Signal transduction pathways that contribute to increased protein synthesis during T-cell activation

Protein synthesis rates were maximally stimulated in human lymphocytes by ionomycin and the phorbol ester PMA (I+P), which promotes proliferation, whereas PMA alone, which does not promote proliferation, stimulated protein synthesis to a lesser degree. Three translation-associated activities, eIF4E phosphorylation, eIF2B activity and 4E-BP1 phosphorylation also increased with stimulation by I+P and PMA, but only 4E-BP1 phosphorylation was differentially stimulated by these conditions. Correspondingly, signaling pathways activated in T cells were probed for their connection to these activities. Immunosuppressants FK506 and rapamycin partially blocked the protein synthesis rate increases by I+P stimulation. FK506 had less of an inhibitory effect with PMA stimulation suggesting that its mechanism mostly affected ionomycin-activated signals. I+P and PMA equally stimulated phosphorylation of ERK1/2, but I+P more strongly stimulated Akt, and p70(S6K) phosphorylation. An inhibitor that blocks ERK1/2 phosphorylation only slightly reduced protein synthesis rates stimulated by I+P or PMA, but greatly reduced eIF4E phosphorylation and eIF2B activity. In contrast, inhibitors of the PI-3 kinase and mTOR pathways strongly blocked early protein synthesis rate stimulated by I+P and PMA and also blocked 4E-BP1 phosphorylation and release of eIF4E suggesting that these pathways regulate protein synthesis activities, which are important for proliferation in T cells.     

Two distinct, calcium-mediated, signal transduction pathways can trigger deflagellation in Chlamydomonas reinhardtii

The molecular machinery of deflagellation can be activated in detergent permeabilized Chlamydomonas reinhardtii by the addition of Ca2+ (Sanders, M. A., and J. L. Salisbury, 1989. J. Cell Biol. 108:1751- 1760). This suggests that stimuli which induce deflagellation in living cells cause an increase in the intracellular concentration of Ca2+, but this has never been demonstrated. In this paper we report that the wasp venom peptide, mastoparan, and the permeant organic acid, benzoate, activate two different signalling pathways to trigger deflagellation. We have characterized each pathway with respect to: (a) the requirement for extracellular Ca2+; (b) sensitivity to Ca2+ channel blockers; and (c) 45Ca influx. We also report that a new mutant strain of C. reinhardtii, adf-1, is specifically defective in the acid-activated signalling pathway. Both signalling pathways appear normal in another mutant, fa-1, that is defective in the machinery of deflagellation (Lewin, R. and C. Burrascano. 1983. Experientia. 39:1397-1398; Sanders, M. A., and J. L. Salisbury. 1989. J. Cell Biol. 108:1751-1760). We conclude that mastoparan induces the release of an intracellular pool of Ca2+ whereas acid induces an influx of extracellular Ca2+ to activate the machinery of deflagellation.     

Integrin-mediated signal transduction pathways.

Integrins serve as adhesion receptors for extracellular matrix proteins and also transduce biochemical signals into the cell. They regulate a variety of cellular functions, including spreading, migration, proliferation and apoptosis. Many signaling pathways downstream of integrins have been identified and characterized and are discussed here. In particular, integrins regulate many protein tyrosine kinases and phosphatases, such as FAK and Src, to coordinate many of the cell processes mentioned above. The regulation of MAP kinases by integrins is important for cell growth or other functions, and the putative roles of Ras and FAK in these pathways are discussed. Phosphatidylinositol lipids and their modifying enzymes, particularly PI 3-kinase, are strongly implicated as mediators of integrin-regulated cytoskeletal changes and cell migration. Similarly, actin cytoskeleton regulation by the Rho family of GTPases is coordinated with integrin signaling to regulate cell spreading and migration, although the exact relationship between these pathways is not clear. Finally, intracellular pH and calcium fluxes by integrins are suggested to affect a variety of cellular proteins and functions.     

Activation of dense human tonsilar B cells. Induction of c-myc gene expression via two distinct signal transduction pathways.

Antibodies to surface Ig or to the B cell marker CD20 trigger resting human B cells in similar yet distinct ways. Either antibody induces five-fold increases in the expression of the protooncogene, c-myc, as detected with semi-quantitative Northern blot assays. The induction of c-myc mRNA by anti-IgM or anti-CD20 is blocked by inhibitors of protein kinase C (PKC) such as staurosporine and by pretreatment of B cells with phorbol esters to reduce cellular PKC levels. This suggests that PKC is involved in the pathways stimulated by both anti-IgM and anti-CD20. However, anti-CD20, unlike anti-IgM, does not activate significant increases in inositol triphosphate or intracellular-free calcium. Further, anti-CD20-triggered elevation of c-myc mRNA is inhibited by pertussis and cholera toxins, whereas the pathway initiated by anti-IgM if anything is stimulated by pertussis toxin and unchanged by cholera toxin. Further differences in the nature of these two signals were seen when the expression of adhesion/recognition molecules were examined. Anti-IgM consistently induces increased expression of the adhesion molecules CD54 (I-CAM-1) and B7/BB-1 on B cells, but anti-CD20 does not. Yet both anti-CD20 and anti-IgM increase class II MHC, CD18 (LFA-1 beta-chain) and LFA-3 levels. These data suggest that the way in which B cells are activated may influence their surface phenotype and possibly subsequent migration or cell-cell interactions.     

Control of cAMP-induced gene expression by divergent signal transduction pathways.

A compilation of literature data and recent experiments led to the following conclusions regarding cyclic adenosine 3':5' monophosphate (cAMP) regulation of gene expression. Several classes of cAMP-induced gene expression can be discriminated by sensitivity to stimulation kinetics. The aggregation-related genes respond only to nanomolar cAMP pulses. The prestalk-related genes respond both to nanomolar pulses and persistent micromolar stimulation. The prespore specific genes respond only to persistent micromolar stimulation. The induction of the aggregation- and prestalk-related genes by nanomolar cAMP pulses may share a common transduction pathway, which does not involve cAMP, while involvement of the inositol 1,4,5-trisphosphate (IP3)/Ca2+ pathway is unlikely. Induction of the expression of prespore and prestalk-related genes by micromolar cAMP stimuli utilizes divergent signal processing mechanisms. cAMP-induced prespore gene expression does not involve cAMP and probably also not cyclic guanosine 3'.5' monophosphate (cGMP) as intracellular intermediate. Involvement of cAMP-induced phospholipase C (PLC) activation in this pathway is suggested by the observation that IP3 and 1,2-diacylglycerol (DAG) can induce prespore gene expression, albeit in a somewhat indirect manner and by the observation that Li+ and Ca2+ antagonists inhibit prespore gene expression. Cyclic AMP induction of prestalk-related gene expression is inhibited by IP3 and DAG and promoted by Li+, and is relatively insensitive to Ca2+ antagonists, which indicates that PLC activation does not mediate prestalk-related gene expression. Neither prespore nor prestalk-related gene expression utilizes the sustained cAMP-induced pHi increase as intracellular intermediate.     

Signalling to translation: how signal transduction pathways control the protein synthetic machinery

Recent advances in our understanding of both the regulation of components of the translational machinery and the upstream signalling pathways that modulate them have provided important new insights into the mechanisms by which hormones, growth factors, nutrients and cellular energy status control protein synthesis in mammalian cells. The importance of proper control of mRNA translation is strikingly illustrated by the fact that defects in this process or its control are implicated in a number of disease states, such as cancer, tissue hypertrophy and neurodegeneration. Signalling pathways such as those involving mTOR (mammalian target of rapamycin) and mitogen-activated protein kinases modulate the phosphorylation of translation factors, the activities of the protein kinases that act upon them and the association of RNA-binding proteins with specific mRNAs. These effects contribute both to the overall control of protein synthesis (which is linked to cell growth) and to the modulation of the translation or stability of specific mRNAs. However, important questions remain about both the contributions of individual regulatory events to the control of general protein synthesis and the mechanisms by which the translation of specific mRNAs is controlled.     

Cytosolic Ca2+ pulses and protein kinase activation in the signal transduction pathways leading to the plant oxidative burst

The oxidative burst, the rapid production of O₂- and H₂O₂ by plant cells in response to pathogens and Stressors, is a critical step in plant disease resistance and is controlled by several different elicitor-initiated signaling pathways. While different defense elicitors appear to activate disparate initial steps in signaling the oxidative burst, all of the elicitors tested thus far appear to stimulate pathways that converge on the same three core signaling intermediates: 1) the Ca₂₊-independent activation of a mitogen-activated protein kinase (MAPK) family member, 2) the influx of Ca₂₊ into the cytosol, deriving most critically from an internal compartment, and 3) the Ca²⁺-dependent activation of additional protein kinases including a second MAPK homologue and possibly calcium dependent protein kinases (CDPKs). Data from several recent reports are summarized to place these signaling events into a complete and updated model of signaling to the plant oxidative burst.     

Two distinct gut microbial pathways contribute to meta-organismal production of phenylacetylglutamine with links to cardiovascular disease

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Heterogeneity of the signal transduction pathways for VEGF-induced MAPKs activation in human vascular endothelial cells.

Vascular endothelial growth factor (VEGF) activates ERK and p38 MAPK in endothelial cells (ECs). The present study was aimed to compare its intracellular signal transduction pathways between three primary cultures of human ECs including human aortic ECs (HAECs), human umbilical vein ECs (HUVECs), and human microvascular ECs (HMVECs). VEGF activated ERK and p38 MAPK in all of three ECs. Isoforms of p38 MAPK that were activated by VEGF in HUVECs were p38-alpha and p38-delta. GF109203X, a specific inhibitor of PKC, markedly inhibited VEGF-induced activation of ERK and p38 MAPK in HAECs and HUVECs, whereas it exhibited little effect in HMVECs. In contrast, dominant negative mutant of Ha-Ras almost completely abrogated VEGF-induced activation of ERK and p38 MAPK in HMVECs. Although dominant negative mutant of Ha-Ras substantially inhibited the basal activities of ERK and p38 MAPK, it exhibited marginal effect on VEGF-induced activation of ERK and p38 MAPK in HUVECs and HAECs. The activation of Ras by VEGF appeared to be most prominent in HMVECs. These results indicate that intracellular signal transduction pathways for VEGF-induced activation of MAPKs are heterogeneous and vary depending on the origin of ECs.Copyright 2001 Wiley-Liss, Inc.     

Quantitative phosphoproteomics strategies for understanding protein kinase-mediated signal transduction pathways

Protein phosphorylation is a central regulatory mechanism of cell signaling pathways. This highly controlled biochemical process is involved in most cellular functions, and defects in protein kinases and phosphatases have been implicated in many diseases, highlighting the importance of understanding phosphorylation-mediated signaling networks. However, phosphorylation is a transient modification, and phosphorylated proteins are often less abundant. Therefore, the large-scale identification and quantification of phosphoproteins and their phosphorylation sites under different conditions are one of the most interesting and challenging tasks in the field of proteomics. Both 2D gel electrophoresis and liquid chromatography-tandem mass spectrometry serve as key phosphoproteomic technologies in combination with prefractionation, such as enrichment of phosphorylated proteins/peptides. Recently, new possibilities for quantitative phosphoproteomic analysis have been offered by technical advances in sample preparation, enrichment, separation, instrumentation, quantification and informatics. In this article, we present an overview of several strategies for quantitative phosphoproteomics and discuss how phosphoproteomic analysis can help to elucidate signaling pathways that regulate various cellular processes.