kinases. In addition, a number of less well characterized members of the MAP kinase family have been identified such as BMK1/ERK5, ERK7, and ERK3 (4 -6). MAP kinase pathways relay, amplify, and integrate complex signals in order to elicit appropriate biological responses. In mammalian cells, these include cellular proliferation, differentiation, inflammatory responses, and apoptosis. These responses are associated with significant alterations in the pattern of cellular gene expression, and transcription factors are a major target of MAP kinase signaling in vivo (7). To phosphorylate these proteins, activated MAP kinases translocate to the cell nucleus, a process that is generally associated with prolonged activation of the MAP kinase (8). Therefore, the magnitude and duration of MAP kinase activation are critical determinants of biological outcome, and regulatory mechanisms governing the activation of MAP kinase are of key importance in both physiological and pathological cell functions.The duration and magnitude of MAP kinase activation can be regulated at many points within the signal transduction pathway. However, it is now clear that the MAP kinase itself is a major target for regulation through the action of specific protein phosphatases. MAP kinase activation is dependent on the phosphorylation of both the threonine and tyrosine residues of the TXY motif found within the "activation loop" of the kinase. Since phosphorylation of both residues is required for activity, dephosphorylation of either residue is sufficient for inactivation. This can be achieved by protein-tyrosine ph...

RING ubiquitin ligases (E3) recruit ubiquitin-conjugate enzymes (E2) charged with ubiquitin (Ub) to catalyze ubiquitination. Non-covalent Ub binding to the backside of certain E2s promotes processive polyUb formation, but the mechanism remains elusive. Here, we show that backside bound Ub (Ub(B)) enhances both RING-independent and RING-dependent UbcH5B-catalyzed donor Ub (Ub(D)) transfer, but with a more prominent effect in RING-dependent transfer. Ub(B) enhances RING E3s' affinities for UbcH5B-Ub, and RING E3-UbcH5B-Ub complex improves Ub(B)'s affinity for UbcH5B. A comparison of the crystal structures of a RING E3, RNF38, bound to UbcH5B-Ub in the absence and presence of Ub(B), together with molecular dynamics simulation and biochemical analyses, suggests Ub(B) restricts the flexibility of UbcH5B's α1 and α1β1 loop. Ub(B) supports E3 function by stabilizing the RING E3-UbcH5B-Ub complex, thereby improving the catalytic efficiency of Ub transfer. Thus, Ub(B) serves as an allosteric activator of RING E3-mediated Ub transfer.

A class of anti-virulence compounds, the salicylidene acylhydrazides, has been widely reported to block the function of the type three secretion system of several Gram-negative pathogens by a previously unknown mechanism. In this work we provide the first identification of bacterial proteins that are targeted by this group of compounds. We provide evidence that their mode of action is likely to result from a synergistic effect arising from a perturbation of the function of several conserved proteins. We also examine the contribution of selected target proteins to the pathogenicity of Yersinia pseudotuberculosis and to expression of virulence genes in Escherichia coli O157.

To initiate infection many viruses enter their host cells by triggering endocytosis following receptor engagement. The mechanisms by which non-enveloped viruses escape the endosome are however poorly understood. Here we present near-atomic resolution cryoEM structures for feline calicivirus (FCV) both undecorated and labelled with a soluble fragment of its cellular receptor feline junctional adhesion molecule A (fJAM-A). We show that VP2, a minor capsid protein encoded by all caliciviruses 1,2 , forms a large portal-like assembly at a unique threefold symmetry axis following receptor engagement. This feature, which was not detected in undecorated virions, is formed of twelve copies of VP2 arranged with their hydrophobic N-termini pointing away from the virion surface. Local rearrangement at the portal site leads to opening of a pore in the capsid shell. We hypothesise that the portal-like assembly functions as a channel for delivery of the calicivirus genome through the endosomal membrane into the cytoplasm of a host cell to initiate infection. While VP2 was known to be critical for the production of infectious virus 3 , its structure and function were hitherto undetermined. Our findings therefore represent a major step forward in our understanding of the Caliciviridae.

Cellular cross-talk between ubiquitination and other posttranslational modifications contributes to the regulation of numerous processes. One example is ADP-ribosylation of the carboxyl terminus of ubiquitin by the E3 DTX3L/ADP-ribosyltransferase PARP9 heterodimer, but the mechanism remains elusive. Here, we show that independently of PARP9, the conserved carboxyl-terminal RING and DTC (Deltex carboxyl-terminal) domains of DTX3L and other human Deltex proteins (DTX1 to DTX4) catalyze ADP-ribosylation of ubiquitin’s Gly76. Structural studies reveal a hitherto unknown function of the DTC domain in binding NAD+. Deltex RING domain recruits E2 thioesterified with ubiquitin and juxtaposes it with NAD+ bound to the DTC domain to facilitate ADP-ribosylation of ubiquitin. This ubiquitin modification prevents its activation but is reversed by the linkage nonspecific deubiquitinases. Our study provides mechanistic insights into ADP-ribosylation of ubiquitin by Deltex E3s and will enable future studies directed at understanding the increasingly complex network of ubiquitin cross-talk.

Understanding the role of coherent electronic motion is expected to resolve general questions of importance in macromolecular energy transfer. We demonstrate a novel nonlinear optical method, angle-resolved coherent wave mixing, that separates out coherently coupled electronic transitions and energy transfers in an instantaneous two-dimensional mapping. Angular resolution of the signal is achieved by using millimeter laser beam waists at the sample and by signal relay to the far field; for this we use a high energy, ultrabroadband hollow fiber laser source. We reveal quantum electronic beating with a time-ordered selection of transition energies in a photosynthetic complex.

SummaryMesenchymal cell motility is driven by polarized actin polymerization [1]. Signals at the leading edge recruit actin polymerization machinery to promote membrane protrusion, while matrix adhesion generates tractive force to propel forward movement. To work effectively, cell motility is regulated by a complex network of signaling events that affect protein activity and localization. H2O2 has an important role as a diffusible second messenger [2], and mediates its effects through oxidation of cysteine thiols. One cell activity influenced by H2O2 is motility [3]. However, a lack of sensitive and H2O2-specific probes for measurements in live cells has not allowed for direct observation of H2O2 accumulation in migrating cells or protrusions. In addition, the identities of proteins oxidized by H2O2 that contribute to actin dynamics and cell motility have not been characterized. We now show, as determined by fluorescence lifetime imaging microscopy, that motile cells generate H2O2 at membranes and cell protrusions and that H2O2 inhibits cofilin activity through oxidation of cysteines 139 (C139) and 147 (C147). Molecular modeling suggests that C139 oxidation would sterically hinder actin association, while the increased negative charge of oxidized C147 would lead to electrostatic repulsion of the opposite negatively charged surface. Expression of oxidation-resistant cofilin impairs cell spreading, adhesion, and directional migration. These findings indicate that H2O2 production contributes to polarized cell motility through localized cofilin inhibition and that there are additional proteins oxidized during cell migration that might have similar roles.

SummaryRING and U-box E3 ubiquitin ligases regulate diverse eukaryotic processes and have been implicated in numerous diseases, but targeting these enzymes remains a major challenge. We report the development of three ubiquitin variants (UbVs), each binding selectively to the RING or U-box domain of a distinct E3 ligase: monomeric UBE4B, phosphorylated active CBL, or dimeric XIAP. Structural and biochemical analyses revealed that UbVs specifically inhibited the activity of UBE4B or phosphorylated CBL by blocking the E2∼Ub binding site. Surprisingly, the UbV selective for dimeric XIAP formed a dimer to stimulate E3 activity by stabilizing the closed E2∼Ub conformation. We further verified the inhibitory and stimulatory functions of UbVs in cells. Our work provides a general strategy to inhibit or activate RING/U-box E3 ligases and provides a resource for the research community to modulate these enzymes.