Diacylglycerol kinase: a key modulator of signal transduction?

Diacylglycerol kinase (DGK) plays a central role in the metabolism of diacylglycerol released as a second messenger in agonist-stimulated cells. The major purified form of the enzyme (80 kDa DGK) is highly abundant in lymphocyte cytosol and may become membrane-associated via phosphorylation by protein kinase C. In addition, there are several kinase subspecies immunologically distinct from the 80 kDa enzyme, which differ markedly in their responses to several compounds such as sphingosine and R59022. Thus, further work on each enzyme species is needed to define the function of DGK in stimulated cells.     

Oxidation of phosphatidylserine: a mechanism for plasma membrane phospholipid scrambling during apoptosis?

Selective oxidation of phosphatidylserine (PS) during apoptosis precedes its externalization in plasma membrane and is essential for the engulfment of apoptotic cells. To experimentally test whether PS oxidation stimulates its externalization via its effects on aminophospholipid translocase (APT) or by enhanced PS scrambling, action of oxidized PS (PSox) was studied using leukemia HL-60 cells and lymphoma Raji cells. Both PS and PSox were equally well recognized by APT. PSox did not inhibit APT. Rate of transmembrane PS diffusion was fourfold higher in cells with integrated PSox than with PS. Thus, PSox acts as a "non-enzymatic scramblase" likely contributing to PS externalization.     

Activation segment exchange: a common mechanism of kinase autophosphorylation?

The crystal structure of the kinase domain from human checkpoint kinase 2 (Chk2) has shown, for the first time, the reciprocal exchange of activation segments between two adjacent molecules and provides the molecular basis for understanding the observed mode of Chk2 kinase activation via trans-autophosphorylation. With further examples of activation segment exchanged kinase domains now publicly available (i.e. Ste20-like kinase, Ser/Thr kinase 10 and Death-associated protein kinase 3), we suggest that this phenomenon represents a common mechanism of activation amongst a particular subset of protein kinases, that is, those that are dimeric (either transiently or constitutively), that undergo activation by autophosphorylation and that have activation segment amino acid sequences that do not resemble those of their substrate consensus sequence.     

Inhibition of neutrophil apoptosis by acrolein: a mechanism of tobacco-related lung disease?

Cigarette smoking is known to contribute to inflammatory diseases of the respiratory tract by promoting recruitment of inflammatory-immune cells such as neutrophils and perhaps by altering neutrophil functional properties. We investigated whether acrolein, a toxic unsaturated aldehyde found in cigarette smoke, could directly affect neutrophil function. Exposure of freshly isolated human neutrophils to acrolein markedly inhibited spontaneous neutrophil apoptosis as indicated by loss of membrane asymmetry and DNA fragmentation and induced increased neutrophil production of the chemokine interleukin-8 (IL-8). Acrolein (1--50 microM) was found to induce marked activation of extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinases (MAPKs), and inhibition of p38 MAPK activation by SB-203580 prevented acrolein-induced IL-8 release. However, inhibition of either ERK or p38 MAPK did not affect acrolein-dependent inhibition of apoptosis. Acrolein exposure prevented the activation of caspase-3, a crucial step in the execution of neutrophil apoptosis, presumably by direct inhibition of the enzyme. Our results indicate that acrolein may contribute to smoke-induced inflammatory processes in the lung by increasing neutrophil recruitment and reducing neutrophil clearance by apoptosis.     

CDPKs - a kinase for every Ca2+ signal?

Numerous stimuli can alter the Ca2+concentration in the cytoplasm, a factor common to many physiological responses in plant and animal cells. Calcium-binding proteins decode information contained in the temporal and spatial patterns of these Ca2+ signals and bring about changes in metabolism and gene expression. In addition to calmodulin, a calcium-binding protein found in all eukaryotes, plants contain a large family of calcium-binding regulatory protein kinases. Evidence is accumulating that these protein kinases participate in numerous aspects of plant growth and development.     

Synergistic activation of caspase-3 by m-calpain after neonatal hypoxia-ischemia: a mechanism of "pathological apoptosis"?

The relative contributions of apoptosis and necrosis in brain injury have been a matter of much debate. Caspase-3 has been identified as a key protease in the execution of apoptosis, whereas calpains have mainly been implicated in excitotoxic neuronal injury. In a model of unilateral hypoxia-ischemia in 7-day-old rats, caspase-3-like activity increased 16-fold 24 h postinsult, coinciding with cleavage of the caspase-3 proenzyme and endogenous caspase-3 substrates. This activation was significantly decreased by pharmacological calpain inhibition, using CX295, a calpain inhibitor that did not inhibit purified caspase-3 in vitro. Activation of caspase-3 by m-calpain, but not mu-calpain, was facilitated in a dose-dependent manner in vitro by incubating cytosolic fractions, containing caspase-3 proform, with calpains. This facilitation required the presence of some active caspase-3 and could be abolished by including the specific calpain inhibitor calpastatin. This indicates that initial cleavage of caspase-3 by m-calpain, producing a 29-kDa fragment, facilitates the subsequent cleavage into active forms. This is the first report to our knowledge suggesting a direct link between the early, excitotoxic, calcium-mediated activation of calpain after cerebral hypoxia-ischemia and the subsequent activation of caspase-3, thus representing a tentative pathway of "pathological apoptosis."     

Beta-arrestins as regulators of signal termination and transduction: how do they determine what to scaffold?

Over the last decade β-arrestins have emerged as pleiotropic scaffold proteins, capable of mediating numerous diverse responses to multiple agonists. Most well characterized are the G-protein-coupled receptor (GPCR) stimulated β-arrestin signals, which are sometimes synergistic with, and sometimes independent of, heterotrimeric G-protein signals. β-arrestin signaling involves the recruitment of downstream signaling moieties to β-arrestins; in many cases specific sites of interaction between β-arrestins and the downstream target have been identified. As more information unfolds about the nature of β-arrestin scaffolding interactions, it is evident that these proteins are capable of adopting multiple conformations which in turn reveal a specific set of interacting domains. Recruitment of β-arrestin to a specific GPCR can promote formation of a specific subset of available β-arrestin scaffolds, allowing for a higher level of specificity to given agonists. This review discusses recent advances in β-arrestin signaling, discussing the molecular details of a subset of known β-arrestin scaffolds and the significance of specific binding interactions on the ultimate cellular response.     

Heat shock protein 10 and signal transduction: a "capsula eburnea" of carcinogenesis?

To date, little is known either about the physical interactions of heat shock protein 10 (Hsp10) with other proteins within the cell or its involvement in signal transduction pathways. Hsp10 has been considered mainly as a partner of Hsp60 in the Hsp60/10 protein folding machine. Only recently, Hsp10 was reported to interact with proteins involved in deoxyribonucleic acid checkpoint inactivation, termination of M-phase, messenger ribonucleic acid export, import of nuclear proteins, nucleocytoplasmic transport, and pheromone signaling pathways. At the same time, Hsp10 expression can be up-regulated in cancer cells, because it accumulates as the cell transformation progresses. Recent data suggest that Hsp10 may be not only a component of the folding machine but also an active player of the cell signaling network, influencing cell cycle, nucleocytoplasmic transport, and metabolism, with putative roles in the lack of cell differentiation and in the inhibition of apoptosis. In this review, we revise the involvement of Hsp10 in signal transduction pathways and its possible role in cancer etiology.     

Sensitization of adenylate cyclase: a general mechanism of neuroadaptation to persistent activation of Galpha(i/o)-coupled receptors?

Acute activation of Galphas-coupled receptors stimulates cyclic AMP accumulation leading to the activation of downstream signaling cascades. These Galphas-mediated events can be countered by acute activation of inhibitory G proteins (Galpha(i/o)), which inhibit the activity of adenylate cyclase, thereby attenuating cyclic AMP accumulation. Furthermore, an additional, less direct mechanism for Galpha(i/o) proteins modulation of cyclic AMP signaling also has been described. Persistent activation of several Galpha(i/o)-coupled receptors has been shown to result in a subsequent paradoxical enhancement of adenylate cyclase activity in response to drug-stimulated cyclic AMP accumulation. This sensitization of adenylate cyclase likely represents a cellular adaptive response following prolonged activation of inhibitory receptors. Recent advances in our knowledge of G protein signaling, adenylate cyclase regulation, and other cellular signaling mechanisms have extensively increased our insight into this phenomenon. It is now thought that sensitization occurs as part of a compensatory mechanism by which the cell adapts to chronic inhibitory input. Such a mechanism may be involved in modulating Galphas-coupled receptor signaling following neurotransmitter elevations that occur in psychiatric disease states or following the administration of many drugs of abuse. This review will focus on recent advances in the understanding of molecular signaling pathways that are involved in sensitization and describe the potential role of sensitization in neuronal cell function.     

Prostaglandins as reducing agents: a model of adenylate cyclase activation?

It has been suggested that adenylate cyclase activation involves reduction of a disulfide linkage. Prostaglandin E1 (PGE1), prostaglandin E2 (PGE2), prostaglandin I2 (PGI2) and prostaglandin F2 alpha (PGF2 alpha) were tested for their ability to act as reducing agents with either cytochrome c, or the disulfide 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), the latter with a catalytic amount of ferric chloride. PGE1, PGE2, and PGI2 significantly reduced cytochrome c while PGF2 alpha did not. PGE1, PGE2 and PGI2 reduced DTNB while PGF2 alpha did not. The results are consistent with the postulate that prostaglandins which are effective in activating adenylate cyclase can act as reducing agents and might be involved in reductive activation of adenylate cyclase.     

Induction of apoptosis in host cells: a survival mechanism for Leishmania parasites?

Leishmania parasites invade host macrophages, causing infections that are either limited to skin or spread to internal organs. In this study, 3 species causing cutaneous leishmaniasis, L. major, L. aethiopica and L. tropica, were tested for their ability to interfere with apoptosis in host macrophages in 2 different lines of human monocyte-derived macrophages (cell lines THP-1 and U937) and the results confirmed in peripheral blood mononuclear cells (PBMC). All 3 species induced early apoptosis 48 h after infection (expression of phosphatidyl serine on the outer membrane). There were significant increases in the percentage of apoptotic cells both for U937 and PBMC following infection with each of the 3 species. Early apoptotic events were confirmed by mitochondrial membrane permeabilization detection and caspase activation 48 and 72 h after infection. Moreover, the percentage of infected THP-1 and U937 macrophages increased significantly (up to 100%) following treatment with an apoptosis inducer. Since phosphatidyl serine externalization on apoptosing cells acts as a signal for engulfment by macrophages, induction of apoptosis in the parasitized cells could actively participate in spreading the infection. In summary, parasite-containing apoptotic bodies with intact membranes could be released and phagocytosed by uninfected macrophages.     

Chick begging as a signal: are nestlings honest?

Begging by dependent avian offspring is known to correlate withhunger level, and parents use this as a signal of brood demandto adjust their chick feeding behavior. While there is informationon how each chick adjusts its begging to its own condition,little is known of how chicks adjust to the state of their nestmates. In two experiments we manipulated the competitive environmentof individual European starling (Sturnus vulgaris) chicks byaltering the state of nest mates while holding the state oftarget chicks constant In the first experiment we placed thetarget chick's nest mates in neighboring nests with brood sizesof two, five, or eight chicks. Following the manipulation wereturned them to their own nests and recorded begging behavioron videotape. In the second experiment we separated a targetchick from its siblings and manipulated feeding level in thelaboratory. The siblings were fed at one of three levels; meanwhile,all the target chicks were fed at the intermediate level. Afterthe manipulation we placed the target chicks with their siblingsand recorded their begging in response to an artificial stimulus.In neither experiment was the begging effort of the unmanipulatedtarget chicks affected by the changes in begging behavior oftheir siblings. This result supports the view that begging isa reliable signal of individual chick state and does not involveresponses to the effort of nest mates.     

The β-scaffold of the LOV domain of the Brucella light-activated histidine kinase is a key element for signal transduction

Light-oxygen-voltage (LOV) domains are blue-light-activated signaling modules present in a wide range of sensory proteins. Among them, the histidine kinases are the largest group in prokaryotes (LOV-HK). Light modulates the virulence of the pathogenic bacteria Brucella abortus through LOV-HK. One of the striking characteristic of Brucella LOV-HK is the fact that the protein remains activated upon light sensing, without recovering the basal state in the darkness. In contrast, the light state of the isolated LOV domain slowly returns to the dark state. To gain insight into the light activation mechanism, we have characterized by X-ray crystallography and solution NMR spectroscopy the structure of the LOV domain of LOV-HK in the dark state and explored its light-induced conformational changes. The LOV domain adopts the α/β PAS (PER-ARNT-SIM) domain fold and binds the FMN cofactor within a conserved pocket. The domain dimerizes through the hydrophobic β-scaffold in an antiparallel way. Our results point to the β-scaffold as a key element in the light activation, validating a conserved structural basis for light-to-signal propagation in LOV proteins.     

Induction and activation of the p53 pathway: a role for the protein kinase CK2?

Protein kinase CK2 has many established in vitro substrates, but it is only within the past few years that we have begun to ascertain which of these are its real physiological targets, how their phosphorylation may contribute towards regulating normal cell physiology, and how phosphorylation of these proteins might influence the development of diseases such as cancer. One of the well-characterised in vitro substrates for CK2 is the tumour suppressor protein, p53. However, the physiological nature of this interaction has never been fully established. In the present article, we summarise a recent study from our laboratory showing that phosphorylation of p53 at Ser392, the sole site modified by CK2 in vitro, is regulated by a novel mechanism where the stoichiometry of phosphorylation is governed by the rate of turnover of the p53 protein. Such a model is entirely consistent with phosphorylation by a constitutively active protein kinase such as CK2. In contrast to this, while there is overwhelming evidence that CK2 phosphorylates p53 in vitro and is the only detectable Ser392 protein kinase in cell extracts, our data raise uncertainty as to whether this interaction truly reflects events underpinning Ser392 phosphorylation in vivo. We consider the possible role of CK2 in regulating the p53 response in a wider context and suggest key issues that should be addressed experimentally to provide a more cohesive picture of the relationship between this important protein kinase and a pivotal anti-cancer surveillance system in cells.     

Host genetics as a cause of overdispersion of parasites among hosts: how general a phenomenon?

In the white-footed mouse, Peromyscus leucopus, the tapeworm Hymenolepis citelli occurs at low (2-3%) prevalence in the field. We found that mature infections (i.e., with egg production) developed in up to 100% of hosts. In the laboratory, a majority of hosts lost their infection by 28 days postintubation. In wild mice infected in the laboratory and returned to the field, infections were more prolonged, with half of the mice still infected at 100 days postintubation. A majority of previously infected hosts resisted challenge infection. Our introduction of laboratory-infected mice into a natural population of hosts appeared to cause infections among previously uninfected mice, leading to an increase in the prevalence of tapeworm infection among mice not intubated. Although genetically based expulsion of tapeworms before maturity is important in causing low prevalence in a similar host-parasite system, such resistance cannot explain low prevalence in the present system. It appears that both heterogeneous distribution and rarity of intermediate hosts as well as short parasite lifespan contribute to low prevalence and overdispersion. Host-parasite dynamics of 2 very similar systems appear to differ markedly.     

ATM kinase promotes both caspase-8 and caspase-9 activation during TNF-α-induced apoptosis of HeLa cells

In this study, we show that atraxia telangiectasia mutated kinase (ATM) activity is generally upregulated by different apoptotic stimuli, i.e. TNF-α, TRAIL, paclitaxel, or UV. Apoptotic progression is markedly attenuated by siATM-RNA through down regulation of caspase-8 and caspase-9 in parallel with decreases in FLIP-S (short form of cellular FLICE inhibitory protein) protein levels and Bid cleavage. In addition, ATM activity is upregulated through t-Cdc6 while caspase-8 and caspase-9 activities increase. Taken together, we suggest that ATM regulates caspase-8 activation by influencing levels of FLIP-S, ATM kinase activity is upregulated by t-Cdc6, and increased ATM activity plays an essential role in the amplification of apoptosis in TNF-α-stimulated HeLa cells.     

Inhibiting astrocytic activation: a novel analgesic mechanism of ketamine at the spinal level?

Although ketamine is widely used as an analgesic agent and has an anti-allodynic effect on neuropathic pain, the underlying analgesic mechanisms are not fully explained by the modern 'neuronal-based' theories. As emerging studies have focused on the critical role of spinal astrocytes in the pathological pain states, we have hypothesized that there exist some 'astrocytes-related' mechanisms in the analgesic function of ketamine. In the present study, using the spinal nerve ligation (SNL) pain model, we investigated the anti-nociceptive effects of intraperitoneal or intrathecal ketamine on SNL-induced neuropathic pain response, meanwhile, we investigated the astrocytic activation after ketamine administration on SNL rats. Behavioral data showed that either intraperitoneal or intrathecal ketamine inhibited SNL-induced allodynia, however, immunohistochemistry showed that SNL induced astrocytic activation was suppressed by intrathecal but not intraperitoneal ketamine. Using quantitative Western blot analysis, our report showed that intrathecal ketamine down-regulated glial fibrillary acidic protein expression, suggesting inhibition of SNL-induced astrocytic activation, which wasn't influenced by intraperitoneal administration. We conclude that intraperitoneal ketamine could alleviate SNL-induced neuropathic pain via the classical 'neuronal-based' mechanisms, but in addition, 'astrocytes-related' mechanisms were also important underlying the anti-allodynic effect of intrathecal ketamine.