Effects of sequestration on signal transduction cascades

The building blocks of most signal transduction pathways are pairs of enzymes, such as kinases and phosphatases, that control the activity of protein targets by covalent modification. It has previously been shown [Goldbeter A & Koshland DE (1981) Proc Natl Acad Sci USA 78, 6840-6844] that these systems can be highly sensitive to changes in stimuli if their catalysing enzymes are saturated with their target protein substrates. This mechanism, termed zero-order ultrasensitivity, may set thresholds that filter out subthreshold stimuli. Experimental data on protein abundance suggest that the enzymes and their target proteins are present in comparable concentrations. Under these conditions a large fraction of the target protein may be sequestrated by the enzymes. This causes a reduction in ultrasensitivity so that the proposed mechanism is unlikely to account for ultrasensitivity under the conditions present in most in vivo signalling cascades. Furthermore, we show that sequestration changes the dynamics of a covalent modification cycle and may account for signal termination and a sign-sensitive delay. Finally, we analyse the effect of sequestration on the dynamics of a complex signal transduction cascade: the mitogen-activated protein kinase (MAPK) cascade with negative feedback. We show that sequestration limits ultrasensitivity in this cascade and may thereby abolish the potential for oscillations induced by negative feedback.     

Insulin signal transduction through protein kinase cascades

This review summarizes the evolution of ideas concerning insulin signal transduction, the current information on protein ser/thr kinase cascades as signalling intermediates, and their status as participants in insulin regulation of energy metabolism. Best characterized is the Ras-MAPK pathway, whose input is crucial to cell fate decisions, but relatively dispensable in metabolic regulation. By contrast the effectors downstream of PI-3 kinase, although less well elucidated, include elements indispensable for the insulin regulation of glucose transport, glycogen and cAMP metabolism. Considerable information has accrued on PKB/cAkt, a protein kinase that interacts directly with Ptd Ins 3OH phosphorylated lipids, as well as some of the elements further downstream, such as glycogen synthase kinase-3 and the p70 S6 kinase. Finally, some information implicates other erk pathways (e.g. such as the SAPK/JNK pathway) and Nck/cdc42-regulated PAKs (homologs of the yeast Ste 20) as participants in the cellular response to insulin. Thus insulin recruits a broad array of protein (ser/thr) kinases in its target cells to effectuate its characteristic anabolic and anticatabolic programs.     

MAP kinase cascades in elicitor signal transduction

 Protein kinases play important roles in elicitor signal transduction. In this article, I describe the current view of the role of mitogen-activated protein kinase (MAPK) cascades in elicitor signal transduction of plant cells based on our own research and recent developments in this field. In the past several years, it has become apparent that MAPK cascades play important roles in elicitor signal transduction in plants. Our early studies demonstrated the identification of p47 MAPK in tobacco as an elicitor-responsive protein kinase and possible involvement of p47 MAPK in elicitor signal transduction to induce defense responses, including defense gene expression and hypersensitive cell death. However, the molecular identity of p47 MAPK is still unclear. Recent important studies suggest that tobacco MAPK cascades that include SIPK, and/or WIPK, and NtMEK2, an upstream kinase for both SIPK and WIPK, have a crucial function in induction of defense responses and hypersensitive cell death. The orthologs of these protein kinases in Arabidopsis and alfalfa are also suggested to have similar functions. Furthermore, the identification of loss-of-function mutation in Arabidopsis reveals a negative regulatory role for putative MAPK cascades in plant defense mechanisms. Received: February 7, 2002 / Accepted: February 25, 2002     

Sequestration shapes the response of signal transduction cascades

Many signal transduction cascades are composed of covalent modification cycles such as kinase/phosphatase cycles. In the 1980s Goldbeter and Koshland showed that such cycles can exhibit non-linear input-output relations when the enzymes are saturated by their substrates, which may facilitate signal processing. Recent papers show that this mechanism is unlikely to cause non-linearity in mammalian signal transduction cascades as sequestration of the target due to enzyme concentrations present in these cascades will hamper this mechanism. However, sequestration due to high-affinity enzymes can shape the dynamics and steady-state behaviour of signal transduction cascades in different ways, some of which are discussed in this review.     

Microsystems for the analysis of cellular signal transduction

Drug development and cell biological research alike are confronted by the complexity of molecular signal transduction networks. These networks integrate information from different stimuli inside the cell. Progress in both disciplines will largely depend on experimental techniques that reflect the complexity of cellular signalling networks. The testing of combinations, either of drug candidates or of stimuli is considered one promising solution of this challenge. However, the systematic testing of combinations introduces a new dimension of experimental complexity. Miniaturization, parallelization and especially a reduction of the number of steps required for the preparation of samples are prerequisites for resolving this dilemma. As a solution for the testing of different combinations of drug candidates, we present the generation of substance mixtures by diffusion. For each combination of compounds only two pipetting steps are required for generating a virtually unlimited number of different test conditions. The potential of testing the response of cells to systematic combinations of different stimuli is demonstrated by using antibody microarrays where each spot carries a different combination of stimulatory antibodies. Both experimental strategies are combinatorial in nature. Through miniaturization and implementation of high-content read-outs a maximum of information is obtained with a minimum of manipulations. These authors contributed equally to this work.     

The interaction of ApoA-I and ABCA1 triggers signal transduction pathways to mediate efflux of cellular lipids

Reverse cholesterol transport (RCT) has been characterized as a crucial step for antiatherosclerosis, which is initiated by ATP-binding cassette A1 (ABCA1) to mediate the efflux of cellular phospholipids and cholesterol to lipid-free apolipoprotein A-I (apoA-I). However, the mechanisms underlying apoA-I/ABCA1 interaction to lead to the lipidation of apoA-I are poorly understood. There are several models proposed for the interaction of apoA-I with ABCA1 as well as the lipidation of apoA-I mediated by ABCA1. ApoA-I increases the levels of ABCA1 protein markedly. In turn, ABCA1 can stabilize apoA-I. The interaction of apoA-I with ABCA1 could activate signaling molecules that modulate posttranslational ABCA1 activity or lipid transport activity. The key signaling molecules in these processes include protein kinase A (PKA), protein kinase C (PKC), Janus kinase 2 (JAK2), Rho GTPases and Ca²⁺, and many factors also could influence the interaction of apoA-I with ABCA1. This review will summarize these mechanisms for the apoA-I interaction with ABCA1 as well as the signal transduction pathways involved in these processes.     

Structural-functional organization of cellular signal transduction systems

The review summarizes recent data and current opinions of the structural and functional organization of the known signalling systems and their functional elements. A possible role of adenylate cyclase, phosphoinositide, guanylate cyclase, tyrosine kinase systems and also of arachidonic acid, its oxygenated derivatives and of other fatty acids in intracellular signalling processes is discussed.     

TGF—β家族的细胞信号转导

转化生长因子β（ＴＧＦ－β）家族成员与各自的膜受体结合后,使通路限制性（ｐａｔｈｗａｙ－ｒｅｓｔｒｉｃｔｅｄ）ＳＭＡＤｓ磷酸化,后者再与Ｓｍａｄ４形成杂聚体并转位至细胞核,调节一些基因的转录而产生生物学效应。抑制性ＳＭＡＤｓ可阻断通路限制性ＳＭＡＤｓ的磷酸化。     

Reactions of 4-hydroxynonenal with proteins and cellular targets

Peroxidative degradation of lipids yields the aldehyde 4-hydroxy-2-nonenal (4HNE) as a major product. The lipid aldehyde is an electrophile, and reactivity of 4HNE toward protein nucleophiles (i.e., Cys, His, and Lys) has been characterized. Through the use of purified enzymes and isolated cells, various pathways for biotransformation of the lipid aldehyde have been identified and include enzyme-mediated oxidation, reduction, and glutathione conjugation. Uncontrolled oxidative stress can yield excessive lipid peroxidation and 4HNE generation, however, and overwhelm these cellular defenses. Indeed, in vitro and in vivo production of 4HNE in response to pro-oxidant exposure has been demonstrated using antibodies to protein adducts of the lipid aldehyde. Recent evidence suggests a role for protein modification by 4HNE in the pathogenesis of several diseases (e.g., alcohol-induced liver disease); however, the precise mechanism(s) is currently unknown but likely results from adduction of proteins involved in cellular homeostasis or biological signaling.     

Human 55-kDa receptor for tumor necrosis factor coupled to signal transduction cascades.

The numerous biological activities of tumor necrosis factor (TNF) appear mediated by two types of receptors of 55 kDa (TR55) and 75 kDa (TR75) molecular mass. To test TR55 for its individual role in signaling across the membrane, a cDNA coding for the human TR55 was stably expressed in murine 70Z/3 pre-B cells, which lack binding sites for, and proved nonresponsive to human TNF. The transfected TR55 showed high affinity ligand binding and active internalization. It is demonstrated that the TNF signaling cascade, i.e. stimulation of protein kinase C, sphingomyelinase, and phospholipase A2, production of the second messengers diacylglycerol and ceramide, can occur completely through exclusive binding of TNF to TR55. The p55 TNF-binding site functions as an autonomous TNF receptor that mediates key signal transduction pathways, which may control the majority of TNF actions.     

Protein tyrosine nitration in cellular signal transduction pathways

How specificity and reversibility in tyrosine nitration are defined biologically in cellular systems is poorly understood. As more investigations identify proteins involved in cell regulatory pathways in which only a small fraction of that protein pool is modified by nitration to affect cell function, the mechanisms of biological specificity and reversal should come into focus. In this review experimental evidence has been summarized to suggest that tyrosine nitration is a highly selective modification and under certain physiological conditions fulfills the criteria of a physiologically relevant signal. It can be specific, reversible, occurs on a physiological time scale, and, depending on a target, can result in either activation or inhibition.     

The role of protein kinase cascades in stress signal transduction in unicellular eukaryotes

The review summarizes current data on transduction mechanisms of stress signals by protein kinase cascades in unicellular eukaryotes. The role of sensor histidine kinases, tyrosine kinases, PKC, and cyclic nucleotid-dependent kinases are reviewed. Special attention is paid to a comparative analysis of transduction mechanisms of stress signals in vertebrates and unicellular eukaryotes.     

A unified model for signal transduction reactions in cellular membranes.

An analytical solution is obtained for the steady-state reaction rate of an intracellular enzyme, recruited to the plasma membrane by active receptors, acting upon a membrane-associated substrate. Influenced by physical and chemical effects, such interactions are encountered in numerous signal-transduction pathways. The generalized modeling framework is the first to combine reaction and diffusion limitations in enzyme action, the finite mean lifetime of receptor-enzyme complexes, reactions in the bulk membrane, and constitutive and receptor-mediated substrate insertion. The theory is compared with other analytical and numerical approaches, and it is used to model two different signaling pathway types. For two-state mechanisms, such as activation of the Ras GTPase, the diffusion-limited activation rate constant increases with enhanced substrate inactivation, dissociation of receptor-enzyme complexes, or crowding of neighboring complexes. The latter effect is only significant when nearly all of the substrate is in the activated state. For regulated supply and turnover pathways, such as phospholipase C-mediated lipid hydrolysis, an additional influence is receptor-mediated substrate delivery. When substrate consumption is rapid, this process significantly enhances the effective enzymatic rate constant, regardless of whether enzyme action is diffusion limited. Under these conditions, however, enhanced substrate delivery can result in a decrease in the average substrate concentration.     

The regulatory circuits for hysteretic switching in cellular signal transduction pathways

Hysteresis can be found in many physical systems, and a hysteretic switch has been used for various mechanical and electrical systems. Such a hysteretic switch can be created by using a single positive feedback loop, as often used in engineering systems. It is, however, intriguing that various cellular signaling systems use coupled positive feedback loops to implement the hysteretic switch. A question then arises about the advantage of using coupled positive feedback loops instead of simple isolated positive feedback for an apparently equivalent hysteretic switch. Through mathematical simulations, we determined that cellular systems with coupled positive feedback loops show enhanced hysteretic switching, and can thereby make a more reliable decision under conditions of noisy signaling. As most intracellular processes are accompanied by intrinsic noise, important cellular decisions such as differentiation and apoptosis need to be highly robust to such noises. The coupled positive feedback loops might have been evolutionarily acquired to enable correct cell fate decisions to be made through enhanced hysteretic switching in noisy cellular environments.     

An investigation of spatial signal transduction in cellular networks

ABSTRACT: BACKGROUND: Spatial signal transduction plays a vital role in many intracellular processes such as eukaryotic chemotaxis, polarity generation, cell division. Furthermore it is being increasingly realized that the spatial dimension to signalling may play an important role in other apparently purely temporal signal transduction processes. It is being recognized that a conceptual basis for studying spatial signal transduction in signalling networks is necessary. RESULTS: In this work we examine spatial signal transduction in a series of standard motifs/networks. These networks include coherent and incoherent feedforward, positive and negative feedback, cyclic motifs, monostable switches, bistable switches and negative feedback oscillators. In all these cases, the driving signal has spatial variation. For each network we consider two cases, one where all elements are essentially non diffusible, and the other where one of the network elements may be highly diffusible. A careful analysis of steady state signal transduction provides many insights into the behaviour of all these modules. While in the non-diffusible case for the most part, spatial signalling reflects the temporal signalling behaviour, in the diffusible cases, we see significant differences between spatial and temporal signalling characteristics. Our results demonstrate that the presence of diffusible elements in the networks provides important constraints and capabilities for signalling. CONCLUSIONS: Our results provide a systematic basis for understanding spatial signalling in networks and the role of diffusible elements therein. This provides many insights into the signal transduction capabilities and constraints in such networks and suggests ways in which cellular signalling and information processing is organized to conform to or bypass those constraints. It also provides a framework for starting to understand the organization and regulation of spatial signal transduction in individual processes.     

Soluble Abeta inhibits specific signal transduction cascades common to the insulin receptor pathway

Numerous studies have now shown that the amyloid beta-protein (Abeta), the principal component of cerebral plaques in Alzheimer disease, rapidly and potently inhibits certain forms of synaptic plasticity. The amyloid (or Abeta) hypothesis proposes that the continuous disruption of normal synaptic physiology by Abeta contributes to the development of Alzheimer disease. However, there is little consensus about how Abeta mediates this inhibition at the molecular level. Using mouse primary hippocampal neurons, we observed that a brief treatment with cell-derived, soluble, human Abeta disrupted the activation of three kinases (Erk/MAPK, CaMKII, and the phosphatidylinositol 3-kinase-activated protein Akt/protein kinase B) that are required for long term potentiation, whereas two other kinases (protein kinase A and protein kinase C) were stimulated normally. An antagonist of the insulin receptor family of tyrosine kinases was found to mimic the pattern of Abeta-mediated kinase inhibition. We then found that soluble Abeta binds to the insulin receptor and interferes with its insulin-induced autophosphorylation. Taken together, these data demonstrate that physiologically relevant levels of naturally secreted Abeta interfere with insulin receptor function in hippocampal neurons and prevent the rapid activation of specific kinases required for long term potentiation.