Pathophysiological concentrations of amyloid beta proteins directly inhibit rat brain and recombinant human type II phosphatidylinositol 4-kinase activity

We previously found that pathophysiological concentrations (< or = 10 nm) of an amyloid beta protein (Abeta25-35) reduced the plasma membrane phosphatidylinositol monophosphate level in cultured rat hippocampal neurons with a decrease in phosphatidylinositol 4-monophosphate-dependent Cl- -ATPase activity. As this suggested an inhibitory effect of Abeta25-35 on plasma membrane phosphatidylinositol 4-kinase (PI4K) activity, in vitro effects of Abetas on PI4K activity was examined using rat brain subcellular fractions and recombinant human type II PI4K (PI4KII). Abeta25-35 (10 nm) inhibited PI4KII activity, but neither PI 3-kinase (PI3K) nor type III PI4K (PI4KIII) activity, in microsomal fractions, while 100 nm Abeta25-35 inhibited PI3K activity in mitochondrial fractions. In plasma membrane-rich fractions, Abetas (> 0.5 nm) dose-dependently inhibited PI4KII activity, the maximal inhibition to 77-87% of control being reached around 10 nm of Abetas without significant changes in apparent Km values for ATP and PI, suggesting non-competitive inhibition by Abetas. The inhibition by 10 nm Abeta25-35 was reversible. In recombinant human PI4KIIalpha, inhibition profiles of Abetas were similar to those in rat brain plasma membranes. Therefore, pathophysiological concentrations of Abetas directly and reversibly inhibited plasma membrane PI4KII activity, suggesting that plasma membrane PI4KII is a target of Abetas in the pathogenesis of Alzheimer's disease.     

Phosphatidylinositol-3 kinase p85 enhances expression from the myelin basic protein promoter in oligodendrocytes

Phosphatidylinositol-3 kinase (PI3K) is a family of enzymes that phosphorylates the D3 position of phosphoinositides in membranes which can then act as a second messenger and affect many essential cellular processes such as survival, proliferation and differentiation. Class IA PI3K is composed of two subunits: a regulatory subunit, p85, and a catalytic subunit, p110. The p85 subunit is composed of several adapter domains which, upon interaction with the appropriate molecules, transmit the signal to activate p110. We have used the spontaneously immortalized oligodendrocyte cell line, CG4, to examine the role of PI3K in maturation of the oligodendrocyte. We show that overexpression of the p85 subunit enhances expression of myelin basic protein (MBP) upon differentiation of CG4 cells and primary oligodendrocytes. In experiments in CG4 cells, neither cotransfection with the tumor suppressor PTEN, which dephosphorylates the D3 position of phosphoinositides, nor inhibition of PI3K activity with wortmannin mimics this effect. Further, we have shown that this effect is dependent on the coexpression of the two SH2 domains within p85. Thus, the p85-mediated enhancement of MBP promoter activity in oligodendrocytes appears to be independent of PI3K activity and dependent on the adapter functions of the p85 subunit's SH2 domains.     

Role of the conserved DRY motif on G protein activation of rat angiotensin II receptor type 1A

To delineate the functional importance of the highly conserved triplet amino acid sequence, Asp-Arg-Tyr (DRY) among G protein-coupled receptors in the second intracellular loop, these residues of rat angiotensin II (Ang II) receptor type 1A (AT(1A)) were changed by alanine or glycine by site-directed mutagenesis. These mutant receptors were stably expressed in CHO-K1 cells, and the binding of Ang II, GTP effect, InsP(3) production, and the acidification of the medium in response to Ang II were determined. The effects of GTPgammaS on Ang II binding in the mutant receptors D125A and D125G were markedly reduced. InsP(3) production of the mutant D125A, D125G, R126A, and R126G was markedly reduced. Extracellular acidification of D125A was not distinguishable from untransfected CHO-K1 cells. Mutant Y127A was able to produce InsP(3) and acidify medium comparable with wild type AT(1A). These results indicate as follows; Asp(125) is essential for intracellular signal transduction involving G protein coupling, Arg(126) is essential for coupling of G(q) protein but not other G proteins, and Tyr(127) is not important for G protein coupling.     

TRAIL-induced apoptosis in gliomas is enhanced by Akt-inhibition and is independent of JNK activation

Patients with malignant gliomas have a poor prognosis and new treatment paradigms are needed against this disease. TRAIL/Apo2L selectively induces apoptosis in malignant cells sparing normal cells and is hence of interest as a potential therapeutic agent against gliomas. To determine the factors that modulate sensitivity to TRAIL, we examined the differences in TRAIL-activated signaling pathways in glioma cells with variable sensitivities to the agent. Apoptosis in response to TRAIL was unrelated to DR5 expression or endogenous p53 status in a panel of 8 glioma cell lines. TRAIL activated the extrinsic (cleavage of caspase-8, caspase-3 and PARP) and mitochondrial apoptotic pathways and reduced FLIP levels. It also induced caspase-dependent JNK activation, which did not influence TRAIL-induced apoptosis. Because the pro-survival PI3K/Akt pathway is highly relevant to gliomas, we assessed whether Akt could protect against TRAIL-induced apoptosis. Pretreatment with SH-6, a novel Akt inhibitor, enhanced TRAIL-induced apoptosis, suggesting a protective role for Akt. Conversely, TRAIL induced caspase-dependent cleavage of Akt neutralizing its anti-apoptotic effects. These results demonstrate that TRAIL-induced apoptosis in gliomas involves both activation of death pathways and downregulation of survival pathways. Additional studies are warranted to determine the therapeutic potential of TRAIL against gliomas.Supported in part by the NIH grant PO1 CA55261     

PI3-Kinase Is Involved in Mitogenic Signaling by the Oncogenic Receptor Tyrosine Kinase Xiphophorus Melanoma Receptor Kinase in Fish Melanoma

Overexpression of the mutationally activated receptor tyrosine kinase Xiphophorus melanoma receptor kinase (Xmrk) initiates formation of hereditary malignant melanoma in the fish Xiphophorus. In melanoma as well as in a melanoma-derived cell line (PSM) this receptor is highly activated resulting in constitutive Xmrk-mediated mitogenic signaling. In order to analyze mitogenic signaling triggered by Xmrk a possible involvement of phosphatidylinositol 3 (PI3)-kinase in Xmrk signal transduction was examined. Constitutive binding of the p85 adapter subunit of PI3-kinase to the Xmrk receptor was detected in PSM melanoma cells. Further analyses in BHK cells expressing a Xmrk chimera (HER-mrk) showed that p85 association with the intracellular part of Xmrk was dependent on autophosphorylation of the receptor. In vitro binding studies revealed that the interaction is mediated mainly through the N-terminal SH2 domain of p85 which directly binds to a sequence motif around phosphorylated Tyr-983 in the Xmrk carboxy-terminus. In accordance with recruitment of p85 by Xmrk in PSM cells, the PI3-kinase downstream target Akt was found to be highly phosphorylated on Ser-473, indicating efficient PI3-kinase signaling in melanoma cells. PI3-kinase activation was also detected in Xiphophorus melanoma. Moreover, malignant melanomas exhibited an increased level of PI3-kinase activity which was about three times higher than that in benign pigmented lesions. Inhibition of PI3-kinase activity in PSM melanoma cells by both Wortmannin and LY294002 blocked entry into S-phase. Together these data demonstrate that PI3-kinase is a substrate of the oncogenic Xmrk receptor and plays a significant role in mitogenic signaling of melanoma cells and the formation of malignant melanoma in Xiphophorus.     

Activation of signaling pathways by putative scavenger receptor class A (SR-A) ligands requires CD14 but not SR-A

Macrophage scavenger class A type I and type II receptors (SR-A) are trimeric, integral membrane glycoproteins that bind an unusually broad array of macromolecular ligands. These ligands include modified proteins and lipoproteins, nucleic acids, and a variety of plant and microbial cell wall constituents, such as fucoidan and lipoteichoic acid. Early studies of SR-A functions indicated that the receptors bound, internalized, and degraded their ligands without provoking any macrophage activating signaling events. More recent studies have provided evidence that several SR-A ligands can activate macrophage gene expression via utilization of a receptor-linked, PI3-kinase pathway. To investigate the role of SR-A in engaging signal transduction events, we employed macrophages taken from mice lacking these receptors. Using either fucoidan or lipoteichoic acid, we confirm that both ligands stimulate tyrosine phosphorylation of PI3-kinase and production of modest levels of the cytokine, TNFalpha. However, macrophages taken from SR-A null mice did not differ from wild type macrophages in these responses, indicating that these signaling events arise independently of SR-A activity. Employing mice lacking CD14, a GPI anchored receptor that binds bacterial lipopolysaccharide and signals via activation of Toll-like receptors, we show that the fucoidan and lipoteichoic acid responses are largely abrogated when CD14 is absent. These data do not provide support for direct SR-A involvement in signal transduction events and suggest that the early characterization of these receptors as initiators of a non-phlogistic, pathogen clearance pathway was correct.     

Identification of ribosomal protein S3a as a candidate for a novel PI 3-kinase target in the nucleus

Phosphatidylinositol 3,4,5-trisphosphate (PIP₃) is an important lipid second messenger that mediates various cell responses. We have searched for the nuclear PIP₃ binding proteins using PIP₃ analogue beads. A 33 kD protein was detected in this method, which was identified as ribosomal protein S3a by the mass spectrometric analysis. The recombinant S3a protein bound specifically to PIP₃. S3a localized not only in the cytosol but also in the nucleus. Interestingly, not cytosolic but nuclear S3a bound to PIP₃, suggesting different roles of S3a in the cytosol and the nucleus. This revised version was published online in August 2006 with corrections to the Cover Date.     

Distinct Roles for the p110α and hVPS34 Phosphatidylinositol 3′-Kinases in Vesicular Trafficking, Regulation of the Actin Cytoskeleton, and Mitogenesis

We have examined the roles of the p85/ p110α and hVPS34 phosphatidylinositol (PI) 3′-kinases in cellular signaling using inhibitory isoform-specific antibodies. We raised anti-hVPS34 and anti-p110α antibodies that specifically inhibit recombinant hVPS34 and p110α, respectively, in vitro. We used the antibodies to study cellular processes that are sensitive to low-dose wortmannin. The antibodies had distinct effects on the actin cytoskeleton; microinjection of anti-p110α antibodies blocked insulin-stimulated ruffling, whereas anti-hVPS34 antibodies had no effect. The antibodies also had different effects on vesicular trafficking. Microinjection of inhibitory anti-hVPS34 antibodies, but not anti-p110α antibodies, blocked the transit of internalized PDGF receptors to a perinuclear compartment, and disrupted the localization of the early endosomal protein EEA1. Microinjection of anti-p110α antibodies, and to a lesser extent anti-hVPS34 antibodies, reduced the rate of transferrin recycling in CHO cells. Surprisingly, both antibodies inhibited insulin-stimulated DNA synthesis by 80%. Injection of cells with antisense oligonucleotides derived from the hVPS34 sequence also blocked insulin-stimulated DNA synthesis, whereas scrambled oligonucleotides had no effect. Interestingly, the requirement for p110α and hVPS34 occurred at different times during the G1–S transition. Our data suggest that different PI 3′-kinases play distinct regulatory roles in the cell, and document an unexpected role for hVPS34 during insulin-stimulated mitogenesis.     

Minocycline prevents glutamate-induced apoptosis of cerebellar granule neurons by differential regulation of p38 and Akt pathways

Minocycline has been shown to have remarkably neuroprotective qualities, but underlying mechanisms remain elusive. We reported here the robust neuroprotection by minocycline against glutamate-induced apoptosis through regulations of p38 and Akt pathways. Pre-treatment of cerebellar granule neurons (CGNs) with minocycline (10-100 microm) elicited a dose-dependent reduction of glutamate excitotoxicity and blocked glutamate-induced nuclear condensation and DNA fragmentations. Using patch-clamping and fluorescence Ca2+ imaging techniques, it was found that minocycline neither blocked NMDA receptors, nor reduced glutamate-caused rises in intracellular Ca2+. Instead, confirmed by immunoblots, minocycline in vivo and in vitro was shown to directly inhibit the activation of p38 caused by glutamate. A p38-specific inhibitor, SB203580, also attenuated glutamate excitotoxicity. Furthermore, the neuroprotective effects of minocycline were blocked by phosphatidylinositol 3-kinase (PI3-K) inhibitors LY294002 and wortmannin, while pharmacologic inhibition of glycogen synthase kinase 3beta (GSK3beta) attenuated glutamate-induced apoptosis. In addition, immunoblots revealed that minocycline reversed the suppression of phosphorylated Akt and GSK3beta caused by glutamate, as were abolished by PI3-K inhibitors. These results demonstrate that minocycline prevents glutamate-induced apoptosis in CGNs by directly inhibiting p38 activity and maintaining the activation of PI3-K/Akt pathway, which offers a novel modality as to how the drug exerts protective effects.     

Effects of phosphatidylserine on p38 mitogen activated protein kinase,cyclic AMP responding element binding protein and nuclear factor-kappaB activation in resting and activated microglial cells

In the last few years, the interaction between phosphatidylserine (PS), a phospholipid that becomes permanently exposed on the external cell surface in the early phases of apoptosis, and its specific receptor (PtdSerR) has emerged as a crucial event for the engulfing of apoptotic cells and for preventing the acquisition of pro-inflammatory functions by peripheral macrophages. Recently, we demonstrated that PtdSerR is expressed in microglial cultures purified from neonatal rat brain, and that PS-liposomes, used to mimic apoptotic cells, strongly reduce the lipopolysaccharide (LPS)-induced release of inflammatory mediators. Here, we show that in resting microglia, PS-liposomes induce cyclic AMP responding element binding protein (CREB) phosphorylation but do not activate nuclear factor-kappaB (NF-kappaB) and p38 mitogen-activated protein kinase (p38), in line with the non-inflammatory consequences of the recognition and removal of apoptotic cells by macrophages. In LPS-activated microglia, PS-liposomes did not affect NF-kappaB activation but inhibited the phosphorylation of p38 and delayed that of CREB. To our knowledge, this is the first biochemical evidence of the molecular signaling evoked by PS/PtdSerR interaction possibly related to repression of pro-inflammatory activities in microglial cells.     

Fatty acid protection from palmitic acid-induced apoptosis is lost following PI3-kinase inhibition

We have previously shown that saturated fatty acids induce DNA damage and cause apoptotic cell death in insulin-producing beta-cells. Here we examine further the effects of single or combined dietary fatty acids on RINm5F survival or cell death signalling. Palmitate and stearate, but not linoleate, oleate or palmitoylmethyl ester, induced growth inhibition and increased apoptosis in RINm5F cells following 24 h exposure. Co-incubation with inhibitors of ceramide synthesis, myriocin or fumonisin B(1), did not improve viability of palmitic acid treated RINm5F cells. The inhibitor of inducible nitric oxide synthase, 1400 W, similarly had no protective effect. However, linoleic acid protected against palmitic acid-induced apoptotic and necrotic cell death. The specific pharmacological inhibitors of phosphatidylinositol 3-kinase, LY294002 and wortmannin, abolished the protective effect of linoleic acid on apoptosis but not on necrosis. These data show that the growth inhibitory and apoptosis-inducing effect of the saturated fatty acid palmitate on RINm5F cells is prevented by co-incubation with the polyunsaturated fatty acid linoleate but not inhibitors of ceramide or nitric oxide generation. A key role for phosphatidylinositol 3-kinase in mediating the linoleic-acid reduction in apoptosis is suggested.     

PI3K‐dependent cross‐talk interactions converge with Ras as quantifiable inputs integrated by Erk

Although it is appreciated that canonical signal‐transduction pathways represent dominant modes of regulation embedded in larger interaction networks, relatively little has been done to quantify pathway cross‐talk in such networks. Through quantitative measurements that systematically canvas an array of stimulation and molecular perturbation conditions, together with computational modeling and analysis, we have elucidated cross‐talk mechanisms in the platelet‐derived growth factor (PDGF) receptor signaling network, in which phosphoinositide 3‐kinase (PI3K) and Ras/extracellular signal‐regulated kinase (Erk) pathways are prominently activated. We show that, while PI3K signaling is insulated from cross‐talk, PI3K enhances Erk activation at points both upstream and downstream of Ras. The magnitudes of these effects depend strongly on the stimulation conditions, subject to saturation effects in the respective pathways and negative feedback loops. Motivated by those dynamics, a kinetic model of the network was formulated and used to precisely quantify the relative contributions of PI3K‐dependent and ‐independent modes of Ras/Erk activation.     

蛋白激酶的研究 (综述)

近年来,蛋白激酶研究进展较快,本文综述蛋白激酶的种类、结构、细胞定位,讨论几种植物蛋白激酶及其与信号转导的关系。     

Induction of Apoptosis and Tyrosine Phosphorylation of Cellular Proteins in T Cells and Non-T Cells by Stimulation with Concanavalin A

A high concentration (30 μg/ml or more) of Con A caused the death of not only thymocytes but also splenic cells of BALB/c mice, whereas a moderate concentration (3 μg/ml) of Con A induced proliferation of these cells. A high concentration of Con A also induced the death of splenic cells of athymic BALB/c-nu/nu mice and the bone marrow cells of BALB/c mice which mainly consist of non-T cells. However, any concentration (1-30 μg/ml) of Con A failed to induce the proliferation of these cells. Specific binding of tetrameric Con A to mannose-containing receptors was required for the induction of cell death. DNA fragmentation was observed by both laser flow cytometry and electrophoresis in Con A-stimulated T cells and non-T cells. This indicated that the mechanism of induction of apoptosis with Con A is not necessarily TCR-dependent. Con A induced tyrosine phosphorylation of a number of proteins in various types of cells. Interestingly, phosphorylation of the 40 kDa protein developed only in the thymocytes and spleen cells that contain T cells, whereas phosphorylation of the 80 and 120 kDa proteins appeared in both T cells and non-T cells. These results suggested that the Con A-induced apoptosis of T cells and non-T cells involves different but possibly mutually related protein tyrosine phosphorylation-linked signals.     

Regulation of neutrophil apoptosis: A role for protein kinase C and phosphatidylinositol-3-kinase

Neutrophils play a central role in host defense and are recruited in vast numbers to sites of infection where they phagocytose and kill invading bacterial pathogens. Neutrophils have a short half-life that is extended at the inflamed site by pro-inflammatory cytokines and contact with bacterial cell walls. Normal resolution of inflammation involves the removal of neutrophils and other inflammatory cells by the induction of apoptosis. Spontaneous neutrophil apoptosis does not require Fas ligation, but is mediated by caspases 3, 8 and possibly caspase 9 and also involves activation of protein kinase C-. With chronic inflammatory disease, neutrophil apoptosis is delayed by pro-inflammatory cytokines, leading to persistence of neutrophils at the inflamed site and non-specific tissue damage. Here we discuss the evidence for inhibition of neutrophil apoptosis via signaling though PI-3-kinase and downstream pathways, including PDK-1 and PKB. Therapeutic strategies to resolve chronic inflammation could therefore usefully target neutrophil apoptosis and the PI-3-kinase or PKC- signaling pathways.     

Reactive oxygen species from mitochondria mediate SW480 cells apoptosis induced by Na2SeO3

A number of selenium compounds have been found to inhibit tumorigenesis in a variety of animal and cell models. In order to explore the molecular mechanism involved in the anticarcinogenesis activity of selenium, we examined the effects of sodium selenite on cell viabilty, generation of reactive oxygen species (ROS), and mitochondrial transmembrane potential (Δω _m ) in human colonic carcinoma cells SW480. The result from MTT test showed that sodium selenite reduced cell viability. Morophologic and flow cytometric results indicated that Na₂SeO₃ induced the apoptosis of SW480 cells. Na₂SeO₃ increased the generation of intracellular ROS, whereas BAPTA-AM, rotenone, and NaCN completely inhibited the increase of ROS induced by Na₂SeO₃. Na₂SeO₃ also caused the disruption of Δω _m . The intracellular ROS increase and apoptosis induced by Na₂SeO₃ were significantly decreased by superoxide dismutase (SOD), catalase. These data suggest that the ROS mediate apoptosis induced by Na₂SeO₃ and mitochondria may be a major source of Na₂SeO₃-induced ROS.     

The involvement of lipid activators of protein kinase C in the induction of ZIF268 in PC12 cells exposed to lead

Lead (Pb) induces the expression of immediate early genes (IEG) in PC12 cells by a mechanism that involves protein kinase C (PKC). To define the mechanisms, the involvement of two commonly observed lipid activators of PKC, diacylglycerols, and phosphatidylinositols, were examined. A dose-dependent increase in the expression of the IEG zif268 was observed in PC12 cells exposed to Pb. The PKC inhibitor Ro-31-8220 blocked the induction. An increase in levels of diacylglycerols was observed in PC12 cells exposed to Pb, but the increase was inhibited by Ro-31-8220. The phosphatidylinositol 3-kinase inhibitor Wortmannin, but not the inhibitor LY 294002, blocked the induction zif268 in Pb-exposed cells. Small increases in phosphatidylinositol 3-kinase activity were observed after exposure to Pb. In summary, diacylglycerols are elevated in PC12 cells exposed to Pb by a mechanism that requires PKC. It is possible that diacylglycerols contribute to the induction of zif268 by Pb by sustaining PKC activation.     

Transmembrane protein 106A is silenced by promoter region hypermethylation and suppresses gastric cancer growth by inducing apoptosis

Inactivation of tumour suppressor genes by promoter methylation plays an important role in the initiation and progression of gastric cancer (GC). Transmembrane 106A gene (TMEM106A) encodes a novel protein of previously unknown function. This study analysed the biological functions, epigenetic changes and the clinical significance of TMEM106A in GC. Data from experiments indicate that TMEM106A is a type II membrane protein, which is localized to mitochondria and the plasma membrane. TMEM106A was down‐regulated or silenced by promoter region hypermethylation in GC cell lines, but expressed in normal gastric tissues. Overexpression of TMEM106A suppressed cell growth and induced apoptosis in GC cell lines, and retarded the growth of xenografts in nude mice. These effects were associated with the activation of caspase‐2, caspase‐9, and caspase‐3, cleavage of BID and inactivation of poly (ADP‐ribose) polymerase (PARP). In primary GC samples, loss or reduction of TMEM106A expression was associated with promoter region hypermethylation. TMEM106A was methylated in 88.6% (93/105) of primary GC and 18.1% (2/11) in cancer adjacent normal tissue samples. Further analysis suggested that TMEM106A methylation in primary GCs was significantly correlated with smoking and tumour metastasis. In conclusion, TMEM106A is frequently methylated in human GC. The expression of TMEM106A is regulated by promoter hypermethylation. TMEM106A is a novel functional tumour suppressor in gastric carcinogenesis.     

Regulation of connexin43 function by activated tyrosine protein kinases

Gap junctions are specialized membrane structures that are involved in the normal functioning of numerous mammalian tissues and implicated in several human disease processes. This mini-review focuses on the regulation of gap junctions through phosphorylation of connexin43 induced by the v-Src or epidermal growth factor receptor tyrosine kinases. These tyrosine kinases markedly disrupt gap junctional communication in mammalian cells. Here, we describe work correlating the alteration of connexin43 function with the ability of the v-Src tyrosine kinase to phosphorylate connexin43 directly on two distinct tyrosine sites in mammalian cells (Y247 and Y265). We also present evidence that proline-rich regions and phosphotyrosine sites of connexin43 may mediate interactions with the SH3 and SH2 domains of v-Src. In contrast to v-Src, the activated epidermal growth factor receptor acts indirectly through activated MAP kinase which may stimulate phosphorylation of connexin43 exclusively on serine. This phosphorylation event is complex because MAP kinase phosphorylates three serine sites in connexin43 (S255, S279, and S282). These findings suggest novel interactions between connexin43, the v-Src tyrosine kinase, and activated MAP kinase that set the stage for future investigations into the regulation of gap junctions by protein phosphorylation.