MAP kinases mediate phagocytosis and melanization via prophenoloxidase activation in medfly hemocytes

E. coli phagocytosis by medfly hemocytes, in contrast to mammalian macrophages, associates with E. coli-challenged hemocyte secretion by mitogen activating protein (MAP) kinases. In the present work, we examined whether this system links with the proteolytic activation of prophenoloxidase (proPO). ProPO and prophenoloxidase-activating proteinases (PAPs) were initially identified within freshly isolated medfly hemocytes. Moreover, flow cytometry and immunocytochemical analysis revealed the constitutive expression of proPO and its stable association with hemocyte surface. The expression level of hemocyte surface proPO is not affected by E. coli infection. In addition, flow cytometry analysis in freshly isolated hemocytes showed that E. coli phagocytosis is markedly blocked by antibodies against proPO or PAPs, as well as by several serine protease inhibitors, strongly supporting the involvement of proPO cascade in the phagocytosis process. Similarly, it was shown that melanization process depends on proPO activation. MAP kinases appeared to control both phagocytosis and melanization, since they regulate PAPs secretion, a prerequisite for the conversion of proPO to active PO. From this and previous studies, hemocytes appear to be central to immune response in medfly.     

Growth signalling pathways in Arabidopsis and the AGC protein kinases

Lipid-derived signals are central to regulating a multitude of cellular processes but, in plants, little is known of the downstream signalling pathways. The Arabidopsis 3-phosphoinositide-dependent protein kinase (PDK1) could couple lipid signals to the activation of several protein kinases of the so-called AGC kinase family. The Arabidopsis AGC kinases contain sequence motives required for the docking of PDK1 and phosphorylation of their activation loop in the kinase catalytic domain. It is becoming evident that specific members of the AGC kinases are implicated in key growth signalling pathways. For example, Arabidopsis p70(S6K) might be a nodal point able to integrate hormonal and developmental signals with nutritional inputs, together with the Arabidopsis Target of Rapamycin (TOR) protein.     

Insulin and 12-O-tetradecanoylphorbol-13-acetate activation of two immunologically distinct myelin basic protein/microtubule-associated protein 2 (MBP/MAP2) kinases via de novo phosphorylation of threonine and tyrosine residues.

Two site-specific antibodies have been prepared by immunizing rabbits with chemically synthesized peptides derived from the partial cDNA-predicted amino acid sequence of extracellular signal-regulated kinase 1 (ERK1), which has been proposed to encode the microtubule-associated protein 2 (MAP2) kinase (Boulton, T. G., Yancopoulos, G. D., Gregory, J. S., Slauer, C., Moomaw, C., Hsu, J., and Cobb, M. H. (1990) Science 249, 64-67). With immunoprecipitation in the presence of sodium dodecyl sulfate (SDS) and Western blotting, an antibody to the peptide containing triple tyrosine residues (alpha Y91) resembling one of the insulin receptor autophosphorylation sites specifically recognized 42- and 44-kDa proteins. On the other hand, an antibody to the peptide corresponding to the COOH terminus portions (alpha C92) of the ERK1 cDNA gene product recognized the 44-kDa protein much more efficiently than the 42-kDa protein. With immunoprecipitation in the absence of SDS, alpha Y91 could barely recognize these two proteins and alpha C92 recognized the 44-kDa protein but failed to recognize the 42-kDa protein. Kinase assays in myelin basic protein (MBP)-containing gel, after SDS-polyacrylamide gel electrophoresis, revealed that insulin or 12-O-tetradecanoylphorbol-13-acetate (TPA)-stimulated MBP kinase activity in alpha Y91 immunoprecipitates comigrated at molecular mass 42 and 44 kDa. On the other hand, the stimulated MBP kinase activity in alpha C92 immunoprecipitates comigrated only at molecular mass 44 kDa. Insulin stimulated the MBP kinase activity in gels and phosphorylation of these two proteins by greater than 10-fold with a maximal level at 5 min. Insulin and TPA rapidly stimulate the phosphorylation of the 42- and 44-kDa proteins via de novo threonine and tyrosine phosphorylation. Tryptic phosphopeptide mapping analysis of the 42- and 44-kDa proteins, respectively, revealed a single major phosphopeptide containing phosphothreonine and phosphotyrosine, which was common to both insulin- and TPA-stimulated phosphoproteins. Protein phosphatase 2A treatment of these two phosphoproteins caused a complete loss of kinase activity with selective dephosphorylation of phosphothreonine. These data strongly suggest that these two proteins are highly related to the mitogen-activated protein (MAP) kinase with an apparent molecular mass of 42 kDa (Ray, L. B., and Sturgill, T. W. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 3753-3757) and that these two immunologically similar but distinct MBP/MAP2 kinases may represent isozymic forms of MBP/MAP2 kinases. These data also demonstrate that insulin and TPA activate MBP/MAP2 kinase activity by de novo phosphorylation of threonine and tyrosine residues via a very similar pathway.     

The genetics of protein secretion in E. coli

Genetic studies have identified six genes whose products comprise the general protein secretion machinery of Escherichia coli. Insights from mutant analysis and the biochemical properties of the purified components allows the secretion pathway to be described in some detail. The picture emerging provides a useful paradigm for similar pathways in other organisms.     

trans-Resveratrol induces apoptosis in human breast cancer cells MCF-7 by the activation of MAP kinases pathways

Polyphenols represent a large class of plant-derived molecules with a general chemical structure that act as potent free radical scavengers. They have long been recognized to possess several therapeutic activities ranging from anti-thrombotic to antioxidant. Moreover, the capability of polyphenols to act as reducing or oxidizing molecules depends on the presence of environmental metals and on the concentrations used. In this work we demonstrated that the stilbene trans-resveratrol was able to commit human breast cancer MCF-7 cells to apoptosis. Mainly, we evidenced a pivotal role of the mitochondria in this phenomenon as cytochrome c release into the cytosol was found after the treatment. We further showed that trans-resveratrol was able to affect cellular redox state. In particular, it induced an early production of ROS and lipid oxidation, and only later compromised the GSH/GSSG ratio. This mode of action was mirrored by a temporally different activation of JNK and p38(MAPK), with the former rapidly induced and the latter weakly activated at long intervals. The results obtained demonstrate a pro-apoptotic activity for trans-resveratrol, and suggest a preferential activation of different classes of MAP kinases in response to different oxidative stimuli (ROS versus GSH/GSSG alteration).     

Sec-independent protein translocation in Escherichia coli. A distinct and pivotal role for the TatB protein

In Escherichia coli, transmembrane translocation of proteins can proceed by a number of routes. A subset of periplasmic proteins are exported via the Tat pathway to which proteins are directed by N-terminal "transfer peptides" bearing the consensus (S/T)RRXFLK "twin-arginine" motif. The Tat system involves the integral membrane proteins TatA, TatB, TatC, and TatE. Of these, TatA, TatB, and TatE are homologues of the Hcf106 component of the DeltapH-dependent protein import system of plant thylakoids. Deletion of the tatB gene alone is sufficient to block the export of seven endogenous Tat substrates, including hydrogenase-2. Complementation analysis indicates that while TatA and TatE are functionally interchangeable, the TatB protein is functionally distinct. This conclusion is supported by the observation that Helicobacter pylori tatA will complement an E. coli tatA mutant, but not a tatB mutant. Analysis of Tat component stability in various tat deletion backgrounds shows that TatC is rapidly degraded in the absence of TatB suggesting that TatC complexes, and is stabilized by, TatB.     

Differential signalling pathways for EGF versus PDGF activation of Erk1/2 MAP kinase and cell proliferation in brown pre-adipocytes

Stimulation by both adrenergic and non-adrenergic pathways can induce proliferation of brown pre-adipocytes. To understand the signalling pathways involved in non-adrenergic stimulation of cell proliferation, we examined Erk1/2 activation. In primary cultures of mouse brown pre-adipocytes, both EGF (epidermal growth factor) and PDGF (platelet-derived growth factor) induced Erk1/2 activation. EGF-stimulated Erk1/2 activation involved Src tyrosine kinases, but not PKC or PI3K, whereas in PDGF-induced Erk1/2 activation, PI3K, PKC (probably the atypical ζ isoform) and Src were involved sequentially. Both EGF and PDGF induced PI3K-dependent Akt activation that was not involved in Erk1/2 activation. By comparing effects of signalling inhibitors (wortmannin, SH-6, TPA, Gö6983, PP2, PD98059) on EGF- and PDGF-induced Erk1/2 activation and cell proliferation (³H-thymidine incorporation), we conclude that while the signal transduction pathways initiated by these growth factors are clearly markedly different, their effects on cell proliferation can be fully explained through their stimulation of Erk1/2 activation; thus Erk1/2 is a common, essential step for stimulation of proliferation in these cells.     

Opposite changes in membrane fluidity mimic cold and heat stress activation of distinct plant MAP kinase pathways

Mitogen-activated protein kinases (MAPKs) appear to be ubiquitously involved in signal transduction during eukaryotic responses to extracellular stimuli. In plants, no heat shock-activated MAPK has so far been reported. Also, whereas cold activates specific plant MAPKs such as alfalfa SAMK, mechanisms of such activation are unknown. Here, we report a heat shock-activated MAPK (HAMK) immunologically related to ERK (Extracellular signal-Regulated Kinase) superfamily of protein kinases. Molecular mechanisms of heat-activation of HAMK and cold-activation of SAMK were investigated. We show that cold-activation of SAMK requires membrane rigidification, whereas heat-activation of HAMK occurs through membrane fluidization. The temperature stress- and membrane structure-dependent activation of both SAMK and HAMK is mimicked at 25 degrees C by destabilizers of microfilaments and microtubules, latrunculin B and oryzalin, respectively; but is blocked by jasplakinolide, a stabilizer of actin microfilaments. Activation of SAMK or HAMK by temperature, chemically modulated membrane fluidity, or by cytoskeleton destabilizers is inhibited by blocking the influx of extracellular calcium. Activation of SAMK or HAMK is also prevented by an antagonist of calcium-dependent protein kinases (CDPKs). In summary, our data indicate that cold and heat are sensed by structural changes in the plasma membrane that translates the signal via cytoskeleton, Ca2+ fluxes and CDPKs into the activation of distinct MAPK cascades.     

Induction of E. coli recA protein via recBC and alternate pathways: quantitation by enzyme-linked immunosorbent assay (ELISA)

Summary An enzyme-linked immunosorbent assay (ELISA) has been adapted to measure E. coli recA protein in the 1 to 10 ng range in whole-cell sonicates, membrane extracts, and osmotic shock fluid from 2x10⁸ cells. The specific activity of recA protein is maintained at a relatively constant basal level (800 to 1,200 molecules per cell for wild-type E. coli in L-broth, salt-depleted broth and minimal media) during early-log and mid-log phase growth, but it increases by two- to ten-fold as the culture approaches saturation density. Nalidixate-induced levels are 20- to 50-fold higher, and 100-fold higher in a constitutive tif ^- spr ^-mutant.Induction of recA protein synthesis by nalidixic acid, which normally requires functional recBC enzyme, also occurs in recB ^-and recC ^-cells by pathways activated by mutation in the sbcA and sbcB indirect suppressors. In recB ^- sbcA ^-mutants, exonuclease VIII, the recE gene product, is required for induction of recA protein. Abolition of exonuclease I activity by mutation in sbcB allows induction of recA protein by nalidixate in recB ^-and recC ^-cells. Mutation in recF does not affect induction by nalidixate in RecBC⁺ cells, but it enables induction to occur in RecBC^- cells, suggesting that recF gene product is involved in regulation of recA protein.     

Echinostoma paraensei and Schistosoma mansoni: adherence of unaltered or modified latex beads to hemocytes of the host snail Biomphalaria glabrata.

Hemocytes derived from a strain (13-16-R1) of Biomphalaria glabrata resistant to Schistosoma mansoni were significantly more likely to bind untreated latex beads than hemocytes from the schistosome-susceptible M line strain. Beads preincubated in 13-16-R1 plasma were more readily bound by both 13-16-R1 and M line hemocytes than beads preincubated in M line plasma. Beads preincubated in plasma derived from snails of either strain infected with the trematode Echinostoma paraensei were more readily bound by hemocytes than beads preincubated in plasma from control snails of the corresponding strain. Plasma from snails exposed to S. mansoni did not have a similar effect. Throughout these experiments, beads receiving a particular treatment were consistently bound at higher rates by 13-16-R1 than M line hemocytes. SDS-PAGE of plasma components eluted from beads revealed differences between treatments, particularly in diffuse bands falling into two groups, of 75-130 and 150-220 kDa. The results indicate that both hemocytes and plasma components from the two host strains differ and identify plasma molecules deserving of additional study as possible modulators of hemocyte effector functions. Also, S. mansoni and E. paraensei provoked different responses in the same host snail.     

Inhibition of neutrophil oxidative burst and granule secretion by Wortmannin: Potential role of MAP kinase and renaturable kinases

Exposure of neutrophils to a variety of agonists including soluble chemoattractant peptides and cytokines results in degranulation and activation of the oxidative burst (effector functions) that are required for bacterial killing. At present, the signaling pathways regulating these important functions are incompletely characterized. Mitogen-activated protein (MAP) kinases (MAPK) as well as members of a family of “renaturable kinases” are rapidly activated in neutrophils in response to diverse physiological agonists, suggesting that they may regulate cell activation. Antagonists of phosphatidyl inositol-3-(OH) kinase (PI3-kinase) such as wortmannin (Wtmn) inhibit these effector responses as well as certain of the above-mentioned kinases, leading to the suggestion that these enzymes lie downstream of PI3-kinase in the pathway regulating the oxidative burst and granule secretion. However, an apparent discrepancy exists in that, while virtually obliterating activity of PI3-kinase and the oxidase at low concentrations (ID₅₀ < 20 nM), Wtmn has only variable inhibitory effects on MAPK even at substantially higher concentrations (75–100 nM). This raises the possibility that the inhibitory effects of Wtmn are mediated via other enzyme systems. The purpose of the current study was therefore to compare the effects of Wtmn on PI3-kinase activity and on the chemoattractant-activated kinases, and to determine the potential relationship of these pathways to microbicidal responses. In human neutrophils, both the oxidative burst and granule secretion induced by fMLP were inhibited by Wtmn but at markedly different concentrations: the oxidative burst was inhibited with an ID₅₀ of <5 nM while granule secretion was only partially inhibited at concentrations exceeding 75 nM. Activation of both MEK-1 and MAPK in response to fMLP was only partially inhibited by high doses of Wtmn (ID₅₀ of > 100 nM and ≡75 nM, respectively). In contrast, Wtmn potently inhibited fMLP-induced activation of the 63 and 69 kDa renaturable kinases (ID₅₀ ≡ 5–10 nM). We speculate that the renaturable kinases may be involved in the regulation of the oxidative burst, whereas the MAPK pathway may play a role in other neutrophil functions such as granule secretion. J. Cell. Physiol. 172:94–108, 1997. © 1997 Wiley-Liss, Inc.     

Involvement of the ERK/MAP kinase signalling pathway in milli-calpain activation and myogenic cell migration

Recent research carried out in our laboratory has shown that IGF-1, TGF-beta1, and insulin were able to strongly stimulate myoblast migration by increasing milli-calpain expression and activity. However, the signalling pathways involved in these phenomena remain unknown. The aim of this study was to identify the signalling pathway(s) responsible for the effects of IGF-1, TGF-beta1, and insulin on myoblast migration and on milli-calpain expression and activity. For this purpose, wound healing assays were carried out in the presence of growth factors with or without specific inhibitors of ERK/MAP kinase and PI3K/Akt pathways. The results clearly showed that the inhibition of the ERK/MAP kinase pathway prevents the effects of growth factors on myoblast migration. Secondly, the expression and the activity of milli-calpain were studied in cells treated with growth factor, alone or with ERK/MAP kinase inhibitor. The results demonstrated that the up-regulation of milli-calpain expression and activity was mediated by the ERK/MAP kinase pathway. Finally, the possible implication of MyoD and myogenin, myogenic regulatory factors able to regulate milli-calpain expression, was studied. Taken together our results clearly showed that the ERK/MAP kinase signalling pathway is responsible for the effects of the three growth factors on myoblast migration and on milli-calpain expression and activity. On the opposite, the PI3K/Akt signalling pathway, MyoD and myogenin seem to be not implicated in these phenomena.     

Human macrophage adhesion on fibronectin: the role of substratum and intracellular signalling kinases.

Fibronectin and Arg-Gly-Asp (RGD)- and/or Pro-His-Ser-Arg-Asn (PHSRN)-containing oligopeptides were immobilized onto physicochemically distinct substrata: polyethyleneglycol-based networks or tissue culture polystyrene (TCPS). The role of selected signalling kinases in the adhesion of human primary blood-derived macrophages on these modified substrata was investigated. We demonstrated that the protein tyrosine kinase (PTK) or protein serine/threonine kinase (PSK) dependency and the PTK-PSK cross-talk compensation for macrophage adhesion varied dynamically with the substratum modification and the culture time. The inhibition of MAPK kinase (MAPKK) decreased macrophage adhesion on TCPS, whereas the inhibition of phosphoinositide-3 kinase (PI3 kinase) decreased macrophage adhesion on networks at 24 h. The PI3 kinase-protein kinase C (PKC)-MAPK cascade was involved in macrophage adhesion on fibronectin-preadsorbed TCPS or networks but not on fibronectin-grafted networks. This fibronectin-mediated adhesion signalling involved both RGD and PHSRN sequences in a form of G(3)RGDG(6)PHSRNG on TCPS but not on networks. Furthermore, G(3)RGDG(6)PHSRNG grafted onto networks evoked unique signalling in macrophage adhesion from that preadsorbed onto networks. Thus, macrophage adhesion and the role of selected signalling kinases were modulated by the substratum and the ligand conjugation method.     

Nitrofurazone-reducing enzymes in E. coli and their role in drug activation in vivo.

Earlier work showed that Escherichia coli contains at least two enzymes which reduce nitrofurazone and other nitrofuran derivatives. One of these enzymes is lacking in some nitrofurazone-resistant mutant strains. We now report that there are three separable nitrofuran reductases in this organism: reductase I (mol. wt. approximately 50 000, insensitive to O2), reductase IIa (mol. wt. approximately 120 000, inhibited by oxygen), reductase IIb (mol. wt. approximately 700 000, inhibited by O2). Unstable metabolites formed during the reduction of nitrofurazone by preparations containing reductases IIa and IIb produce breaks in DNA in vitro. In vivo experiments with nitrofurazone-resistant strains, which lack reductase II but contain reductases IIa and IIb, demonstrated that lethality, mutation, and DNA breakage are all greatly increased when cultures are incubated under anaerobic conditions, i.e., conditions such that reductase II is active. These results provide further evidence for the importance of reductive activation of nitrofurazone.     

Uptake and remodeling of exogenous phosphatidylethanolamine in E. coli

The fate of exogenous short-chain analogues of phosphatidylethanolamine and phosphatidylserine was studied in a deep-rough derivative of E. coli mutant strain AD93 that cannot synthesize phosphatidylethanolamine de novo. Using mass spectrometry, it was shown that dicaproyl(di 6:0)-phosphatidylethanolamine is extensively remodeled, eventually adopting the phosphatidylethanolamine species profile of the parental wild-type strain of AD93. Dicaproyl-phosphatidylserine was decarboxylated to form phosphatidylethanolamine, and yielded a species profile, which strongly resembled that of the introduced phosphatidylethanolamine. This demonstrates transport of phosphatidylserine to the cytosolic leaflet of the inner membrane. The changes of the species profile of phosphatidylethanolamine indicate that the short-chain phospholipids are most likely remodeled via two consecutive acyl chain substitutions, and at least part of this remodeling involves transport to the inner membrane.     

Spermine signalling in tobacco: activation of mitogen-activated protein kinases by spermine is mediated through mitochondrial dysfunction

Polyamines (PAs) play important roles in cell proliferation, growth and environmental stress responses of all living organisms. In this study, we examine whether these compounds act as signal mediators. Spermine (Spm) specifically activated protein kinases of tobacco leaves, which were identified as salicylic acid (SA)-induced protein kinase (SIPK) and wound-induced protein kinase (WIPK), using specific antibodies. Upon Spm treatment, upregulation of WIPK, but not SIPK, was observed. Spm-induced mitogen-activated protein kinases (MAPKs) activation and WIPK upregulation were prevented upon pre-treatment with antioxidants and Ca2+ channel blockers. Additionally, Spm specifically stimulated expression of the alternative oxidase (AOX) gene, which was disrupted by these antioxidants and Ca2+ channel blockers. Bongkrekic acid (BK), an inhibitor of the opening of mitochondrial permeability transition (PT) pores, suppressed MAPKs activation and accumulation of WIPK and AOX mRNA. Our data collectively suggest that Spm causes mitochondrial dysfunction via a signalling pathway in which reactive oxygen species and Ca2+ influx are involved. As a result, the phosphorylation activities of the two MAPK enzymes SIPK and WIPK are stimulated.     

Expression and secretion of proteins in E. coli

This review outlines approaches to the cloning and expression of proteins in Escherichia coli. The expression vectors described here (pIN-III derivatives) utilize the strong lipoprotein promoter, which is controlled by the lac-UV5 promoter-operator. These vectors provide the means for targeting a protein to any of the four subcellular compartments of the bacterial cell: cytoplasm, cytoplasmic membrane, periplasm, and outer membrane. Of particular importance is that secretion of proteins into the E. coli periplasm (using the OmpA signal peptide) is applicable for the production of both prokaryotic and eukaryotic proteins thereby enhancing protein activity and stability.