Incorporation Of 32Pi Research Articles

Differential regulation of the glutamate transporters GLT-1 and GLAST by GSK3β

The glutamate transporters GLAST and GLT-1 are mainly expressed in glial cells and regulate glutamate levels in the synapses. GLAST and GLT-1 are the targets of several signaling pathways. In this study we explore the possible functional interaction between these transporters and GSK3β. This kinase is involved in multiple cellular processes including neuronal development and synaptic plasticity. To evaluate whether GLT-1 and GLAST were regulated by GSK3β, we coexpresed these proteins in heterologous expression systems. In both COS-7 cells and Xenopus laevis oocytes, GSK3β stimulated the activity of GLT-1 and reduced that of GLAST. These effects were associated with corresponding changes in the amounts of GLT-1 or GLAST in the plasma membrane. These effects were suppressed by inhibitors of GSK3β or a catalytically inactive form of the kinase. GSK3β also decreases the incorporation of 32Pi into GLT-1 and increases GLAST phosphorylation. Pharmacological inhibition of endogenous GSK3β in primary cultures of rat brain cortex also leads to a differential modulation of GLT-1 and GLAST. Our results suggest that constitutively active GSK3β is important in controlling the expression of functional glutamate transporters on the plasma membrane. This regulation might be relevant in physiological and pathological conditions in which glutamate transporters and GSK3β signaling are involved.

Glycine transporters GlyT1 and GlyT2 are differentially modulated by glycogen synthase kinase 3β

Inhibitory glycinergic neurotransmission is terminated by the specific glycine transporters GlyT1 and GlyT2 which actively reuptake glycine from the synaptic cleft. GlyT1 is associated with both glycinergic and glutamatergic pathways, and is the main regulator of the glycine levels in the synapses. GlyT2 is the main supplier of glycine for vesicle refilling, a process that is vital to preserve the quantal glycine content in synaptic vesicles. Therefore, to control glycinergic neurotransmission efficiently, GlyT1 and GlyT2 activity must be regulated by diverse neuronal and glial signaling pathways. In this work, we have investigated the possible functional modulation of GlyT1 and GlyT2 by glycogen synthase kinase 3 (GSK3β). This kinase is involved in mood stabilization, neurodegeneration and plasticity at excitatory and inhibitory synapses. The co-expression of GSK3β with GlyT1 or GlyT2 in COS-7 cells and Xenopus laevis oocytes, leads to inhibition and stimulation of GlyT1 and GlyT2 activities, respectively, with a decrease of GlyT1, and an increase in GlyT2 levels at the plasma membrane. The specificity of these changes is supported by the antagonism exerted by a catalytically inactive form of the kinase and through inhibitors of GSK3β such as lithium chloride and TDZD-8. GSK3β also increases the incorporation of 32Pi into GlyT1 and decreases that of GlyT2. The pharmacological inhibition of the endogenous GSK3β in neuron cultures of brainstem and spinal cord leads to an opposite modulation of GlyT1 and GlyT2.Our results suggest that GSK3β is important for stabilizing and/or controlling the expression of functional GlyTs on the neural cell surface.

180 Can Neutrophil Apoptosis Be Targeted Therapeutically to Modify Inflammation During Myocardial Infarct Healing?

Neutrophils are rapidly recruited to infarct site in response to cardiomyocyte death. Apoptosis and phagocytosis by macrophages prevents further damage to host tissues, but can also polarise macrophages towards an...

Genistein induces apoptosis and autophagy in human breast MCF-7 cells by modulating the expression of proapoptotic factors and oxidative stress enzymes

Breast cancer is one of the common tumors occurring in woman and despite treatment, the prognostic is poor. Genistein, a soy isoflavone, has been reported to have chemopreventive\chemotherapeutic potential in multiple tumor types. Here, we investigated the genistein antiproliferative effect in MCF-7 breast cancer, underlying the molecular mechanisms involved in this effect. MCF-7 cancer and CCD1059sK fibroblast cells were treated with estradiol (10 nM) or genistein (0.01-100 μM) for 24, 48, and 72 h and the cell proliferation was investigated by MTT; membrane cell permeability was evaluated by LDH and PI incorporation; apoptosis was investigated by externalization of phosphatidylserine by FACS; and presence of autophagy was detected by LC3A/B immunostaining. The expression of apoptotic proteins and antioxidant enzymes was evaluated by qPCR. The results demonstrate that genistein (100 μM) for 72 h of treatment selectively reduced MCF-7 cell proliferation independent of estrogen receptor activation, while no cytotoxicity was observed in fibroblast cells. Further experiments showed that genistein induced phosphatidylserine externalization and LC3A/B immunopositivity in MCF-7 cells, indicating apoptosis and autophagy cell death. Genistein increased in three times proapoptotic BAX/Bcl-2 ratio and promoted a parallel downregulation of 20 times of antiapoptotic survivin. In addition, genistein promoted a decrease of 5.5, 9.3, and 3.6 times of MnSOD, CuZnSOD, and TrxR mRNA expression, respectively, while the GPx expression was increased by 6.5 times. These results suggest that the antitumor effect of genistein involved the modulation of antioxidant enzyme and apoptotic signaling expression, which resulted in apoptosis and progression of autophagy.

Separation of Electrical and Optical Energy Gaps: Selectively Adjusting the Electrical and Optical Properties for a Highly Efficient Blue Emitter

The design concept of separation of optical and electrical bandgap for wide bandgap materials is further developed in DCzSiPI. The HOMO/LUMO levels can be tuned by incorporation of PI and DCz substituents. The tetraphenylsilane core avoids the intramolecular charge transfer from DCz to PI (DCz = dimer carbazole, PI = phenanthro[9,10-d]imidazole). The allowed transitions are found to be from HOMO-1 to LUMO providing DCzSiPI with sufficient bandgap.

Thermodynamic Effects of the Alteration of the Axial Ligand on the Unfolding of Thermostable Cytochrome c

The role the axial methionine plays in the conformational properties and thermostability of the heme active site has been investigated with the help of site-specific mutations at the axial Met69 position with His (M69H) and Ala (M69A) in thermostable cytochrome c(552) from Thermus thermophilus. Detailed circular dichroism, direct electrochemistry, and other spectroscopic studies have been employed to investigate the thermally induced and GdnHCl-induced unfolding properties of the heme active site of the wild type and the mutants of cytochrome c(552). We observed an unusually high thermodynamic and thermal stability of the M69A mutant compared to that of wild-type cytochrome c(552). However, the M69H mutant exhibited a slightly lower unfolding free energy compared to that of the wild-type protein. The high conformational stability of the M69A mutant was attributed to the presence of residual structure in the unfolded state as well as to the altered conformation in the folded state of this mutant of cytochrome c(552). This study thus supports the view that apart from the folded state, the unfolded state of a protein may also make a significant contribution to the stability of a protein.

Stable polyphosphate accumulation by a pseudo-revertant of an Escherichia coli phoU mutant

phoU mutants of bacteria are potentially useful for the removal of inorganic phosphate (Pi) from sewage because they can accumulate a large amounts of polyphosphate (polyP). However, the growth of phoU mutants is severely defective and is easily outgrown by revertant(s) that have lost the ability to accumulate polyP during growth in a nutrient-rich medium. We found that a pseudo-revertant, designated LAP[+], that appeared in a culture of an Escherichia coli phoU mutant that could accumulate polyP even after ten serial passages. Reduction in the expression of the Pi-specific transporter Pst in LAP[+] may contribute to relieving stresses such as excess Pi incorporation that could stimulate reversions. The discovery of a LAP[+] provides a clue to generate phoU mutants that accumulate polyP in a stable manner.

Regulatory activity of azabisphosphonate-capped dendrimers on human CD4+ T cell proliferation enhances ex-vivo expansion of NK cells from PBMCs for immunotherapy

BackgroundAdoptive cell therapy with allogenic NK cells constitutes a promising approach for the treatment of certain malignancies. Such strategies are currently limited by the requirement of an efficient protocol for NK cell expansion. We have developed a method using synthetic nanosized phosphonate-capped dendrimers allowing such expansion. We are showing here that this is due to a specific inhibitory activity towards CD4+ T cell which could lead to further medical applications of this dendrimer.MethodsMononuclear cells from human peripheral blood were used to investigate the immunomodulatory effects of nanosized phosphonate-capped dendrimers on interleukin-2 driven CD4+T cell expansion. Proliferation status was investigated using flow cytometry analysis of CFSE dilution and PI incorporation experiments. Magnetic bead cell sorting was used to address activity towards individual or mixed cell sub-populations. We performed equilibrium binding assay to assess the interaction of fluorescent dendrimers with pure CD4+ T cells.ResultsPhosphonate-capped dendrimers are inhibiting the activation, and therefore the proliferation; of CD4+ T cells in IL-2 stimulated PBMCs, without affecting their viability. This allows a rapid enrichment of NK cells and further expansion. We found that dendrimer acts directly on T cells, as their regulatory property is maintained when stimulating purified CD4+ T cells with anti-CD3/CD28 microbeads. Performing equilibrium binding assays using a fluorescent analogue, we show that the phosphonate capped-dendrimers are specifically interacting with purified CD4+ T cells. Ultimately, we found that our protocol prevents the IL-2 related expansion of regulatory T cells that would be deleterious for the activity of infused NK cells.ConclusionHigh yield expansion of NK cells from human PBMCs by phosphonate-capped dendrimers and IL-2 occurs through the specific inhibition of the CD4+ lymphocyte compartment. Given the specificity of the interaction of dendrimers with CD4+ T cell, we hypothesize that regulatory activity may signal through a specific receptor that remains to be indentified. Therefore phosphonate-capped dendrimers constitute not only tools for the ex-vivo expansion of NK cells in immunotherapy of cancers but their mode of action could also lead to further medical applications where T cell activation and proliferation need to be dampened.

Phosphate (Pi) starvation effect on the cytosolic Pi concentration and Pi exchanges across the tonoplast in plant cells: an in vivo 31P-nuclear magnetic resonance study using methylphosphonate as a Pi analog.

In vivo (31)P-NMR analyses showed that the phosphate (Pi) concentration in the cytosol of sycamore (Acer pseudoplatanus) and Arabidopsis (Arabidopsis thaliana) cells was much lower than the cytoplasmic Pi concentrations usually considered (60-80 mum instead of >1 mm) and that it dropped very rapidly following the onset of Pi starvation. The Pi efflux from the vacuole was insufficient to compensate for the absence of external Pi supply, suggesting that the drop of cytosolic Pi might be the first endogenous signal triggering the Pi starvation rescue metabolism. Successive short sequences of Pi supply and deprivation showed that added Pi transiently accumulated in the cytosol, then in the stroma and matrix of organelles bounded by two membranes (plastids and mitochondria, respectively), and subsequently in the vacuole. The Pi analog methylphosphonate (MeP) was used to analyze Pi exchanges across the tonoplast. MeP incorporated into cells via the Pi carrier of the plasma membrane; it accumulated massively in the cytosol and prevented Pi efflux from the vacuole. This blocking of vacuolar Pi efflux was confirmed by in vitro assays with purified vacuoles. Subsequent incorporation of Pi into the cells triggered a massive transfer of MeP from the cytosol to the vacuole. Mechanisms for Pi exchanges across the tonoplast are discussed in the light of the low cytosolic Pi level, the cell response to Pi starvation, and the Pi/MeP interactive effects.

Modulation of expression and cellular distribution of p21 by macrophage migration inhibitory factor

BackgroundThe pleiotropic protein MIF, (macrophage migration inhibitory factor), has been demonstrated to modulate several key proteins governing cell cycle control and is considered to contribute to cell growth and differentiation. In this study we investigated the effect of MIF on the expression and cellular distribution of the CDK inhibitor p21.MethodsThe effect of endogenous MIF on p21 expression and distribution was examined by comparing murine dermal fibroblasts derived from wt and MIF -/- mice. The effect of MIF on cell growth and apoptotic rates was compared using 3H-Thymidine incorporation assays and annexin V/PI assays respectively. Total p21 protein levels were compared using flow cytometry and western blotting. p21 mRNA was assessed by RT-PCR. Intracellular p21 staining was performed to assess cellular distribution of total protein. To further confirm observations siRNA was used to knockdown MIF protein in wt cells. Cell cycle analysis was performed using PI incorporation assays.ResultsMIF-/- murine dermal fibroblasts exhibited reduced proliferative responses and were more susceptible to apoptosis. This was associated with reduced p21 expression and nuclear distribution. Treatment with recombinant MIF protein was demonstrated to reduce both basal and induced apoptosis and increase nuclear p21 expression. Reduced nuclear p21 expression was also observed in MIF siRNA treated wt cells.ConclusionThe results demonstrate that in the absence of MIF p21 expression and nuclear distribution is reduced which is associated with a reduction in cell growth and increased apoptosis. MIF may therefore play a role in maintaining homeostatic control of p21.

Phosphatidylinositol-4,5-bisphosphate Affects Ceramide 1-phosphate Phase Behavior

Ceramide is a well-characterized sphingolipid metabolite and second messenger that participates in numerous biological processes. When ceramide is phosphorylated by ceramide kinase (CERK), ceramide-1-phosphate (Cer1P) is obtained. It has recently been proposed that Cer1P is involved in cell survival, cell proliferation, inflammation and phagocytosis. It has been observed that the CERK activity is dependent on the interaction with phosphatidyl inositol-4,5-bisphosphate (PI(4,5)P2). In turn, Cer1P was found to affect the PI3K/AKT pathway. This suggests that PI(4,5)P2 and Cer1P might co-localize and interact. To address this issue, giant unilamellar vesicles (GUVs) composed of POPC and 10% Cer1P were made with different concentrations of brain PI(4,5)P2 (from 2%, 5%, 10% to 20 mol%), labeled with fluorescent lipids and analyzed by fluorescence microscopy. GUVs composed of POPC and 10% Cer1P showed irregular branch-shaped domains, which are characteristic for the Cer1P gel phase. In the presence of 2% brain PI(4,5)P2, the irregularly branched domains took on a shape of beads on strings, i.e., the domains were round indicating increased fluidity. With increasing amount of PI(4,5)P2 added, the bead region became larger and larger. In the presence of 10% PI(4,5)P2; the gel type string regions can barely be seen. For 20% PI(4,5)P2 only one fluid type region can be seen. Since the portion of Cer1P in the GUVs was fixed, (10% of the total lipids) the increasingly larger domains have to be the result of the incorporation of brain PI(4,5)P2 in the Cer1P phase. This incorporation of PI(4,5)P2 in the Cer1P phase leads to an increasing fluidity and increasing size of the domain. In conclusion, Cer1P and PI(4,5)P2 co-localize into a domain when mixed with POPC and this domain exhibits fluid like properties at high PI(4,5)P2 concentrations.

Proteomic approach to studying the cytotoxicity of YC‐1 on U937 leukemia cells and antileukemia activity in orthotopic model of leukemia mice

To evaluate the effects of YC-1 on leukemia cell lines, PI incorporation was used to determine cell viability. YC-1 induced a dose- and time-dependent decrease in viability and apoptosis in YC-1-treated U937 cells. YC-1-induced apoptosis is a cyclic guanosine monophosphate (cGMP)-independent pathway. Proteomic analysis showed that the altered proteins include the significant regulation of HSP70, chaperonin, ATP synthase beta chains, and Chain F. Western blotting and immuno-cytochemistry stain showed that YC-1 treatment caused a time-dependent increase in cytosolic Cytochrome c, pro-caspase-9, Apaf-1, and the activation of caspase-9 and -3. Importantly, the in vivo antileukemia effects of YC-1 were evaluated in BALB/c mice inoculated with WEHI-3B orthotopic model. YC-1 enhanced survival rate and prevented the body weight loss in leukemia mice. The enlargement of spleen and lymph nodes were reduced in YC-1 treated than that in leukemia mice. H-E stain of spleen sections revealed that infiltration of immature myeloblastic cells into red pulp was reduced in YC-1-treated group. The apoptotic cells of splenocyte were significantly increased in YC-1 treated than that in leukemia mice by Tdt-mediated deoxyuridine triphosphate nick end labeling (TUNEL) assay. Taken together, we conclude that YC-1 acted against U937 cells in vitro via a mitochondrial-dependent apoptosis pathway, and in orthotopic leukemia model, YC-1 administered antileukemia activity.

Nitric Oxide Is Critical for Inducing Phosphatidic Acid Accumulation in Xylanase-elicited Tomato Cells

Nitric Oxide (NO) is a second messenger related to development and (a)biotic stress responses in plants. We have studied the role of NO in signaling during plant defense responses upon xylanase elicitation. Treatment of tomato cell cultures with the fungal elicitor xylanase resulted in a rapid and dose-dependent NO accumulation. We have demonstrated that NO is required for the production of the lipid second messenger phosphatidic acid (PA) via the activation of the phospholipase C (PLC) and diacylglycerol kinase (DGK) pathway. Defense-related responses downstream of PA were studied. PA and, correspondingly, xylanase were shown to induce reactive oxygen species production. Scavenging of NO or inhibition of either the PLC or the DGK enzyme diminished xylanase-induced reactive oxygen species production. Xylanase-induced PLDbeta1 and PR1 mRNA levels decreased when NO or PA production were compromised. Finally, we have shown that NO and PA are involved in the induction of cell death by xylanase. Treatment with NO scavenger cPTIO, PLC inhibitor U73122, or DGK inhibitor R59022 diminished xylanase-induced cell death. On the basis of biochemical and pharmacological experimental results, we have shown that PLC/DGK-derived PA represents a novel downstream component of NO signaling cascade during plant defense.

Biocatalytic and semisynthetic studies of the anticancer tobacco cembranoids

Historically, tobacco use was primarily recreational and has been smoked in cigarettes, cigars and pipes, as well as smokeless forms of consumption, all of which are associated with adverse health effects. However, recent scientific investigations indicate that tobacco also contains several diverse secondary metabolites that may provide potential health benefits in the treatment of cancer and neurodegenerative diseases. Specifically, the leaf and flower of Nicotiana tabacum contain high amounts of the key flavor ingredient, cembranoids. Tobacco α- and β-2,7,11-cembratriene-4,6-diols display potent anticancer activity. These compounds have also been found to inhibit phorbol ester-stimulated 32Pi incorporation into phospholipids, induction of Epstein–Barr virus early antigen and protein phosphorylation. This review focuses on the biocatalytic and semisynthetic studies of tobacco cembanoids and their anticancer activity.

Increasing Plasma Membrane Phosphatidylinositol(4,5)Bisphosphate Biosynthesis Increases Phosphoinositide Metabolism inNicotiana tabacum

A genetic approach was used to increase phosphatidylinositol(4,5)bisphosphate [PtdIns(4,5)P2] biosynthesis and test the hypothesis that PtdInsP kinase (PIPK) is flux limiting in the plant phosphoinositide (PI) pathway. Expressing human PIPKIalpha in tobacco (Nicotiana tabacum) cells increased plasma membrane PtdIns(4,5)P2 100-fold. In vivo studies revealed that the rate of 32Pi incorporation into whole-cell PtdIns(4,5)P2 increased >12-fold, and the ratio of [3H]PtdInsP2 to [3H]PtdInsP increased 6-fold, but PtdInsP levels did not decrease, indicating that PtdInsP biosynthesis was not limiting. Both [3H]inositol trisphosphate and [3H]inositol hexakisphosphate increased 3-and 1.5-fold, respectively, in the transgenic lines after 18 h of labeling. The inositol(1,4,5)trisphosphate [Ins(1,4,5)P3] binding assay showed that total cellular Ins(1,4,5)P3/g fresh weight was >40-fold higher in transgenic tobacco lines; however, even with this high steady state level of Ins(1,4,5)P3, the pathway was not saturated. Stimulating transgenic cells with hyperosmotic stress led to another 2-fold increase, suggesting that the transgenic cells were in a constant state of PI stimulation. Furthermore, expressing Hs PIPKIalpha increased sugar use and oxygen uptake. Our results demonstrate that PIPK is flux limiting and that this high rate of PI metabolism increased the energy demands in these cells.

Sphingosine Kinase Type 2 Activation by ERK-mediated Phosphorylation

Sphingosine 1-phosphate (S1P), a potent lipid mediator, is a ligand for a family of five G protein-coupled receptors (S1P(1-5)) that have been shown to regulate a variety of biological responses important for cancer progression. The cellular level of S1P is low and tightly regulated in a spatio-temporal manner through its synthesis catalyzed by two sphingosine kinases, denoted SphK1 and SphK2. Many stimuli activate and translocate SphK1 to the plasma membrane by mechanisms that are dependent on its phosphorylation. Much less is known about activation of SphK2. Here we demonstrate that epidermal growth factor (EGF) as well as the protein kinase C activator, phorbol ester, induce rapid phosphorylation of hSphK2 which was markedly reduced by inhibition of MEK1/ERK pathway. Down-regulation of ERK1 blocked EGF-induced phosphorylation of SphK2. Recombinant ERK1 phosphorylated hSphK2 in vitro and increased its enzymatic activity. ERK1 also was found to be in a complex with hSphK2 in vivo. Site-directed mutagenesis indicated that hSphK2 is phosphorylated on Ser-351 and Thr-578 by ERK1 and that phosphorylation of these residues is important for EGF-stimulated migration of MDA-MB-453 cells. These studies provide the first clues to the mechanism of agonist-mediated SphK2 activation and enhance understanding of the regulation of SphK2 activity by phosphorylation and its role in movement of human breast cancer cells toward EGF.

Protein tyrosine phosphorylation of a heparin-binding sperm membrane mitogen (HBSM) is associated with capacitation and acrosome reaction

Protein tyrosine phosphorylation in spermatozoa is associated with epididymal maturation and though to be central for attainment of a capacitated state and expression of hyperactivated motility. Heparin, the most highly sulfated glycosaminoglycans, was also the most potent at stimulating the acrosomal reaction in bovine epididymal spermatozoa. Studies using radiolabeled inorganic phosphate showed 11-fold increase (32)Pi incorporation in heparin-binding sperm membrane protein (HBSM) during spermatozoal capacitation, and the phosphorylation occurs at the tyrosine residue. Epididymal spermatozoa were induced to undergo capacitation and acrosome reaction by 70% when the cells were incubated in BWW medium supplemented with heparin. The spermatozoa pre-treated with anti-HBSM antibody showed 46% reduction in the hyperactivated motility and lowers the acrosome reaction. This was confirms by measuring the hydrolysis of benzoyl-l-arginine ethyl ether (BAEE) by the acrosomal enzyme; acrosin. The preliminary finding suggests that HBSM may play an important role in the sperm capacitation and acrosome reaction.

Calyculin and okadaic acid promote perilipin phosphorylation and increase lipolysis in primary rat adipocytes

Lipolysis is primarily regulated by protein kinase A (PKA), which phosphorylates perilipin and hormone-sensitive lipase (HSL), and causes translocation of HSL from cytosol to lipid droplets in adipocytes. Perilipin coats lipid droplet surface and assumes to prevent lipase access to triacylglycerols, thus inhibiting basal lipolysis; phosphorylated perilipin facilitates lipolysis on PKA activation. Here, we induced lipolysis in primary rat adipocytes by inhibiting protein serine/threonine phosphatase with specific inhibitors, okadaic acid and calyculin. The incubation with calyculin promotes incorporation of 32Pi into perilipins, thus, confirming that perilipin is hyperphosphorylated. The lipolysis response to calyculin is gradually accompanied by increased accumulation of phosphorylated perilipin A in a concentration- and time-responsive manner. When perilipin phosphorylation is abrogated by the addition of N-ethylmaleimide, lipolysis ceases. Different from a considerable translocation of HSL upon PKA activation with isoproterenol, calyculin does not alter HSL redistribution in primary or differentiated adipocytes, as confirmed by both immunostaining and immunoblotting. Thus, we suggest that inhibition of the phosphatase by calyculin activates lipolysis via promoting perilipin phosphorylation rather than eliciting HSL translocation in adipocytes. Further, we show that when the endogenous phosphatase is inhibited by calyculin, simultaneous PKA activation with isoproterenol converts most of the perilipin to the hyperphosphorylated species, and induces enhanced lipolysis. Apparently, as PKA phosphorylates perilipin and stimulates lipolysis, the phosphatase acts to dephosphorylate perilipin and attenuate lipolysis. This suggests a two-step strategy governed by a kinase and a phosphatase to modulate the steady state of perilipin phosphorylation and hence the lipolysis response to hormonal stimulation.

Regulation of Neuroprotective Activity of Myocyte-enhancer Factor 2 by cAMP-Protein Kinase A Signaling Pathway in Neuronal Survival

The transcription factor myocyte-enhancer factor 2 (MEF2) has been shown to be required for the survival of different types of neurons. However, the death- or survival-inducing second messenger pathways that regulate MEF2 activity remain to be fully elucidated. Membrane depolarization by KCl induces neuronal survival that is dependent upon MEF2-mediated gene transactivation. Here we report that membrane depolarizationinduced activation of MEF2 requires the cAMP-protein kinase A (PKA) pathway. Inhibition of the activity of cAMP-PKA pathway attenuates membrane depolarization-induced activation of MEF2 activity and neuronal survival, whereas enhancing the activity of this pathway prevents KCl withdrawal-induced inhibition of MEF2 and neuronal apoptosis. Moreover, PKA directly phosphorylates MEF2 at Thr-20 in vitro to increase MEF2 DNA binding activity. A mutation of Thr-20 to Ala renders MEF2 resistant to PKA phosphorylation in vitro and reduces its DNA binding activity. Transfection of this T20A mutant blocks survival and induces apoptosis in cultured cortical and cerebellar granule neurons. This study identifies the transcription factor MEF2 as a target of cAMP-PKA pathway and demonstrates that PKA phosphorylation of MEF2 is a key step in modulating its DNA binding activity and ability to promote neuronal survival.

Distinct Conformations of the Corticotropin Releasing Factor Type 1 Receptor Adopted following Agonist and Antagonist Binding Are Differentially Regulated

The corticotropin releasing factor (CRF) type 1 receptor (CRF1) is a class B family G protein-coupled receptor that regulates the hypothalamic-pituitary-adrenal stress axis. Astressin is an amino-terminal truncated analog of CRF that retains high affinity binding to the extracellular domain of the receptor and is believed to act as a neutral competitive antagonist of receptor activation. Here we show that despite being unable to activate the CRF1 receptor, astressin binding results in the internalization of the receptor. Furthermore, entirely different pathways of internalization of CRF1 receptors are utilized following CRF and astressin binding. CRF causes the receptor to be phosphorylated, recruit beta-arrestin2, and to be internalized rapidly, likely through clathrin-coated pits. Astressin, however, fails to induce receptor phosphorylation or beta-arrestin2 recruitment, and internalization is slow and occurs through a pathway that is insensitive to inhibitors of clathrin-coated pits and caveolae. The fate of the internalized receptors also differs because only CRF-induced internalization results in receptor down-regulation. Furthermore, we present evidence that for astressin to induce internalization it must interact with both the extracellular amino terminus and the juxtamembrane domain of the receptor. Astressin binds with 6-fold higher affinity to full-length CRF1 receptors than to a chimeric protein containing only the extracellular domain attached to the transmembrane domain of the activin IIB receptor, yet two 12-residue analogs of astressin have similar affinities for both proteins but are unable to induce receptor internalization. These data demonstrate that agonists and antagonists for CRF1 receptors promote distinct conformations, which are then differentially regulated.