The membrane and lipids as integral participants in signal transduction: lipid signal transduction for the non-lipid biochemist

Reviews of signal transduction have often focused on the cascades of protein kinases and protein phosphatases and their cytoplasmic substrates that become activated in response to extracellular signals. Lipids, lipid kinases, and lipid phosphatases have not received the same amount of attention as proteins in studies of signal transduction. However, lipids serve a variety of roles in signal transduction. They act as ligands that activate signal transduction pathways as well as mediators of signaling pathways, and lipids are the substrates of lipid kinases and lipid phosphatases. Cell membranes are the source of the lipids involved in signal transduction, but membranes also constitute lipid barriers that must be traversed by signal transduction pathways. The purpose of this review is to explore the magnitude and diversity of the roles of the cell membrane and lipids in signal transduction and to highlight the interrelatedness of families of lipid mediators in signal transduction.     

Involvement of the histidine protein (HPr) of the phosphotransferase system in chemotactic signaling of Escherichia coli K-12.

It is known that in mutants of Escherichia coli lacking the histidine protein (HPr) of the carbohydrate: phosphotransferase system, all substrates of the system can be taken up in the presence of the fructose-regulated HPr-like protein FPr (gene fruF). Although this protein fully substituted for HPr in transport and phosphorylation, we found that it was not able to complement efficiently for HPr in mediating chemotaxis toward phosphotransferase system substrates. Furthermore, transport activity and chemotaxis could be genetically dissected by the exchange of single amino acids in HPr. The results suggest a specific role of HPr in chemotactic signaling. We propose a possible link of signal transduction pathways for phosphotransferase system- and methyl chemotaxis protein-dependent substrates via HPr.     

Mechanisms through which sulfur amino acids control protein metabolism and oxidative status

Amino acids regulate protein synthesis and breakdown (i.e., protein turnover) and consequently protein deposition, which corresponds to the balance between the two processes. Elucidating the mechanisms involved in such regulation is important from fundamental and applied points of view since it can provide a basis to optimize amino acid requirements and to control protein mass, body composition and so forth. Amino acids, which have long been considered simply as precursors of protein synthesis, are now recognized to exert other significant influences; that is, they are precursors of essential molecules, act as mediators or signal molecules and affect numerous functions. For example, amino acids act as mediators of metabolic pathways in the same manner as certain hormones. Thus, they modulate the activity of intracellular protein kinases involved in the regulation of metabolic pathways such as mRNA translation. We provide here an overview of the roles of amino acids as regulators of protein metabolism, by focusing particularly on sulfur amino acids. The potential importance of methionine as a "nutrient signal" is discussed in the light of recent findings. Emphasis is also placed on mechanisms controlling oxidative status since sulfur amino acids are involved in the synthesis of intracellular antioxidants (glutathione, taurine etc.) and in the methionine sulfoxide reductase antioxidant system.     

Regulation of protein synthesis by branched-chain amino acids in vivo

Recent advances in the understanding of mRNA translation have facilitated molecular studies on the regulation of protein synthesis by nutrients and the interplay between nutrients and hormonal signals. Numerous reports have established that, in skeletal muscle, the branched-chain amino acids (BCAAs) have the unique ability to initiate signal transduction pathways that modulate translation initiation. Of the BCAAs, leucine is the most potent. Oral administration of leucine to food-deprived rats enhances muscle protein synthesis, in part, through activation of the mRNA binding step of translation initiation. Interestingly, leucine signaling in skeletal muscle differs from that in liver, suggesting that the responses may be tissue specific. The purpose of this paper was to briefly review the current knowledge of how BCAAs act as regulators of protein synthesis in physiologically important tissues, with particular focus on the mechanisms by which BCAAs regulate translation initiation.     

Networking of phospholipases in plant signal transduction

Phospholipases are activated in response to various cellular and environmental cues. Their activation can affect many cellular processes through their roles in signal transduction. Recent advances in the biochemical and molecular understanding of phospholipase D (PLD) have provided insights into potential networks of PLDs and other phospholipases in plants. PLDs are a family of heterogeneous enzymes, and the activities of the multiple types of PLDs are regulated in distinctly different manners. Phosphoinositides, free fatty acids, lysophospholipids, and calcium are differential modulators of PLDs. Since these modulators are substrates, products, or downstream targets of phospholipase As and phospholipase Cs, there are many potential regulatory and metabolic interrelationships among the various PLDs and other phospholipases.     

Amino acid signalling and the integration of metabolism

It has become clear in recent years that amino acids are not only important as substrates for various metabolic pathways but that they can also activate a nutrient-sensitive, mTOR-mediated, signalling pathway in synergy with insulin. Leucine is the most effective amino acid in this regard. The signalling pathway is antagonised by AMP-activated protein kinase. Amino acid signalling stimulates protein synthesis and inhibits (autophagic) proteolysis. In addition, many amino acids cause an increase in cell volume. Cell swelling per se stimulates synthesis of protein, glycogen, and lipid, in part by further stimulating signalling and in part by unrelated mechanisms. Amino acids also stimulate signalling in beta-cells and stimulate beta-cell growth and proliferation. This results in increased production of insulin, which enhances the anabolic (and anti-catabolic) properties of amino acids. Finally, amino acid-dependent signalling controls the production of leptin by adipocytes, and thus contributes to the regulation of appetite.     

Novel forms of chemical protein diversity -- in nature and in the laboratory

Novel chemical variants of proteins have been found in nature, including potent 'microprotein' natural products and folded protein molecules that contain a cyclic polypeptide chain. Researchers have used chemical synthesis and genetic methods to make these proteins and more: protein catenanes, neoglycoproteins, and artificial protein molecules with novel architectures or made from novel building blocks. De novo design has taken a big step forward with the accurate design and construction of proteins with complex molecular structure. A variety of non-coded amino acids and other building blocks has been used to make increasingly sophisticated protein molecular devices for use as biosensors and for the study of signal transduction inside living cells.     

Protein turnover in pulmonary macrophages. Utilization of amino acids derived from protein degradation.

Conditions were defined under which rates of protein synthesis and degradation could be estimated in alveolar macrophages isolated from rabbits by pulmonary lavage and incubated in the presence of plasma concentrations of amino acids and 5.6 mM-glucose. Phenylalanine was validated as suitable precursor for use in these studies: it was not metabolized appreciably, except in the pathways of protein synthesis and degradation; it entered the cells rapidly; it maintained a stable intracellular concentration; and it was incorporated into protein at measurable rates. When extracellular phenylalanine was raised to a concentration sufficient to minimize dilution of the specific radioactivity of the precursor for protein synthesis with amino acid derived from protein degradation, the specific radioactivity of phenylalanyl-tRNA was only 60% of that of the extracellular amino acid. This relationship was unchanged in cells where proteolysis increased 2.5-fold after uptake and degradation of exogenous bovine serum albumin. In contrast, albumin prevented the decrease in phenylalanine incorporation observed in macrophages deprived of an exogenous source of amino acids. These observations suggested that macrophages preferentially re-utilized amino acids derived from the degradation of endogenous, but not from exogenous (albumin), protein. However, when the extracellular supply of amino acids was restricted, substrates derived from albumin catabolism could support the protein-synthetic pathway.     

Editing of errors in selection of amino acids for protein synthesis.

All living cells must conduct protein synthesis with a high degree of accuracy maintained in the transmission and flow of information from gene to finished protein product. One crucial "quality control" point in maintaining a high level of accuracy is the selectivity by which aminoacyl-tRNA synthetases furnish correctly activated amino acids, attached to tRNA species, as the building blocks for growing protein chains. During selection of amino acids, synthetases very often have to distinguish the cognate substrate from a homolog having just one fewer methyl group in its structure. The binding energy of a methyl group is estimated to contribute only a factor of 100 to the specificity of binding, yet synthetases distinguish such closely related amino acids with a discrimination factor of 10,000 to 100,000. Examples of this include methionine versus homocysteine, isoleucine versus valine, alanine versus glycine, and threonine versus serine. Many investigators have demonstrated in vitro the ability of certain aminoacyl-tRNA synthetases to edit, that is, correct or prevent incorrect attachment of amino acids to tRNA molecules. Several major editing pathways are now established from in vitro data. Further, at least some aminoacyl-tRNA synthetases have recently been shown to carry out the editing function in vivo. Editing has been demonstrated to occur in both Escherichia coli and Saccharomyces cerevisiae. Significant energy is expended by the cell for editing of misactivated amino acids, which can be reflected in the growth rate. Because of this, cellular levels of aminoacyl-tRNA synthetases, as well as amino acid biosynthetic pathways which yield competing substrates for protein synthesis, must be carefully regulated to prevent excessive editing. High-level expression of recombinant proteins imposes a strain on the biosynthetic capacity of the cell which frequently results in misincorporation of abnormal or wrong amino acids owing in part to limited editing by synthetases. Unbalanced amino acid pools associated with some genetic disorders in humans may also lead to errors in tRNA aminoacylation. The availability of X-ray crystallographic structures of some synthetases, combined with site-directed mutagenesis, allows insights into molecular details of the extraordinary selectivity of synthetases, including the editing function.     

Sequence of a rat cDNA encoding the ERK1-MAP kinase

Mitogen-activated protein (MAP) kinases are cytoplasmic and/or nuclear protein kinases which are activated by one or several signal transduction pathways from the cell surface into the nucleus. Their activity is regulated by phosphorylation on Tyr as well as on Ser/Thr residues. A cDNA encoding the rat ERK1 member of the MAP kinase family was isolated and sequenced. The longest cDNA consisted of 1875 nucleotides and coded for a polypeptide of 380 amino acids with a predicted M(r) of 42987.     

Metal interaction with redox regulation: an integrating concept in metal carcinogenesis?

The carcinogenicity of cadmium, arsenic, and chromium(VI) compounds has been recognized for some decades. However, the underlying molecular mechanisms seem to be complex and are not completely understood at present. Although, with the exception of chromium(VI), direct DNA damage seems to be of minor importance, interactions with DNA repair processes, tumor suppressor functions, and signal transduction pathways have been described in diverse biological systems. In addition to the induction of damage to cellular macromolecules by reactive oxygen species, the interference with cellular redox regulation by reaction with redox-sensitive protein domains or amino acids may provide one plausible mechanism involved in metal carcinogenicity. Consequences are the distortion of zinc-binding structures and the activation or inactivation of redox-regulated signal transduction pathways, provoking metal-induced genomic instability. Nevertheless, the relevance of the respective mechanisms depends on the actual metal or metal species under consideration and more research is needed to further strengthen this hypothesis.     

Integration of P2Y receptor-activated signal transduction pathways in G protein-dependent signalling networks

The role of nucleotides in intracellular energy provision and nucleic acid synthesis has been known for a long time. In the past decade, evidence has been presented that, in addition to these functions, nucleotides are also autocrine and paracrine messenger molecules that initiate and regulate a large number of biological processes. The actions of extracellular nucleotides are mediated by ionotropic P2X and metabotropic P2Y receptors, while hydrolysis by ecto-enzymes modulates the initial signal. An increasing number of studies have been performed to obtain information on the signal transduction pathways activated by nucleotide receptors. The development of specific and stable purinergic receptor agonists and antagonists with therapeutical potential largely contributed to the identification of receptors responsible for nucleotide-activated pathways. This article reviews the signal transduction pathways activated by P2Y receptors, the involved second messenger systems, GTPases and protein kinases, as well as recent findings concerning P2Y receptor signalling in C6 glioma cells. Besides vertical signal transduction, lateral cross-talks with pathways activated by other G protein-coupled receptors and growth factor receptors are discussed.     

Amino acids conserved in interleukin-1 receptors (IL-1Rs) and the Drosophila toll protein are essential for IL-1R signal transduction.

The cytoplasmic domain of the human T cell-type interleukin-1 receptor (hIL-1R) is not involved in the binding, internalization, or nuclear localization of interleukin-1 (IL-1), but is essential for signal transduction. We have previously localized a 50-amino acid region (residues 477-527) critical for IL-1-mediated activation of the interleukin-2 promoter in T cells. This region displays a striking degree of amino acid conservation in human, murine, and chicken IL-1Rs. Here we report the results of a site-directed mutational analysis of the cytoplasmic domain of the hIL-1R. We have introduced single-amino acid substitutions at positions conserved in all three receptors and at nonconserved positions and identified key amino acids for IL-1R function in signal transduction. Three basic (Arg431, Lys515, and Arg518) and 3 aromatic (Phe513, Trp514, and Tyr519) amino acids that are conserved in human, murine, and chicken IL-1Rs could not be replaced without abolishing IL-1R-mediated signal transduction. A substitution at another conserved position (Pro521) reduces significantly the ability of the IL-1R to transmit the IL-1 signal. Nonconserved residues could be replaced without affecting signal transduction. The cytoplasmic domain of the IL-1R is related to that of the Drosophila Toll protein, with a 26% identity and a 43% similarity in amino acid sequence. The amino acids shown to be essential for IL-1R function are conserved in the Toll protein. Our experimental data indicate that the amino acid sequence similarity between the IL-1R and the Drosophila toll protein reflects a functional homology between the two proteins.     

Structure of a novel phosphotyrosine-binding domain in Hakai that targets E-cadherin

Phosphotyrosine-binding domains, typified by the SH2 (Src homology 2) and PTB domains, are critical upstream components of signal transduction pathways. The E3 ubiquitin ligase Hakai targets tyrosine-phosphorylated E-cadherin via an uncharacterized domain. In this study, the crystal structure of Hakai (amino acids 106-206) revealed that it forms an atypical, zinc-coordinated homodimer by utilizing residues from the phosphotyrosine-binding domain of two Hakai monomers. Hakai dimerization allows the formation of a phosphotyrosine-binding pocket that recognizes specific phosphorylated tyrosines and flanking acidic amino acids of Src substrates, such as E-cadherin, cortactin and DOK1. NMR and mutational analysis identified the Hakai residues required for target binding within the binding pocket, now named the HYB domain. ZNF645 also possesses a HYB domain but demonstrates different target specificities. The HYB domain is structurally different from other phosphotyrosine-binding domains and is a potential drug target due to its novel structural features.