Project description:v-Crk induces cellular tyrosine phosphorylation and transformation of chicken embryo fibroblasts (CEF). We studied the molecular mechanism of the v-Crk-induced transformation. Experiments with Src homology (SH)2 and SH3 domain mutants revealed that the induction of tyrosine phosphorylation of cellular proteins requires only the SH2 domain, but both the SH2 and SH3 domains are required for complete transformation. Analysis of three well defined signaling pathways, the mitogen-activated protein kinase (MAPK) pathway, the Jun N-terminal kinase (JNK) pathway, and the phosphoinositide 3-kinase (PI3K)/AKT pathway, demonstrated that only the PI3K/AKT pathway is constitutively activated in v-Crk-transformed CEF. Both the SH2 and SH3 domains are required for this activation of the PI3K/AKT pathway in CEF. We also found that the colony formation of CEF is strongly induced by a constitutively active PI3K mutant, and that a PI3K inhibitor, LY294002, suppresses the v-Crk-induced transformation. These results strongly suggest that constitutive activation of the PI3K/AKT pathway plays an essential role in v-Crk-induced transformation of CEF.
Project description:Starvation activates MAPK in the pharyngeal muscles of C. elegans through a muscarinic acetylcholine receptor, Gqalpha, and nPKC as shown by the following results: (1) Starvation causes phosphorylation of MAPK in pharyngeal muscle. (2) In a sensitized genetic background in which Gqalpha signaling cannot be downregulated, activation of the pathway by a muscarinic agonist causes lethal changes in pharyngeal muscle function. Starvation has identical effects. (3) A muscarinic antagonist blocks the effects of starvation on sensitized muscle. (4) Mutations and drugs that block any step of signaling from the muscarinic receptor to MAPK also block the effects of starvation on sensitized muscle. (5) Overexpression of MAPK in wild-type pharyngeal muscle mimics the effects of muscarinic agonist and of starvation on sensitized muscle. We suggest that, during starvation, the muscarinic pathway to MAPK is activated to change the pharyngeal muscle physiology to enhance ingestion of food when food becomes available.
Project description:The virulence factor IpgD, delivered into nonphagocytic cells by the type III secretion system of the pathogen Shigella flexneri, is a phosphoinositide 4-phosphatase generating phosphatidylinositol 5 monophosphate (PtdIns5P). We show that PtdIns5P is rapidly produced and concentrated at the entry foci of the bacteria, where it colocalises with phosphorylated Akt during the first steps of infection. Moreover, S. flexneri-induced phosphorylation of host cell Akt and its targets specifically requires IpgD. Ectopic expression of IpgD in various cell types, but not of its inactive mutant, or addition of short-chain penetrating PtdIns5P is sufficient to induce Akt phosphorylation. Conversely, sequestration of PtdIns5P or reduction of its level strongly decreases Akt phosphorylation in infected cells or in IpgD-expressing cells. Accordingly, IpgD and PtdIns5P production specifically activates a class IA PI 3-kinase via a mechanism involving tyrosine phosphorylations. Thus, S. flexneri parasitism is shedding light onto a new mechanism of PI 3-kinase/Akt activation via PtdIns5P production that plays an important role in host cell responses such as survival.
Project description:Weak organic acids such as sorbic acid are important food preservatives and powerful fungistatic agents. These compounds accumulate in the cytosol and disturb the cellular pH and energy homeostasis. Candida glabrata is in many aspects similar to Saccharomyces cerevisiae. However, with regard to confrontation to sorbic acid, two of the principal response pathways behave differently in C. glabrata. In yeast, sorbic acid stress causes activation of many genes via the transcription factors Msn2 and Msn4. The C. glabrata homologs CgMsn2 and CgMsn4 are apparently not activated by sorbic acid. In contrast, in C. glabrata the high osmolarity glycerol (HOG) pathway is activated by sorbic acid. Here we show that the MAP kinase of the HOG pathway, CgHog1, becomes phosphorylated and has a function for weak acid stress resistance. Transcript profiling of weak acid treated C. glabrata cells suggests a broad and very similar response pattern of cells lacking CgHog1 compared to wild type which is over lapping with but distinct from S. cerevisiae. The PDR12 gene was the highest induced gene in both species and it required CgHog1 for full expression. Our results support flexibility of the response cues for general stress signaling pathways, even between closely related yeasts, and functional extension of a specific response pathway.
Project description:cAMP is an important second messenger that executes diverse physiological function in living cells. In this study, we investigated the effect of cAMP on canonical TRPC6 (transient receptor potential channel 6) channels in TRPC6-expressing HEK293 cells and glomerular mesangial cells. The results showed that 500 ?m 8-Br-cAMP, a cell-permeable analog of cAMP, elicited [Ca(2+)](i) increases and stimulated a cation current at the whole-cell level in TRPC6-expressing HEK293 cells. The effect of cAMP diminished in the presence of the PI3K inhibitors wortmannin and LY294002 or the MEK inhibitors PD98059, U0126, and MEK inhibitor I. 8-Br-cAMP also induced phosphorylation of MEK and ERK1/2. Conversion of serine to glycine at an ERK1/2 phosphorylation site (S281G) abolished the cAMP activation of TRPC6 as determined by whole-cell and cell-attached single-channel patch recordings. Experiments based on a panel of pharmacological inhibitors or activators suggested that the cAMP action on TRPC6 was not mediated by PKA, PKG, or EPAC (exchange protein activated by cAMP). Total internal fluorescence reflection microscopy showed that 8-Br-cAMP did not alter the trafficking of TRPC6 to the plasma membrane. We also found that, in glomerular mesangial cells, glucagon-induced [Ca(2+)](i) increases were mediated through the cAMP-PI3K-PKB-MEK-ERK1/2-TRPC6 signaling pathway. In summary, this study uncovered a novel TRPC6 activation mechanism in which cAMP activates TRPC6 via the PI3K-PKB-MEK-ERK1/2 signaling pathway.
Project description:Upregulation of Smad7, an inhibitor of transforming growth factor-β1 (TGF-β1), occurs in sporadic colorectal cancer (CRC) and knockdown of Smad7 inhibits CRC cell growth, a phenomenon that associates with decreased expression of cell division cycle 25 homolog A and arrest of cells in the S phase of the cell cycle. These findings occur in CRC cells unresponsive to TGF-β1, thus suggesting the existence of a Smad7-mediated TGF-β1-independent mechanism that controls CRC cell behavior. Here we show that Smad7 inhibition with a specific Smad7 antisense oligonucleotide upregulates eukaryotic translation initiation factor 2α (eIF2α) phosphorylation, a transcription factor involved in the regulation of cell cycle arrest and induction of cell death, and induces activating transcription factor 4 (ATF4) and CCAAT/enhancer binding protein homology protein (CHOP), two downstream targets of eIF2α. Among the upstream kinases that control eIF2α phosphorylation, the serine-threonine protein kinase RNA (PKR), but not general control non-derepressible 2 (GCN2) and protein kinase RNA-like endoplasmic reticulum kinase (PERK), is activated by Smad7 knockdown. PKR silencing abolishes Smad7 antisense-induced eIF2α phosphorylation and ATF4/CHOP induction, thereby preventing Smad7 antisense-driven cell death. Smad7 inhibition diminishes interaction of PKR with protein kinase inhibitor p58 (p58IPK), a cellular inhibitor of PKR, but does not change the expression and/or activity of other factors involved in the control of PKR activation. These findings delineate a novel mechanism by which Smad7 knockdown promotes CRC cell death.
Project description:Xylazine is a potent analgesic extensively used in veterinary and animal experimentation. Evidence exists that the analgesic effect can be inhibited using adenosine 5'-monophosphate activated protein kinase (AMPK) inhibitors. Considering this idea, the aim of this study was to investigate whether the AMPK signaling pathway is involved in the central analgesic mechanism of xylazine in the rat. Xylazine was administrated via the intraperitoneal route. Sprague-Dawley rats were sacrificed and the cerebral cortex, cerebellum, hippocampus, thalamus and brainstem were collected for determination of liver kinase B1 (LKB1) and AMPKα mRNA expression using quantitative real-time polymerase chain reaction (qPCR), and phosphorylated LKB1 and AMPKα levels using western blot. The results of our study showed that compared with the control group, xylazine induced significant increases in AMPK activity in the cerebral cortex, hippocampus, thalamus and cerebellum after rats received xylazine (P < 0.01). Increased AMPK activities were accompanied with increased phosphorylation levels of LKB1 in corresponding regions of rats. The protein levels of phosphorylated LKB1 and AMPKα in these regions returned or tended to return to control group levels. However, in the brainstem, phosphorylated LKB1 and AMPKα protein levels were decreased by xylazine compared with the control (P < 0.05). In conclusion, our data indicates that xylazine alters the activities of LKB1 and AMPK in the central nervous system of rats, which suggests that xylazine affects the regulatory signaling pathway of the analgesic mechanism in the rat brain.
Project description:In mammals, the well-organized activation of quiescent primordial follicles is pivotal for female reproductive reserve. In the present study, we examined the mechanisms underlying primordial follicle activation in mice. We found that endothelial nitric oxide synthase (eNOS) and its downstream effectors, cyclic guanosine monophosphate (cGMP) and cGMP-dependent protein kinase G (PKG), were expressed in pre-granulosa cells and promoted primordial follicle activation, oocyte growth and granulosa cell proliferation in neonatal ovaries. Mammalian target of rapamycin (mTOR) colocalized with PKG in pre-granulosa cells and was essential for eNOS/cGMP/PKG pathway-induced primordial follicle activation. The eNOS/cGMP/PKG pathway was found to stabilize mTOR protein. The mRNA levels of F-box and WD repeat domain containing 7 (FBXW7), an E3 ubiquitin ligase, correlated negatively with mTOR protein levels in neonatal ovaries. FBXW7 bound to and destabilized mTOR protein in pre-granulosa cells in a ubiquitin/proteasome-dependent manner. However, agonists of the eNOS/cGMP/PKG pathway reduced FBXW7 mRNA levels. FBXW7 overexpression suppressed primordial follicle activation and prevented the eNOS/cGMP/PKG pathway from activating primordial follicles and stabilizing mTOR protein. These findings demonstrate that the eNOS/cGMP/PKG pathway activates primordial follicles by suppressing FBXW7-induced ubiquitination of mTOR in mice.
Project description:Peroxynitrite is a potent oxidizing and nitrating species formed in a diffusion-limited reaction between nitrogen monoxide and superoxide. It induces apoptosis through unknown mechanisms and is believed to interfere with receptor tyrosine kinase signalling through nitration of tyrosine residues. One pathway emanating from receptor tyrosine kinases is that leading to activation of the anti-apoptotic kinase Akt. In the present study we provide evidence that peroxynitrite, administered to cells using two different delivery systems, results in the dose- and time-dependent activation of Akt. Akt activation is rapid and followed by phosphorylation of glycogen synthase kinase-3, an established substrate of Akt. Akt activation is inhibited in the presence of the phosphoinositide 3-kinase (PI-3K) inhibitors wortmannin and LY294002, and by treatment with the platelet-derived growth factor (PDGF) receptor (PDGFR) inhibitor AG1295, indicating a requirement for PDGFR and PI-3K in mediating peroxynitrite-induced Akt activation. Accordingly, the PDGFR-A and PDGFR-B isoforms were shown to undergo rapid tyrosine phosphorylation on treatment with peroxynitrite. Prior exposure of cells to peroxynitrite interferes with PDGF-induced Akt phosphorylation. Our findings suggest that Akt activation occurs as an acute response to peroxynitrite treatment and could play an important role in influencing cell survival and/or alter the cellular response to other growth regulatory signals.
Project description:Methylglyoxal is a typical 2-oxoaldehyde derived from glycolysis. We show here that methylglyoxal activates the Pkc1-Mpk1 mitogen-activated protein (MAP) kinase cascade in a target of rapamycin complex 2 (TORC2)-dependent manner in the budding yeast Saccharomyces cerevisiae. We demonstrate that TORC2 phosphorylates Pkc1 at Thr(1125) and Ser(1143). Methylglyoxal enhanced the phosphorylation of Pkc1 at Ser(1143), which transmitted the signal to the downstream Mpk1 MAP kinase cascade. We found that the phosphorylation status of Pkc1(T1125) affected the phosphorylation of Pkc1 at Ser(1143), in addition to its protein levels. Methylglyoxal activated mammalian TORC2 signaling, which, in turn, phosphorylated Akt at Ser(473). Our results suggest that methylglyoxal is a conserved initiator of TORC2 signaling among eukaryotes.