The chapter lists the superfamily of protein phosphatases, tyrosine (PTP), and serine/threonine (PPP, PPM, and CTD). It provides information about domain architecture, catalytic sites, and catalytic mechanisms. It shows how the tyrosine phosphatase PTPN1 controls insulin signaling and how this affects glucose transport and food-seeking behavior. It shows how it can be inhibited by reactive oxygen species (cyclic sulfenamide bond) and how this boosts growth-factor signaling. It shows the role of SH2-containing phosphatases in the control of inflammation (PTPN6) and in Noonan and LEOPARD syndrome (PTPN11). It describes the receptor-like tyrosine phosphatases and their role in T-cell activation (CD45) and synapse development. It then switches to dual-specificity phosphatases and their role in down-regulation of the MAPK pathway, with the example of dorsal closure in Drosophila, and their role as tumor suppressors (PTEN). The last section deals with serine/threonine phosphatases, how subunits determine substrate selectivity and how they inhibit smooth muscle contraction, control glycogen metabolism, and activate NFAT.
Here we deal primarily with signaling events initiated by phosphatidylinositol 3-kinase (PIK3), and we place it in the context of insulin signaling. We describe in detail the insulin receptor, its mode of activation, phosphoinositide metabolites, the interaction protein domains (among which the PH domain), and the members of the family of PIK3. We show structural details of PIK3CG, AKT (protein kinase B), and how it is primed by the mTORC1 complex at the ribosome and activated by PDK1 at the membrane. We then show the pathway leading to inactivation of GSK3B and activation of the mTORC2 complex, involving TSC2, RHEB, and RRAG, which has subsequent effects on the translation-initiation process of ribosomes. We show how amino-acid depletion and ATP shortage counteract this activation pathway by placing mTORC beyond the control of RHEB and by activating TSC2 through the STK11 and PRKAA2 kinases, respectively.
This chapter deals primarily with non-receptor tyrosine kinases (NRTK) and places them in the context of T-cell activation. After listing the members of the NRTK, it shows the composition of MHCII and TCR and how the two interact. It describes the role of tyrosine kinases (LCK, ZAP70) and LAT in the process of signaling complex formation. From here two pathways emerge, one leading to the activation of NFAT1 and involving phospholipase-Cγ (PLCG1), IP3, Ca2+, and calcineurin (PPP3CA), and the other involving protein kinase Cθ (PRKCQ), activation of CARD11, and the subsequent CARD domain-mediated filamentous assembly of a signaling complex composed of BCL10 (adaptor), MALT1 (protease), and TRAF6 (E3-ubiquitin ligases). The chapter then elaborates how INFα brings about a change in gene transcription through receptor-mediated activation of the non-receptor tyrosine kinases TYK2 and JAK1, leading to phosphorylation and nuclear translocation of STAT transcription factors. The chapter ends with an excursion on the activation process of SRC.
In the biological world, Ca2+ may be considered to exist in three main forms: free, bound, and trapped. In vertebrates, calcified tissues such as bones and teeth account for the major proportion of body calcium. Ca2+ has special roles in the signaling mechanisms that regulate the activities of eukaryotic cells and that this depends upon the sensitivity of some proteins to the concentration of free Ca2+ in their surroundings. The concentration of free Ca2+ within prokaryotic cells is kept very low by powerful ion translocation mechanisms that expel Ca2+ ions. While nature seems to have taken advantage of the presence of Ca2+, its suitability for signaling depends upon its ability to form stable complexes with particular biological molecules. Conversely, Mg2+ forms complexes in which the ligand atoms are nearly always arranged in an octahedral formation (a coordination number of 6). For small anions that can be accommodated in such a structure, the strength of association with Mg2+ is greater than with Ca2+. To detect and follow the progress of changes in Ca2+ in living cells has required the introduction of a Ca2+-sensing agent. It is possible to measure changes in total cell Ca2+ by this means, but impossible to determine how concentrations have changed within particular intracellular compartments, such as the cytosol. To make matters worse, the bulk of cell calcium is retained within organelles and there is very little in the cytosolic pool. Although Ca2+ ions are distributed uniformly in aqueous solutions, the spatial distribution of free Ca2+ within the cytosolic compartment is often far from uniform. During activation the pattern can fluctuate rapidly.
This chapter deals with mitogen-activated protein kinases and how they are activated by receptor tyrosine protein kinases. It starts with a structural description of tyrosine versus serine/threonine protein kinases, lists the large family of receptor tyrosine kinases, and then concentrates on the EGF receptor family (EGFR and ERBB). It shows structural detail of EGF-mediated conformational changes, both in the extra- and intracellular segment, and how these render the protein kinase domain catalytically competent (allosteric regulation). The ensuing transphosphorylation leads to recruitment of SH2- and PTB-containing adaptor, docking, and effector proteins which, by forming signaling complexes, transmit the signal into the cell. We describe in detail, at the level of structure and function, the SOS–RAS–RAF–MEK–ERK cascade and show how ERK activates other protein kinases and transcription factors, thereby preparing the cell for a round of division. The chapter ends with an overview of components of three distinct cascades: the ERK, p38, and JNK pathway.
Of all the hormones, sex steroids have the longest history of fierce public interest dating back to the origins of our great affair with domestic animals. Steroids bind intracellular receptors, a large family of proteins with diverse ligands and named nuclear receptors. We list the members of the family and their respective ligands and discuss in detail the structure of the estrogen receptor, how it binds ligand and DNA, and how ligand binding leads to the recruitment of transcriptional co-activators. This chapter describes how agonists, inverse agonists, and antagonists modify transcriptional activity of the estrogen and retinoic-acid receptor. It discusses the role of chaperone proteins in the process of ligand loading and how nuclear receptors contribute to the action of other transcription factors. We discuss the action of steroids in the context of pregnancy: how progesterone prepares spermatozoid for fertilization, how cortisol helps to reinforce memory, and how estrogen and progesterone prepare the mammary gland for milk production.
This chapter is about transcription and how it is regulated by protein kinase A (PKA) and its substrate cAMP response element-binding protein (CREB) in the context of glucagon-driven gluconeogenesis. The chapter elaborates on PKA, its synthesis, posttranslational modifications, structure and function, the phosphosite sequences of its substrates, and how cAMP causes its activation. It shows the structure of the regulatory subunit (PRKAR), its association with the protein kinase and with anchor proteins (AKAPs), and how these contribute to the formation and subcellular location of signaling complexes. The chapter then switches to a brief overview of gene transcription, transcription factors, the transcription initiation complex, and the role of histone acetylation and methylation in rendering DNA accessible. This is followed by a brief description of the glucagon-signaling pathway, and a detailed description of how CREB recruits histone-acetyltransferases, CRTC2, the SWI/SNF-, promoter-core, and mediator complex. The chapter concludes by explaining how CREB brings about, in cooperation with nuclear receptors, expression of enzymes that control gluconeogenesis.
This chapter brings to light a very short signaling pathway where the intracellular segment of the receptor is cleaved and then acts as a transcription factor. This mechanism is employed by the Notch receptor, upon binding of its ligands Delta-like or Jagged. Ligand binding causes a set of cleavages, first in the LNR-HD region, S2-site cleaved by ADAM, followed by an intracellular cleavage (S3) by the γ-secretase complex. We then show how the intracellular segment forms a transcription complex with RBPJ and drives expression of HES and HEY proteins which play a role in cell fate decisions. We place the action of Notch in the context of Drosophila sensory organ precursor cell differentiation, in maintaining undifferentiated stem cells in the intestine and in arresting cell migration. Mutations in the LNR-HD regions are associated with leukemia.
This chapter deals with serine/threonine kinase receptors which basically are members of the TGFβ-receptor family. We place the subject in the context of epidermal–mesenchymal transition, immunosurveillance, and the Spemann organizer (development). We list the members of the TGFβ family (ligands), their traps, and their accessory receptors. We detail the activation mechanism of TGFβ receptors, formation of type-1 and type-2 receptor dimers, how phosphorylation of the GS domain removes an inhibitory wedge from the N-lobe, and how this leads to phosphorylation of receptor-regulated SMAD proteins. We describe the SMAD proteins, including domains, structure, phosphorylation, and ubiquitinylation sites, and how they form heterotrimeric complexes that bind DNA. We show how they recruit co-activators and contribute to the assembly of the transcription pre-initiation complex and how linker-phosphorylation and subsequent ubiquitinylation plays a role in terminating their action. We show how TRIM33 and cell lineage-dependent (or master) transcription factors prepare the road for SMADS and how they determine cell-specific cell responses.
Introduction to signal transduction provides an overview of the many mechanisms that cells employ to create a symbolic representation of their environment. It deals with first messengers (ligands) and receptors and how they interact, their basic pharmacology, and their modes of signal transmission into the cell. It deals with transducers, effectors, and second messengers. It provides an overview of allosteric regulation and posttranslational mechanisms including methylation, acetylation, glycosylation, ubiquitinylation, sumoylation, and phosphorylation. It focuses on nucleotide exchange and GTP-binding proteins, and on protein phosphorylation, from bacteria to men, with special attention to phosphate donors and the structure and catalytic mechanisms of protein kinases and phosphatases. The distinctive role of the different signaling mechanisms is presented in the context of the most “Nobel” of signaling pathways, adrenaline-mediated activation of glycogenolysis in striated muscle (adrenoceptors, G-proteins, adenylyl cyclase, protein kinase A, phosphorylase kinase, glycogen phosphorylase). It shows how different pathways can interfere with each other (concept of signaling nodes).
Epithelial–mesenchymal transition is an essential process in development (planar polarity, migration, and cell fate) and in the maintenance of a stem cell compartment (cell fate), but it also contributes to oncogenic transformation. Indeed Wnt was discovered independently as a developmental factor (Wg) and a proto-oncogene (Int). This chapter highlights how Wnt affects the subcellular localization of newly synthesized β-catenin (CTNBB1), a protein that normally is a component of the zonula adherens, but may also act as a transcription factor. We highlight how β-catenin interacts with cadherin, AXIN, APC, and members of the TCF family. We describe how the Wnt receptors, Frizzled and LRP, recruit and disable the AXIN/APC complex thereby preventing phosphorylation and destruction of β-catenin. Once in the nucleus and bound to TCF7, β-catenin paves the way for the formation of the transcription initiation complex. We describe how WNT3 plays an essential role in maintaining a stem cell compartment in the crypts of the small intestine and how it is involved in planar cell polarity in Drosophila.
In this chapter, we deal with G-protein-coupled receptors (GPCRs) that operate as sensory receptors for light (vision) and odorants (olfaction). We detail the composition of the retina, the different light receptors (visual pigments), and the role of 11-cis-retinal. We describe the photoisomerization process and how it changes the conformation of the helix bundle of rhodopsin, allowing binding of transducin (Gαt). We describe the activation of phosphodiesterase and how the removal of cGMP leads to a lowering of the membrane potential and a subsequent inhibition of glutamate release. We describe the action of the transducin GAP complex, GRK1 and arrestin and how they terminate the activation cycle. We give a brief overview of signaling in the Drosophila compound eye. We then describe the olfactory epithelium and how odorant receptors activate adenylyl cyclase and how this, through an increase in cAMP, increases permeability of the CNGA2 channel and subsequent Ca2+-mediated opening of the ANO2 channel. The chapter ends with an excursion about the GPCR family of receptors.
This chapter deals with the tumor necrosis factor (TNF) receptor and how it employs two types of ubiquitin chains in the process of signaling complex assembly and its subsequent activation of nuclear-factor kappa-B and RELA. We place the subject in the context of extravasation where TNF-mediated expression of chemokines and up-regulation of ICAM and VCAM play important roles in leukocyte recruitment. We then elaborate on chemokines, their composition, their receptors, and signaling pathways, how they activate integrin adhesion molecules, allowing blood-borne cells to arrest on the vascular bed. We show how they mobilize the actin cytoskeleton, allowing cells to migrate into the tissue (process of extravasation). We highlight the numerous E3-ubiquitin ligases involved in TNF signaling, which give rise to a network of both M1 (linear) and K63-type of ubiquitin chains. These networks play essential roles in both recruitment and stabilization of signaling complexes. We briefly hint on how TNF may also induce apoptosis or necroptosis.
In the context of muscle contraction, we explore the signaling mechanisms of acetylcholine, a neurotransmitter that binds both ionotropic (nicotinic) and metabotropic receptors (muscarinic). We also deal with nitric oxide, a second messenger produced in vascular endothelial cells exposed to acetylcholine. The composition of both the nicotinic and muscarinic receptor, their ligand-binding pockets, and conformational changes that lead to receptor activation are discussed in great detail. We describe how the nicotinic receptor drives skeletal muscle contraction and how the muscarinic receptor slows down rhythmicity and reduces contractile force of the heart. We highlight the action of cAMP-phosphodiesterase. We then turn to bronchi under asthmatic conditions and show how acetylcholine causes smooth muscle constriction and mucus production and how the signaling events are blocked by salbutamol. The last section describes nitric oxide synthase, the activation of guanylyl cyclase by nitric oxide, and how cGMP-mediated activation of protein kinase G causes vasodilation and penile erection.
This chapter considers the adherence of cells to surfaces or to other cells and discusses how this affects their responses to soluble agonists such as growth factors. The binding of cells to the extracellular matrix and their attachment to other cells occurs through specific adhesion molecules. The molecules that effect adhesion behave both as targets for signals that are generated within cells (inside-out signaling) and as receptors for extracellular signals (outside-in signaling). Adhesion molecules not only link cells to surfaces but they also make the connection between the extracellular matrix and the cytoskeleton. The chapter describes the structure of several adhesion molecules including platelet endothelial cell adhesion molecule (PECAM), neuronal cell adhesion molecule (NCAM), intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1(VCAM-1), endothelium leukocyte adhesion molecule-1 (ELAM-1), very late antigen-4 (VLA-4), integrins, cadherins, selectins, and CD44. The chapter discusses of the two roles of adhesion molecules—signal targets and receptors—in the regulation of survival, proliferation, differentiation, and in leukocyte trafficking. Adhesion molecules are also of prime importance in the functioning of synapses and in neuronal cell outgrowth, differentiation of keratinocytes, gene expression in mammary epithelial cells, thymic selection, and the activation of T lymphocytes.
With respect to the transduction of signals from cell surface receptors, there are two main classes of protein tyrosine kinases (PTKs). This chapter considers those PTKs that exist as integral domains of transmembrane receptors, with focus on the signal transduction pathway initiated by binding of the growth factors epidermal growth factor (EGF) and platelet-derived growth factor (PDGF), to their receptors. Additionally, it describes a number of principles that also apply for other tyrosine kinase containing receptors. Tyrosine kinase containing receptors come in several different forms, but they all have a single membrane-spanning domain and an intracellular tyrosine protein kinase catalytic domain. A general feature is that ligand binding results in dimerization of these receptors, and the crosslinking of receptors by growth factors can be achieved in a number of ways. Dimerization allows the kinase activity of both intracellular chains to encounter target sequences on the other, linked receptor molecule. This enables the intermolecular cross-phosphorylation of several tyrosine residues. The phosphorylated dimer then constitutes the active receptor. It possesses an array of phosphotyrosines that enable it to bind proteins (adapters and enzymes) bearing SH2 domains to form receptor signaling complexes. Additionally, the dimerized and phosphorylated receptor has the potential of phosphorylating its targets. A number of signal transduction pathways branch out from the receptor signaling complex, which are described in the chapter.
