Happyhour, a Ste20 family kinase, implicates EGFR signaling in ethanol-induced behaviors.

The consequences of alcohol use disorders (AUDs) are devastating to individuals and society, yet few treatments are currently available. To identify genes regulating the behavioral effects of ethanol, we conducted a genetic screen in Drosophila and identified a mutant, happyhour (hppy), due to its increased resistance to the sedative effects of ethanol. Hppy protein shows strong homology to mammalian Ste20 family kinases of the GCK-1 subfamily. Genetic and biochemical experiments revealed that the epidermal growth factor (EGF)-signaling pathway regulates ethanol sensitivity in Drosophila and that Hppy functions as an inhibitor of the pathway. Acute pharmacological inhibition of the EGF receptor (EGFR) in adult animals altered acute ethanol sensitivity in both flies and mice and reduced ethanol consumption in a preclinical rat model of alcoholism. Inhibitors of the EGFR or components of its signaling pathway are thus potential pharmacotherapies for AUDs.

The dependence receptor UNC5H2/B triggers apoptosis via PP2A-mediated dephosphorylation of DAP kinase.

The UNC5H dependence receptors promote apoptosis in the absence of their ligand, netrin-1, and this is important for neuronal and vascular development and for limitation of cancer progression. UNC5H2 (also called UNC5B) triggers cell death through the activation of the serine-threonine protein kinase DAPk. While performing a siRNA screen to identify genes implicated in UNC5H-induced apoptosis, we identified the structural subunit PR65ß of the holoenzyme protein phosphatase 2A (PP2A). We show that UNC5H2/B recruits a protein complex that includes PR65ß and DAPk and retains PP2A activity. PP2A activity is required for UNC5H2/B-induced apoptosis, since it activates DAPk by triggering its dephosphorylation. Moreover, netrin-1 binding to UNC5H2/B prevents this effect through interaction of the PP2A inhibitor CIP2A to UNC5H2/B. Thus we show here that, in the absence of netrin-1, recruitment of PP2A to UNC5H2/B allows the activation of DAPk via a PP2A-mediated dephosphorylation and that this mechanism is involved in angiogenesis regulation.

Resilience emotions and acute stress reactions in the population of Dimona and the general population of Israel two days after the first suicide bombing attack in Dimona.

BACKGROUND: On 4 February 2008, two terrorists armed with suicide bombs arrived atthe open market in the southern Israeli city of Dimona. One detonated his bomb at approximately 10:30 a.m. causing multiple casualties. Short-term emotional effects and acute stress reactions usually appear among survivors after such incidents. OBJECTIVES: To compare the differences in emotions and in disturbances of daily life activities that emerge a couple of days following such an event and to identify patterns of stress development among resilient and low-resilient members of the population in Dimona and in the general population of Israel. METHODS: A telephone survey of two randomly selected representative samples of adults (428 Israeli residents and 250 Dimona residents) was conducted 2 days afterthe event. RESULTS: A higher prevalence of stress and fear and a lower prevalence of joy were reported among the population of Dimona compared to the general population in Israel (P < 0.05). Differences were also recorded when the population of Dimona was categorized by its personal degree of resilience (P < 0.05). A higher prevalence of disturbances in daily life activities and changes in leisure activity was found in the low-resilient population in Dimona (P < 0.01). CONCLUSIONS: This study demonstrates that following a public terror event, self-reported low-resilient subjects have a higher prevalence of disturbances in daily life activities, as well as adverse emotional responses. These differences must be addressed by the relevant social service agencies for immediate public intervention.

The effect of different educational interventions on schoolchildren's knowledge of earthquake protective behaviour in Israel.

Knowledge of appropriate behaviour during an earthquake is crucial for prevention of injury and loss of life. The Israeli Home Front Command conducts a yearly earthquake education programme in all Israeli schools, using three types of educational interventions: lectures, drills and a combination of the two. The aim of this study was to evaluate the effectiveness of these interventions in providing students with knowledge. We distributed a questionnaire to 2,648 children from the 5th and 6th grades in 120 schools nationwide. Knowledge scores for both 5th and 6th grades were increased, regardless of type of intervention, compared to the non-exposure group. A combined intervention of lectures and drills resulted in the highest knowledge scores. Our findings suggest that for the age group studied a combination of lectures and drills will likely prepare students best for how to behave in the event of an earthquake.

Differences in the sources of information and acquaintance with instructions between Dimona and the general population after a suicide bomber event.

A telephone survey among two randomly selected, representative samples of adults was conducted two days after a suicide bomber event in Dimona, Israel. Television, radio, Internet, and newspapers were more common sources of information in the general population, whereas friends, family, and the local authorities were the more common sources of information in Dimona. Higher acquaintance with police instructions and higher knowledge of the exact location of the event were found in the population of Dimona. Authorities must pay attention to this phenomenon and use the correct sources of information in each area in order to achieve better exposure of the target population to the police instructions after a terrorist event.

Death-associated protein kinase phosphorylates ZIP kinase, forming a unique kinase hierarchy to activate its cell death functions.

The death-associated protein (DAP) kinase family includes three protein kinases, DAP kinase, DAP kinase-related protein 1, and ZIP kinase, which display 80% amino acid identity within their catalytic domains and are functionally linked to common subcellular changes occurring during cell death, such as the process of membrane blebbing. Here we show physical and functional cross talk between DAP kinase and ZIP kinase. The two kinases display strong synergistic effects on cell death when coexpressed and physically bind each other via their catalytic domains. Furthermore, DAP kinase phosphorylates ZIP kinase at six specific sites within its extracatalytic C-terminal domain. ZIP kinase localizes to both the nucleus and the cytoplasm and fractionates as monomeric and trimeric forms. Significantly, modification of the DAP kinase phosphorylation sites influences both the localization and oligomerization status of ZIP kinase. A mutant ZIP kinase construct, in which the six serine/threonine residues were mutated to aspartic acid to mimic the phosphorylated state, was found predominantly in the cytoplasm as a trimer and possessed greater cell death-inducing potency. This suggests that DAP kinase and ZIP kinase function in a biochemical pathway in which DAP kinase activates the cellular function of ZIP kinase through phosphorylation, leading to amplification of death-promoting signals.

The DAP-kinase family of proteins: study of a novel group of calcium-regulated death-promoting kinases.

DAP-kinase (DAPk) is a Ca(2+)/calmodulin (CaM)-regulated Ser/Thr kinase that functions as a positive mediator of programmed cell death. It associates with actin microfilament and has a unique multidomain structure. One of the substrates of DAPk was identified as myosin light chain (MLC), the phosphorylation of which mediates membrane blebbing. Four additional kinases have been identified based on the high homology of their catalytic domain to that of DAPk. Yet, they differ in the structure of their extracatalytic domains and in their intracellular localization. One member of this family, DRP-1, also shares with DAPk both the property of activation by Ca(2+)/CaM and a specific phosphorylation-based regulatory mechanism. The latter involves an inhibitory type of autophosphorylation on a conserved serine at position 308, in the CaM regulatory domains of these two kinases. This phosphorylation, which occurs in growing cells, restrains the death-promoting effects of these kinases, and is specifically removed upon exposure of cells to various apoptotic stimuli. The dephosphorylation at this site increases the binding and sensitivity of each of these two kinases to their common activator-CaM. In DAPk, the dephosphorylation of serine 308 also increases the Ca(2+)/CaM-independent substrate phosphorylation. In DPR-1, it also promotes the formation of homodimers necessary for its full activity. These results are consistent with a molecular model in which phosphorylation on serine 308 stabilizes a locked conformation of the CaM regulatory domain within the catalytic cleft and simultaneously also interferes with CaM binding. In DRP-1, it introduces an additional locking device by preventing homodimerization. We propose that this unique mechanism of autoinhibition, evolved to keep these death-promoting kinases silent in healthy cells and ensures their activation only in response to apoptotic signals.

Protein tyrosine phosphatase epsilon inhibits signaling by mitogen-activated protein kinases.

Mitogen-activated protein kinases (MAPKs) mediate signaling from the cell membrane to the nucleus following their phosphorylation at conserved threonine and tyrosine residues within their activation loops. We show that protein tyrosine phosphatase epsilon (PTP epsilon) inhibits ERK1 and ERK2 kinase activity and reduces their phosphorylation; in agreement, ERK phosphorylation is increased in fibroblasts and in mammary tumor cells from mice genetically lacking PTP epsilon. PTP epsilon inhibits events downstream of ERKs, such as transcriptional activation mediated by Elk1 or by the serum response element. PTP epsilon also inhibits transcriptional activation mediated by c-Jun and C/EBP binding protein (CHOP) but not that mediated by the unrelated NFkB, attesting that it is broadly active within the MAPK family but otherwise specific. The effect of PTP epsilon on ERKs is at least in part indirect because phosphorylation of the threonine residue in the ERK activation loop is reduced in the presence of PTP epsilon. Nonetheless, PTP epsilon is present in a molecular complex with ERK, providing PTP epsilon with opportunity to act on ERK proteins also directly. We conclude that PTP epsilon is a physiological inhibitor of ERK signaling. Slow induction of PTP epsilon and its lack of nuclear translocation following mitogenic stimulation suggest that PTP epsilon functions to prevent inappropriate activation and to terminate prolonged, rather than acute, activation of ERK in the cytosol.

The regulation of death-associated protein (DAP) kinase in apoptosis.

DAP-kinase is a calcium/calmodulin (Ca2+/CaM) serine/threonine kinase which positively mediates programmed cell death in a variety of cell systems. The kinase is localized to the actin microfilament and has a unique, multidomain structure consisting of ankyrin repeats and a death domain. One of the substrates of DAP-kinase was identified as myosin light chain (MLC), the phosphorylation of which mediates membrane blebbing. Another arm in its mode of action leads to the formation of autophagic vesicles. Recent work addressed its mode of regulation and identified a mechanism which restrains its apoptotic function in growing cells and enables its activation during cell death. It involves an inhibitory type of autophosphorylation on serine 308 within the CaM regulatory domain. This negative phosphorylation takes place in growing cells and is strongly reduced upon their exposure to the apoptotic stimulus of C6-ceramide. The substitution of serine 308 to alanine, which mimics the ceramide-induced dephosphorylation at this site, increases Ca2+/CaM-independent substrate phosphorylation, as well as binding and overall sensitivity of the kinase to CaM. At the cellular level, it strongly enhances the death-promoting activity of the kinase. These results are consistent with a molecular model in which phosphorylation on serine 308 stabilizes a locked conformation of the CaM regulatory domain within the catalytic cleft and, simultaneously, also interferes with CaM binding. We propose that this unique mechanism of auto-inhibition evolved to impose a locking device which keeps DAP-kinase silent in healthy cells and ensures its activation only in response to apoptotic signals.

Differences in public emotions, interest, sense of knowledge and compliance between the affected area and the nationwide general population during the first phase of a bird flu outbreak in Israel.

OBJECTIVE: In March 2006, 298,000 cases of birds infected with bird flu were destroyed in nine rural settlements in Israel, out of around 1.2 million birds that were destroyed within these settlements and in a radius of 3 km. The nationwide population was instructed to take preventive measures against the spread of infection. This study aims to compare the emotions, interest, sense of knowledge and compliance, of the population in the affected area with the nationwide general population, during the first phase of a bird flu outbreak in Israel. METHODS: We conducted a telephone survey among two randomly selected, representative samples of adults. One sample involved 500 adult residents of the nationwide area; and the other sample involved 103 adult residents of the affected area during the first phase of the outbreak. We measured perceived emotions, interest, sense of knowledge and compliance. We analyzed the differences in these parameters between the affected area and the nationwide population using chi-square and t-test analysis. A p value of less than 0.05 was considered to be statistically significant. RESULTS: The compliance for using measures of precaution was high and not significantly different between the affected area and the nationwide population. The interest in bird flue and the sense of knowledge were significantly higher in the affected area compared to the nationwide population (p<0.05). A misconception of a high human to human transmission was significantly higher in the nationwide population compared with the affected area (p<0.05). The levels of stress and fear perception were significantly lower in the affected area compared to the nationwide population (p<0.05). CONCLUSION: Interest, sense of knowledge and emotions of the population are different in the affected area during the early phase of bird flu outbreak compared with the general population in the same country. Authorities must consider these differences while planning the strategy of population education during the early phase of a bird flu outbreak.

Hsp90 recognizes a common surface on client kinases.

Hsp90 is a highly abundant chaperone whose clientele includes hundreds of cellular proteins, many of which are central players in key signal transduction pathways and the majority of which are protein kinases. In light of the variety of Hsp90 clientele, the mechanism of selectivity of the chaperone toward its client proteins is a major open question. Focusing on human kinases, we have demonstrated that the chaperone recognizes a common surface in the amino-terminal lobe of kinases from diverse families, including two newly identified clients, NFkappaB-inducing kinase and death-associated protein kinase, and the oncoprotein HER2/ErbB-2. Surface electrostatics determine the interaction with the Hsp90 chaperone complex such that introduction of a negative charge within this region disrupts recognition. Compiling information on the Hsp90 dependence of 105 protein kinases, including 16 kinases whose relationship to Hsp90 is first examined in this study, reveals that surface features, rather than a contiguous amino acid sequence, define the capacity of the Hsp90 chaperone machine to recognize client kinases. Analyzing Hsp90 regulation of two major signaling cascades, the mitogen-activated protein kinase and phosphatidylinositol 3-kinase, leads us to propose that the selectivity of the chaperone to specific kinases is functional, namely that Hsp90 controls kinases that function as hubs integrating multiple inputs. These lessons bear significance to pharmacological attempts to target the chaperone in human pathologies, such as cancer.

The caspase-cleaved DAP5 protein supports internal ribosome entry site-mediated translation of death proteins.

Apoptosis is characterized by a translation switch from cap-dependent to internal ribosome entry site (IRES)-mediated protein translation. During apoptosis, several members of the eukaryotic initiation factor (eIF)4G family are cleaved specifically by caspases. Here we investigated which of the caspase-cleaved eIF4G family members could support cap-independent translation through IRES elements that retain activity in the dying cell. We focused on two major fragments arising from the cleavage of eIF4GI and death-associated protein 5 (DAP5) proteins (eIF4GI M-FAG/p76 and DAP5/p86, respectively), because they are the only potential candidates to preserve the minimal scaffold function needed to mediate translation. Transfection-based experiments in cell cultures indicated that expression of DAP5/p86 in cells stimulated protein translation from the IRESs of c-Myc, Apaf-1, DAP5, and XIAP. In contrast, these IRESs were refractory to the ectopically expressed eIF4GI M-FAG/p76. Furthermore, our study provides in vivo evidence that the caspase-mediated removal of the C-terminal tail of DAP5/p97 relieves an inhibitory effect on the protein's ability to support cap-independent translation through the DAP5 IRES. Altogether, the data suggest that DAP5 is a caspase-activated translation factor that mediates translation through a repertoire of IRES elements, supporting the translation of apoptosis-related proteins.