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Signal transduction

Whenever I've had to resort to adrenaline to the heart it has never worked, Hema said to herself. Not once. Maybe I do it as a way to signal to myself that the patient is dead. But surely it must have worked, somewhere, with someone. Why else was it taught to us?

Cutting for stone. Abraham verghese

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The Structure and Function of Signal Pathways • The enormous structural variety and functional

capacity of multicellular organisms is due to their ability to coordinate the biochemical reactions of the various cells of the total organism

• The basis for this coordination is the intercellular communication, which allows a single cell to influence the behavior of other cells in a specific manner

• Cells can communicate in different ways:1. Chemical Messengers: Cells send out signals in

the form of specific chemical messengers that the target cell converts into a biochemical reaction

2. Gap Junctions: Communication between bordering cells is possible via direct contact in the form of “gap junctions”

3. Cell-cell interaction via cell surface proteins: A cell surface protein of one cell binds a specific complementary protein (or carbohydrate chain) on another cell

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ation • A further intercellular

communication mechanism relies on electrical processes. The conduction of electrical impulses by nerve cells is based on changes in the membrane potential

• Signal transduction is the process by which cells of a particular type receive and transform a biochemical signal into a physiological reaction

• Signaling pathways are involved in the coordination of metabolite flux, the regulation of cell division, differentiation and development of an organism, processing of sensory information

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• In the communication between cells of an organism, the signals (chemical messengers or electrical signals) are produced in specialized cells

• The signal-producing function of these cells is itself regulated, so that the signal is only produced upon a particular stimulus

• The following steps are involved in intercellular communication: Formation of a signal in the signal-producing cell

as a result of an external trigger Transport of the signal to the target cell Registration of the signal in the target cell Further transmission of the signal into the target

cell Transformation of the signal into a biochemical or

electrical reaction in the target cell Termination of the signal• Specialized proteins, termed receptors, are utilized

for the reception of signals

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• There are two principal ways by which target cells can process incoming signals:

a) Cell surface receptors receive the signal at the outside of the cell, become activated and initiate a signaling chain in the interior of the cell.

• In such signaling pathways the membrane-bound receptor transduces the signal at the cell membrane so that it is not necessary for the signal to actually enter the cell

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b) The messenger enters into the target cell and binds and activates the receptor localized in the cytosol or nucleus

• Upon receiving a signal, a receptor becomes activated to transmit the signal further

• The activated receptor passes the signal onto components, usually proteins, further downstream in the signaling pathway, which then become activated themselves for further signal transmission

• A chain of serially operating, intracellular signal transduction processes results

• Finally, a specific biochemical process is triggered in the cell, which represents the endpoint of the signaling pathway

• Cells possess multiple mechanisms to regulate the intercellular communication as well as the intracellular signal transduction

• This allows a specific termination of communication between cells. Often feedback mechanisms are used to adapt the cellular response to the needs of the organism

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• The key components of intracellular signal transduction include receptors, protein kinases and protein phosphatases, regulatory GTPases, adaptor proteins and second messengers

• The regulatory GTPases function as switches that can exist in an active or inactive form

• Adaptor proteins mediate the signal transmission between proteins of a signaling chain by bringing these proteins together

• They function as clamps to co-localize proteins for an effective and specific signaling

• Furthermore, adaptor proteins help to target signaling proteins to specific sub-cellular sites

• Second messengers are chemical signaling substances produced or released due to the intracellular activation of enzymes in a signaling chain

• Extracellular signaling molecules are released either by exocytosis or passive diffusion into the extracellular space

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• In special cases, membrane-bound proteins are also used as signaling molecules

• Signaling molecules for the communication between cells are known as hormones

• Hormones that are proteins and regulate cell proliferation are known as growth factors

• The chemical nature of hormones is extremely variable

• Hormones can be proteins, peptides, amino acids and amino acid derivatives, derivatives of fatty acids, nucleotides, steroids, retinoids, small inorganic molecules, such as NO

• The modification of hormones can lead to compounds that are known as agonists or antagonists

• Antagonists are hormone derivatives that bind to a receptor but do not initiate signal transduction

• Antagonists block the receptor and thus terminate signal transduction. They have broad medical application since they specifically interfere with certain signal transduction pathways

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• Various forms of intercellular communication can be discerned based on the range of the signal transmission

• In endocrine signaling, the hormone is synthesized in specific signaling, or endocrine cells and exported via exocytosis into the extracellular medium (e.g., blood or lymphatic fluid)

• The hormone is then distributed throughout the entire body via the circulatory system so that remote regions of an organism can be reached

• Paracrine signal transduction occurs over medium range.

• The producing cell must be found in the vicinity of the receiving cells for this type of communication

• The signaling is local, and the participating signaling molecules are termed tissue hormones or local mediators

• A special case of paracrine signal transduction is synaptic neurotransmission in which a nerve cell communicates with either another nerve cell or with a muscle cell

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• In autocrine signaling, cells of the same type communicate with one another

• If an autocrine hormone is secreted simultaneously by many cells then a strong response occurs in the cells

• Autocrine mechanisms are of particular importance in the immune response

• A special case of signal transduction is represented by a class of small, reactive signaling molecules, such as NO

• NO is synthesized in a cell in response to an external signal and is delivered to the extracellular fluid

• Either by diffusion or in a protein-bound form, the NO reaches neighboring cells, and modification of target enzymes ensues, resulting in a change in the activity of these enzymes

• NO is characterized as a mediator that lacks a receptor in the classical sense

o Signal pathways commonly amplify the initial signal received by the receptor during the course of the signal transduction

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SIGNALING BY NUCLEAR RECEPTORS

• Nuclear receptors regulate gene expression in response to binding lipophilic molecules and are thereby involved in the control of a diversity of cellular processes

• These proteins are ligand-activated transcription factors that are localized in the cytoplasm and/or in the nucleus

• The ligands pass the cell membrane by simple diffusion and bind to the receptors

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• By binding to DNA elements in the control regions of target genes the ligand-bound receptor influences the transcription of these genes and thus transmits hormonal signals into a change of gene expression

• The naturally occurring ligands of nuclear receptors are lipophilic hormones, among which the steroid hormones, the thyroid hormone T3, and derivatives of vitamin A and D have long been known as central regulators

• In recent years it has been recognized that intracellularly formed lipophilic metabolites can also serve as ligands for nuclear receptors and can regulate gene expression through their binding to nuclear receptors

• These compounds include prostaglandins and leukotrienes

• They also include other molecules synthesized intracellularly as normal metabolites such as fatty acids and bile acids and substances derived from foreign lipophilic substances like drugs

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• The receptors such as PPAR are quite promiscuous with respect to the nature of the ligand and can bind a broad range of lipophilic ligands

• This type of receptors is thought to be involved in the regulation of metabolism and in detoxification

• In comparison to signaling pathways which utilize transmembrane receptors , signaling via nuclear receptors is of relatively simple structure

• The pathways lead directly, with only a few participating protein components, from the extracellular space to the level of transcription in the nucleus

• Nuclear receptors have got separate ligand-binding and DNA-binding domains

• That part of the DNA bound specifically by nuclear receptors is known as hormone response element (HRE)

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• Based on the receptor activation mechanism, the nuclear receptors may be divided into two basic groups

• In the first group (those including most of the steroid hormone receptors), the receptors can be localized in the nucleus or in the cytoplasm

• The receptors of the other group are always localized in the nucleus. Representative ligands of these receptors are the derivatives of retinoic acid, the T3 hormone and Vit D

• The transport of steroid hormones occurs in the form of a complex with a specific binding protein

• An example of such a binding protein is transcortin, which is responsible for the transport of the corticosteroids

• The steroid hormones enter the cell by diffusion and activate the cytosolic receptors. In the absence of steroid hormones, the receptors remain in an inactive complex- aporeceptor complex

• In the aporeceptor complex the receptor is bound to chaperones

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• The binding of the hormone to the aporeceptor complex leads to activation of the receptor and initiates the translocation of the receptor into the nucleus where it binds its HRE

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• In contrast to signal transduction by the steroid hormone receptors, there are multiple pathways by which the ligands of retinoic acid group are made available for receptor activation

• They can follow the classical endocrinological pathway (like vit D), be synthesized inside the cell from inactive precursors (like retinoic acid) or their full synthesis could take place inside a cell (like prostaglandin J2)

• In addition, the receptors in this group are found bound with the corresponding HRE in the absence of hormone, acting as repressors of gene activity

• In the presence of the hormone an activation of gene expression is usually observed

• In general, HREs are composed primarily of two copies of a hexamer DNA sequence

• The hexamers can be inverted (palindromic), everted or direct repeats

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Signal Transduction by RXRHeterodimers

Inverted

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• The receptors bind to the cognate HRE mainly as dimers, allowing the formation of homodimers as well as heterodimers between various receptor monomers

• Very few nuclear receptors are known whose HRE contains only a single copy of the recognition sequence. These receptors bind as monomers to the HRE

• The HREs of the steroid hormone receptors possess a palindromic structure; homodimers of receptors are formed

• The HRE of the nuclear receptors for all-trans retinoic acid, 9-cis retinoic acid, the T3 hormone and vitamin D usually exhibit a direct repeat of the recognition sequence, resulting in the formation of heterodimers on the DNA

• One of the partners in the heterodimer is always the receptor for 9-cis retinoic acid, RXR

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SIGNAL TRANSMISSION THROUGH TRANSMEMBRANE RECEPTORS

• Another way of transducing signals into the interior of the cell is through transmembrane receptors

• Transmembrane receptors are integral membrane proteins, i.e., they possess a structural portion that spans the membrane

• An extracellular domain, a transmembrane domain and an intracellular or cytosolic domain can be differentiated within the structure

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• In many receptors, the extracellular domain contains the ligand-binding site

• The function of the transmembrane domain is to pass the signal on to the cytosolic domain of the receptor

• Two basic mechanisms are used for conduction of the signal to the cytosol:1. Specific protein-protein interactions –the

next protein component in the signal transmission pathway, the effector protein, is activated

• The conformational change that accompanies the perception of the signal by the receptor creates a new interaction surface for proteins that are located downstream of the receptor2. Activation of Enzymes –the arrival of the signal

triggers enzyme activity in the cytosolic domain of the receptor, which, in turn, pulls other reactions along with it

• The enzyme usually has Tyrosine kinase activity

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• However, there are other examples where tyrosine phosphatase or Ser/Thr-specific protein kinase activity is activated

• The enzyme activity may be an integral part of the receptor, or it may also be a separate enzyme associated with the receptor on the inner side of the membrane

G-Protein Coupled Receptors (GPCR)• Of the transmembrane receptors, the G protein-

coupled receptors form the largest single family• GPCR can be activated by extracellular ligands or

sensory signals• Extracellular ligands include biogenic amines,

such as adrenaline and noradrenaline, histamine, serotonin, lipid derivatives, nucleotides, retinal derivatives, peptides such as bradykinin and large glycoproteins such as luteinizing hormone, and parathormone

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• Physical stimuli such as light signals are registered and converted into intracellular signals by GPCR; they are also involved in perception of taste and smell

• A characteristic structural feature of the GPCR is the presence of 7-transmembrane helices

• For the vast majority of 7-helix transmembrane receptors the next downstream located signaling protein is a heterotrimeric GTP/GDP binding protein (G-protein)

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• When a ligand binds to a GPCR, the structure of the transmembrane part is altered and this change is passed on to the cytoplasmic loops of the receptor

• As a consequence, a high-affinity surface is created for binding of the G protein

• The G protein, which exists as the inactive GDP form, now binds to the activated receptor and is itself activated

• An exchange of GDP for GTP takes place, and the βγ-subunit of the G protein dissociates • Once the G protein is activated, it frees itself from

the complex with the receptor, which either returns to its inactive ground state or activates further G proteins

• A phenomenon often seen in transmembrane receptors in general, and in G protein-coupled receptors in particular, is desensitization

• Desensitization means a weakening of the signal transmission under conditions of long-lasting stimulation

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• Despite the persistent effect of extracellular stimuli, the signal is no longer passed into the cell interior, or only in a weakened form, during desensitizing conditions

• A common way of desensitization is the phosphorylation of the receptor at the cytoplasmic side by specific protein kinases

• Phosphorylation of the receptor can be carried out by protein kinase A or protein kinase C

• This is a feedback mechanism since PKA and PKC are activated by GPCR

• Another way of phosphorylation is through G protein coupled receptor protein kinases (GRK)

• The phosphate residues introduced by GRK serve as attachment sites for arrestin which serves as a trigger for internalization of the receptor to endosomes

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• The superfamily of GTPases includes the heterotrimeric G proteins, the Ras family of small GTPases and the family of initiation and elongation factors

• The defining feature of this group is that its members have got a “switch function”

• The binding of GTP brings about the transition into the active form (turned on)

• Hydrolysis of the bound GTP by the intrinsic GTPase activity converts the protein into the inactive, GDP-bound form (turned off)

• Thus, the GTPase activity is one way of terminating signaling

• In the case of heterotrimeric G-proteins, the α-subunit has a binding site for GTP or GDP and carries the GTPase activity

• Based on comparison of the amino acid sequences, the heterotrimeric G-proteins are divided into four families

• The members of the Gs subfamily are activated by hormone receptors, by odor receptors and by taste receptors

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• Examples include signal transmission by type β-adrenaline receptors and by glucagon receptors

• During perception of smell, the smell receptors are activated, and these then pass the signal on via the olfactory G protein Golf

• Perception of sweet taste is also mediated via a Gs protein

• Transmission of the signal further involves an adenylyl cyclase in all cases, the activity of which is stimulated by the Gs-proteins

• The first members of the Gi subfamily to be discovered displayed an inhibitory effect on adenylyl cyclase, hence the name Gi, for inhibitory G proteins

• Other members of the Gi subfamily have phospholipase C as the corresponding effector molecule

• Type α-2 adrenergic receptors fall into this group• Signal transmission in the vision process is

mediated via G proteins known as transducins (Gt)

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• Perception of bitter taste also takes place via Gi• The Gq subfamily includes G-proteins associated

with type α-1 adrenergic receptors• There is also a fourth subfamily known as G12• Two bacterial toxins, namely pertussis toxin and

cholera toxin, were of great importance in determining the function of G- proteins

• Both toxins catalyze ADP ribosylation of proteins. During ADP ribosylation, an ADP-ribose residue is transferred from NAD+ to an amino acid residue of a substrate protein

• Cholera toxin is an enterotoxin made up of one A subunit (composed of one A1 and one A2 peptide joined by a disulfide link) and five B subunits and has a molecular mass of approximately 84 kDa

• In the small intestine, the toxin attaches by means of the B subunits binding to the ganglioside GM1 present in the plasma membrane of mucosal cells

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• The A subunit then dissociates, and the A1 peptide passes across to the inner aspect of the plasma membrane

• It then catalyzes the ADP-ribosylation of the GTP-binding regulatory component (Gs) of adenylyl cyclase, upregulating the activity of this enzyme

• Thus, adenylyl cyclase becomes chronically activated resulting in an elevation of cAMP

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• PKA then phosphorylates the regulatory domains of the cystic fibrosis transmembrane conductance regulator (CFTR) and the Na+-H+ exchanger • This leads to the inhibition of Na+ absorption and

the enhancement of the secretion of Cl-

• Thus, massive amounts of NaCl accumulate inside the lumen of the intestine, attracting water by osmosis and contributing to the liquid stools characteristic of cholera• Pertussis toxin is a protein secreted by the

bacterium Bordetella pertussis which causes whooping cough• Pertussis toxin carries out an ADP-ribosylation at a

cysteine residue of a Gi protein that inhibits adenylyl cyclase, closes Ca2+ channels, and opens K+ channels• The effect of this modification, however, is to lower

the G protein's affinity for GTP, effectively trapping it in the "off" conformation. The pulmonary symptoms have not yet been traced to a particular target of the Gi protein

32Mechanism of Action of

Cholera Toxin

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Effector Molecules of G-Proteins• Activated G proteins pass the signal on to

subsequent effector molecules that have enzyme activity or function as ion channels

• Important effector molecules are adenylyl cyclase, phospolipases, and cGMP-specific phosphodiesterases

• The activation of these enzymes leads to concentration changes of diffusible signal molecules such as cAMP, cGMP, diacylglycerol or inositol triphosphate (IP3), and Ca2+

, which

trigger further specific reactions cAMP• 3’-5’-cyclic AMP is a central intracellular second

messenger that influences many cellular functions, such as gluconeogenesis, glycolysis, lipogenesis, muscle contraction, membrane secretion, learning processes, ion transport, differentiation,…

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• Concentration of cAMP is controlled primarily by two means, namely via new synthesis by adenylyl cyclase and degradation by phosphodiesterases

• Cyclic AMP binds to and activates different signaling proteins

• It can regulate ion passage through cAMP-gated ion channels

• The majority of the biological effects of cAMP are mediated by the activation of protein kinases. Protein kinases regulated by cAMP are classified as protein kinase A

• In the absence of cAMP, protein kinase A exists as a tetramer, composed of two regulatory (R) and two catalytic (C) subunits

• In the tetrameric R2C2 form, protein kinase A is inactive since the catalytic center of the C subunit is blocked by the R subunit

• Upon binding of four molecules of cAMP, the enzyme dissociates into an R subunit dimer with four molecules of cAMP bound and two free C subunits which are now released from inhibition by the regulatory subunits and can thus phosphorylate Ser/Thr residues on specific substrate proteins

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KA Cyclic-GMP• Like cAMP, 3’-5’-cGMP is a

widespread second messsenger

• Analogous to cAMP, cGMP is formed by catalysis via guanylyl cyclase from GTP

• While adenylyl cyclase is an integral membrane protein, guanylyl cyclase can be found either associated with membranes or as a soluble cytosolic form

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Cytosolic Signaling via cAMP

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*cAMP and Gene Transcription• Whereas some responses mediated by cyclic AMP

occur within seconds and do not depend on changes in gene transcription, others do depend on changes in the transcription of specific genes

• When PKA is activated by cAMP, it enters into the nucleus and phosphorylates a specific gene regulatory protein called cyclic AMP response element-binding (CREB) protein

• Phosphorylated CREB then recruits a transcriptional coactivator called CREB-binding protein (CBP)

• The CREB/CBP complex binds to CRE on specific genes and activates transcription

• This signaling pathway controls many processes in cells, ranging from hormone synthesis (e.g. somatostatin) in endocrine cells to the production of proteins required for the induction of long-term memory in the brain

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*Signaling through the βγ –subunit • In some other cases, G proteins directly activate or

inactivate ion channels in the plasma membrane of the target cell

• Acetylcholine reduces both the rate and strength of heart muscle cell contraction

• This effect is mediated by a special class of acetylcholine receptors that activate the Gi protein

• Once activated, the α subunit of Gi inhibits adenylyl cyclase while the βγ subunits bind to K+ channels in the heart muscle cell plasma membrane and open them

• The opening of these K+ channels makes it harder to depolarize the cell and thereby contributes to the inhibitory effect of acetylcholine on the heart

• These acetylcholine receptors, which can be activated by the fungal alkaloid muscarine, are called muscarinic acetylcholine receptors to distinguish them from nicotinic acetylcholine receptors

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• Nicotinic receptors are ion-channel-coupled receptors on skeletal muscle and nerve cells that can be activated by the binding of nicotine, as well as by acetylcholine

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• The second messenger function of cGMP is directed towards three targets: cGMP-dependent protein kinases (protein kinase G, PKG), ion channels and cAMP phosphodiesterases

• Cyclic GMP carries different messages in different tissues. In the kidney and intestine it triggers changes in ion transport and water retention; in cardiac muscle, it signals relaxation; in the brain it may be involved both in development and in adult brain function

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• Guanylyl cyclase in the kidney is activated by the hormone atrial natriuretic factor (ANF), which is released by cells in the atrium of the heart when the heart is stretched by increased blood volume

• Carried in the blood to the kidney, ANF activates guanylyl cyclase in cells of the collecting ducts

• The resulting rise in [cGMP] triggers increased renal excretion of Na+ and, consequently, of water, driven by the change in osmotic pressure

• Water loss reduces the blood volume, countering the stimulus that initially led to ANF secretion

• Vascular smooth muscle also has an ANF receptor—guanylyl cyclase; on binding to this receptor, ANF causes vasodilation, which increases blood flow while decreasing blood pressure

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• A similar receptor guanylyl cyclase in the plasma membrane of intestinal epithelial cells is activated by an intestinal peptide, guanylin, which regulates Cl- secretion in the intestine

• This receptor is also the target of a heat-stable peptide endotoxin produced by E. coli and other gram-negative bacteria

• The elevation in [cGMP] caused by the endotoxin increases Cl- secretion and consequently decreases reabsorption of water by the intestinal epithelium, producing diarrhea

• The soluble guanylyl cyclases are regulated by the second messenger NO

• They have a heme group that confers NO-sensitivity. NO binding to the heme group results in activation of the guanylyl cyclase activity

• Acetylcholine is a vasodilator that acts by causing relaxation of the smooth muscle of blood vessels

• However, it does not act directly on smooth muscle

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• If endothelial cells are stripped away from underlying smooth muscle cells, acetylcholine no longer exerts its vasodilator effect

• This indicates that vasodilators such as acetylcholine initially interact with the endothelial cells of small blood vessels via receptors

• The receptors are coupled to the phosphoinositide cycle, leading to the intracellular release of Ca2+

through the action of inositol trisphosphate• In turn, the elevation of Ca2+ leads to the liberation

of NO also known as endothelium-derived relaxing factor (EDRF), which diffuses into the adjacent smooth muscle

• This leads to the elevation of intracellular levels of cGMP which in turn stimulates the activities of certain PKG, which probably phosphorylate specific muscle proteins, causing relaxation; however, the details are still being clarified

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• In the heart, cGMP reduces the forcefulness of contractions by stimulating the ion pump(s) that expel Ca+2 from the cytosol

• This NO-induced relaxation of cardiac muscle is the same response brought about by nitroglycerin tablets and other nitrovasodilators taken to relieve angina, the pain caused by contraction of a heart deprived of O2 because of blocked coronary arteries

• Another important cardiovascular effect of NO is that by increasing synthesis of cGMP, it acts as an inhibitor of platelet aggregation

• NO is unstable and its action is brief; within seconds of its formation, it undergoes oxidation to nitrite, nitrate or peroxynitrite

• Nitrovasodilators produce long-lasting relaxation of cardiac muscle because they break down over several hours, yielding a steady stream of NO

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• The effects of increased cGMP synthesis diminish after the stimulus ceases, because a specific phosphodiesterase (cGMP PDE) converts cGMP to the inactive 5-GMP

• Humans have several isoforms of cGMP PDE, with different tissue distributions

• The isoform in the blood vessels of the penis is inhibited by the drug sildenafil citrate (Viagra), which therefore causes cGMP levels to remain elevated once raised by an appropriate stimulus, accounting for the usefulness of this drug in the treatment of erectile dysfunction

• NO is inhibited by hemoglobin and other heme proteins, which bind it tightly

• Administration of NO synthase inhibitors to animals and humans leads to vasoconstriction and a marked elevation of blood pressure, indicating that NO is of major importance in the maintenance of blood pressure in vivo

47The Synthesis and (One) Action of NO

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Inositol phospholipids and inositol phosphates• Inositol-containing phospholipids of the plasma

membrane are the starting compounds for the formation of various inositol messengers in response to various signals

• These messengers include the central second messengers diacylglycerol (DAG) and inositol trisphosphate (IP3) as well as membrane-bound phosphatidyl inositol phosphates (e.g. PIP3)

• Phosphatidylinositol is first phosphorylated by specific kinases at the 4’ and 5’ positions of the inositol residue, leading to the formation of phosphatidyl inositol-4,5-bisphosphate (PIP2)

• From PIP2, two paths lead to physiologically important messenger substances

• One path is phosphorylation to yield PIP3 , which functions as a membrane-localized messenger

• The other option is cleavage by phospholipase C, forming the second messengers DAG and IP3

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• IP3 activates the release of Ca2+ , while DAG acts primarily by stimulation of protein kinase C (PKC)

• Phospholipase C can be activated by G-proteins or by transmembrane receptors with intrinsic or associated enzymatic activity

• Ca2+ is a ubiquitous signaling molecule whose signaling function is activated by its release from intracellular stores or through Ca2+ entry channels from the extracellular side

• The concentration of free Ca2+ in the cytosol of resting cells is very low, about 10–7 M

• One reason that the cell tries to keep the free Ca2+

concentration low is the ability of these ions to form poorly soluble complexes with inorganic phosphate

• The low concentration of free cytosolic Ca2+ is opposed by a large storage capacity for Ca2+ in specific organelles and vesicles and by a high concentration in the extracellular region where Ca2+

is present at millimolar concentration

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• Storage sites include endoplasmic reticulum, mitochondria and calciosomes

• In the endoplasmic reticulum, Ca2+ exists in complex with the storage protein calreticulin

• In the protein-bound and compartmentalized form, Ca2+ is not freely available but may be released in the process of signal transduction

• In muscle cells, Ca2+ is stored in the sarcoplasmic reticulum

• The storage takes place particularly by binding to the storage protein calsequestrin. Ca2+ is released from storage by a neural stimulus and initiates muscle contraction

• Mobilization of Ca2+ from the Ca2+ stores of the endoplasmic reticulum takes place with the help of Ca2+ channels, of which two types stand out: the IP3 receptors and the ryanodine receptors

• Ca2+ enters from the extracellular space through either voltage-gated or ligand-gated channels

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• One of the primary functions of Ca2+ entry into cells is to charge up the internal stores, which can then release an internal Ca2+ signal

• Ca+2 that has entered from the extracellular space plays a role in the opening of IP3 and ryanodine receptors

• Overall, multiple pathways can be used for mobilizing Ca2+ from the internal stores

• A Ca2+ signaling ‘toolkit’ is available from which cells can select specific components to activate the internal Ca2+ stores and to generate a variety of different Ca2+ signals that suit their physiology

• The cytosolic Ca2+ concentration is generally increased only temporarily and is often only locally increased during stimulation of cells

• The cell possesses efficient Ca2+ transport systems, which can rapidly transport Ca2+ back into the extracellular region or into the storage organelles

53Tools for Ca2+ Release

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• Ca2+ -ATPases, in particular, are involved in draining the cytosol of Ca2+ back into the extracellular region

• The Ca2+ -ATPases perform active transport of Ca2+ against its concentration gradient, using the hydrolysis of ATP as an energy source

• Other transport systems in the plasma membrane exchange Na+ ions for Ca2+ (use the energy of the Na+ gradient)

• These Na+-Ca2+

exchange proteins are located especially in muscle cells and in neurons

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• Examples of Ca2+ -dependent reactions are numerous and affect many important processes of the organism, including muscle contraction, vision process, cell proliferation, secretion, cell motility, formation of the cytoskeleton, exocytosis, gene expression, reactions of intermediary metabolism,…

• The information encoded in transient Ca2+ signals is deciphered by various intracellular Ca2+-binding proteins that convert the signal into a wide variety of biochemical changes

• There are two principle mechanisms by which Ca2+

can perform a regulatory function:1. Direct Activation of Proteins • There are many enzymes that have a specific Ca2+

-binding center in the active site and for which Ca2+ has an essential role in catalysis

• Examples of Ca2+ -dependent enzymes are phospholipase A2 and PKC (which is activated by the joint action of DAG and Ca2+ )

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2. Binding of Ca2+ Receptors • The Ca2+ receptors are Ca2+ sensing proteins that

activate target proteins in response to changes in Ca2+ concentration

• The most widespread Ca2+ receptor is calmodulin • Calmodulin is a small protein of ca. 150 amino

acids. It has got two globular domains each capable of binding 2 calcium ions

• The Ca2+/calmodulin complex is involved in in regulation of mitosis, neuronal signal transduction, muscle contraction, glucose metabolism,….

• Ca2+ receptors related to calmodulin include troponin C (which can only bind 2 Ca2+ ) and recoverin (which is involved in vision)

The Mechanism of Vision • The molecule that absorbs light in the eyes is cis-

retinal, which is linked covalently (via a lysine residue) to a protein, opsin

• In the rods, the complex of retinal and opsin is known as rhodopsin, which is also referred to as the photopigment

• There are also red, blue and green sensitive opsins in the cons

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Ca2+/Calmodulin Activation of Different Proteins

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• The absorption of photons by 11-cis-retinal converts it to the all trans-form which induces a conformational change in opsin• Upon this change, the retinal dissociates from the

opsin. Opsin is now free to initiate the sequence of events that leads to the detection of light• The opsin interacts with a membrane trimeric G-

protein, known as transducin, which results in an exchange GDP for GTP on the transducin• This activates the trimeric G-protein in the usual

way, i.e. by dissociation of the α-β-γ complex, which releases the α-subunit • The α-subunit activates an enzyme, cyclic GMP phosphodiesterase, which decreases the concentration of cGMP• In the dark, cGMP binds to a channel that allows the

entry of Na+ and Ca+2 and leads to depolarization• When the level of cGMP falls, the channels are

closed and the membrane is hyperpolarized

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Light-Induced Hyperpolarization of Rod

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• Hyperpolarization decreases the release of the neurotransmitter glutamate into the synapse that connects the photoreceptor cell to the bipolar neurons

• The decreased release of glutamate leads to the depolarization of the bipolar neurons

• The action potential created in the bipolar neurons is relayed to the visual cortex

• The visual signal is terminated through a combination of ways

The intrinsic GTPase activity of transducin means the cGMP phosphodiesterase is no longer activated; cGMP levels rise

When the levels of Ca2+ fall, the inhibition on guanylyl cyclase is relieved while cGMP PDE is inhibited

Rhodopsin is desensitized by rhodopsin kinase. The phosphorylated rhodopsin is bound by the protein arrestin

Recoverin inhibits rhodopsin kinase at high [Ca2+ ], but the inhibition is relieved when [Ca2+ ] drops after illumination

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62

• On a relatively long time scale (seconds to minutes), the all-trans-retinal of an excited rhodopsin molecule is removed and replaced by 11-cis-retinal, to produce rhodopsin that is ready for another round of excitation

• If properly dark adapted, the eye can detect a single photon

• Vitamin A deficiency reduces the amount of rhodopsin in the retina, increasing the minimum amount of illumination that can be detected (the visual threshold) and causing night blindness

The

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Transmembrane Receptors and Tyrosine Kinase Activity

• PKA, PKC, PKG, Ca2+/calmodulin-dependent kinases, MLCK, glycogen phosphorylase kinase, rhodopsin kinase and numerous other kinases are Ser/Thr kinases

• In addition, multicellular organisms communicate through the binding of protein ligands to transmembrane receptors that are dependent of Tyr kinase activity

• Some transmembrane receptors possess intrinsic tyrosine kinase activity; these receptors are known as receptor tyrosine kinases

• Ligand binding to an extracellular domain of the receptor is coupled to the stimulation of tyrosine kinase activity localized on a cytoplasmic receptor domain

• The ligand-binding domain and the tyrosine kinase domain are part of one and the same protein

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• Another type of transmembrane receptor is associated, on the cytoplasmic side, with a tyrosine kinase that is activated when a ligand binds to the extracellular receptor

• The tyrosine kinase and the receptor are not located on the same protein in this case

Intr

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• Activation of tyrosine-specific protein kinase activity is triggered, in particular, by signals that control cell growth and differentiation

• Ligand binding to the extracellular portions of the receptor tyrosine kinase induces the non-covalent oligomerization – mostly dimerization – of monomeric receptors, or it induces a structural rearrangement in a preassembled oligomeric receptor facilitating tyrosine autophosphorylation

• For ligand-induced dimerization, two pathways have been described

• In the first pathway, which applies for the growth hormone receptor and the erythropoietin receptor, the ligand has two binding sites for the receptor molecule and brings about a dimerization of the receptor

• In the absence of the ligand, the receptor exists in a monomeric form

• In another pathway, two ligands are needed (e.g. EGF)

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• In the case of the insulin receptor, the bound ligand appears to stabilize a distinct conformation of the pre-assembled oligomeric form of the receptor, fixing the receptor in a catalytically active state

• The insulin receptor is a heterotetrameric protein composed of two αβ-units linked by disulfide bridges

• The binding of insulin brings about a change in the relative configuration of the two tyrosine kinase domains, in such a way that mutual Tyr phosphorylation is enabled

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• In the absence of the ligand, the two active sites are thought to be too distant for this trans-phosphorylation

Effector Proteins of Receptor Tyrosine Kinases • Autophosphorylation of receptor tyrosine kinases

has a double effect• The tyrosine kinase activity undergoes

autoactivation by phosphorylation of Tyr residues localized in the catalytic domain

• In addition, Tyr residues that lie outside the active center are phosphorylated

Aut

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• The phosphotyrosine residues thereby created serve as binding sites for effector molecules next in the sequence of the signal transduction pathway

• The phosphotyrosine residues of the activated receptors are attachment points for effector proteins that possess a phosphotyrosine-binding domain, such as the SH2 domain (the Src homology 2 domain, named for the first protein in which it was found, the src protein of the Rous sarcoma virus)

• The docking of signaling proteins to autophosphorylation sites provides a mechanism for assembly and recruitment of signaling complexes

• Many of the signaling pathways activated by receptor tyrosine kinases ultimately lead to the activation of transcription factors, influencing central differentiation and developmental programs of the cell

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Insulin Signaling- example of intrinsic tyrosine kinase activity

• The activated phosphorylated insulin receptor binds a protein called IRS-1 (insulin receptor substrate-1)

• The activated receptor kinase phosphorylates IRS-1 at multiple sites, creating multiple binding sites for different proteins with SH2 domains

• One of the phosphotyrosine sites of IRS binds the SH2 domain of Grb2

• Grb2 is also anchored to PIP3 in the plasma membrane through its PH (pleckstrin homology) domain

• Grb 2 has got a third domain (SH3) which binds with Sos

• Sos catalyzes the replacement of bound GDP by GTP on Ras, a G protein

• When GTP is bound, Ras can activate a protein kinase, Raf-1 , the first of three protein kinases—Raf-1, MEK, and ERK—that form a cascade in which each kinase activates the next by phosphorylation

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Regulation of Gene

Expression by Insulin

71

• When Erk is activated, it mediates some of the biological effects of insulin by entering the nucleus and phosphorylating proteins such as Elk1, which modulates the transcription of about 100 insulin-regulated genes

• ERK is a member of the MAPK family (mitogen-activated protein kinases; mitogens are signals that act from outside the cell to induce mitosis and cell growth)

• The above is the pathway insulin follows to exert its effects on gene transcription

• Below are the ways insulin affects cytosolic processes

• At other phosphotyrosine sites on IRS, phospholipase C and PI 3-kinase bind using their SH2 domains

• Phospholipase C leads to Ca2+ signaling • The signal pathway initiated by the insulin receptor

complex involving PI 3-kinase leads to activation of protein kinase B (PKB), a serine-threonine kinase that mediates many of the downstream effects of insulin

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Cytosolic Effects of Insulin

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• PI 3-kinase synthesizes PIP3 which serves as a docking site for PKB and PDK1 (phosphoinositide-dependent kinase-1)

• PDK1 phosphorylates and activates PKB• PKB phosphorylates Ser and Thr residues on target

proteins like glycogen synthase kinase (GSK ) 3, and Glut 4; this favors glycogen synthesis and entry of glucose into the cells

• PKB also functions in several other signaling pathways, including that triggered by 9-tetrahydrocannabinol (THC), the active ingredient of marijuana and hashish

• THC activates the CB1 receptor in the plasma membrane of neurons in the brain

• One consequence of CB1 activation is the stimulation of appetite, one of the well-established effects of marijuana use

• The normal ligands for the CB1 receptor are endocannabinoids such as anandamide, which serve to protect the brain from the toxicity of excessive neuronal activity-as in an epileptic seizure

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The Cytokine Receptors -examples of associated tyrosine kinase activity

• The large family of cytokine receptors includes receptors for many kinds of local mediators (collectively called cytokines), as well as receptors for some hormones, such as growth hormone and prolactin

• These receptors are stably associated with cytoplasmic tyrosine kinases called Janus kinases (JAKs) (after the two-faced Roman god), which phosphorylate and activate gene regulatory proteins called STATs (signal transducers and activators of transcription)

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• STAT proteins are located in the cytosol and are referred to as latent gene regulatory proteins because they only migrate into the nucleus and regulate gene transcription after they are activated

• Although many intracellular signaling pathways lead from cell-surface receptors to the nucleus, where they alter gene transcription, the JAK–STAT signaling pathway provides one of the more direct routes

• Cytokine receptors are dimers or trimers and are stably associated with one or two of the four known JAKs (JAK1, JAK2, JAK3, and Tyk2)

• Cytokine binding alters the arrangement so as to bring two JAKs into close proximity so that they transphosphorylate each other, thereby increasing the activity of their tyrosine kinase domains

• The JAKs then phosphorylate tyrosines on the cytokine receptors, creating phosphotyrosine docking sites for STATs

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• Some adaptor proteins can also bind to some of these sites and couple cytokine receptors to the MAPK pathway

• There are at least six STATs in mammals. Each has an SH2 domain that performs two functions

• First, it mediates the binding of the STAT protein to a phosphotyrosine docking site on an activated cytokine receptor

• Once bound, the JAKs phosphorylate the STAT on tyrosines, causing the STAT to dissociate from the receptor

• Second, the SH2 domain on the released STAT now mediates its binding to a phosphotyrosine on another STAT molecule, forming either a STAT homodimer or a heterodimer

• The STAT dimer then translocates to the nucleus, where, in combination with other gene regulatory proteins, it binds to a specific DNA response element in various genes and stimulates their transcription

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Receptor Serine/Threonine Kinases• The transforming growth factor-β (TGF β)

superfamily consists of a large number of structurally related, secreted, dimeric proteins

• They act either as hormones or, more commonly, as local mediators to regulate a wide range of biological functions in all animals

• During development, they regulate pattern formation and influence various cell behaviors, including proliferation, differentiation, extracellular matrix production, and cell death

• In adults, they are involved in tissue repair and in immune regulation, as well as in many other processes

• All of these proteins act through enzyme-coupled receptors that have a serine/threonine kinase domain on the cytosolic side of the plasma membrane

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• There are two classes of these receptor serine/threonine kinases—type I and type II—which are structurally similar homodimers

• Each member of the TGFβ superfamily binds to a characteristic combination of type-I and type-II receptor dimers, bringing the kinase domains together so that the type-II receptor can phosphorylate and activate the type-I receptor, forming an active tetrameric receptor complex

• Once activated, the receptor complex uses a strategy for rapidly relaying the signal to the nucleus that is very similar to the JAK–STAT strategy used by cytokine receptors

• The activated type-I receptor directly binds and phosphorylates a latent gene regulatory protein of the Smad family (named after the first two identified, Sma in C. elegans and Mad in Drosophila)

• The Smads bound by receptors are Smads 1, 2, 3, 5 and 8 and are known as receptor Smads (R-Smads)

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The Smad Signaling Pathway

81

• Once one of these R-Smads has been phosphorylated, it dissociates from the receptor and binds to Smad4 (called a co-Smad), which can form a complex with any of the five R-Smads

• The Smad complex then translocates into the nucleus, where it associates with other gene regulatory proteins and regulates the transcription of specific target genes

SIGNAL TRANSDUCTION IN CELL BIRTH, DEATH AND CANCER

• Cells execute their reproduction in a cyclic process, in which at least two phases, S phase and M phase, can be differentiated on the basis of biochemical and morphological features

• The biochemical characteristic of S (synthesis) phase is the replication of nuclear DNA and thus doubling of the genetic information

• In M (mitosis) phase, division of the chromosomes between the daughter cells is prepared and carried out

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• In most cell types, two further phases can be distinguished, G1 and G2 phase

• G1 phase covers the period between M phase and S phase while G2 phase covers the period between S phase and M phase

• From G1 phase, the cell may transfer into a quiescent state known as G0 phase

• Appropriate signals (e. g., addition of growth factors) can induce the cell to return from G0 into G1 phase and proceed with the cell cycle

• Rapidly dividing cells in mammals require 12–24 h for completion of a cell cycle

• In some cell types, such as early embryonic cells, the period between the S and the M phases is reduced to the extent that discrete G1 and G2 phases cannot be identified. The duration of the cell cycle is then only 8–60 min

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• The cell-cycle control system is based on a connected series of biochemical switches, each of which initiates a specific cell-cycle event

• The cell cycle has three major regulatory transition points known as checkpoints

• The first checkpoint is Start (or the restriction point) in late G1, where the cell commits to cell-cycle entry and chromosome duplication

• The second is the G2/M checkpoint, where the control system triggers the early mitotic events that lead to chromosome alignment on the spindle in metaphase

• The third is the metaphase-to-anaphase transition, where the control system stimulates sister-chromatid separation, leading to the completion of mitosis and cytokinesis

• The control system blocks progression through each of these checkpoints if it detects problems inside or outside the cell

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• If the control system senses problems in the completion of DNA replication, for example, it will hold the cell at the G2/M checkpoint until those problems are solved

• Similarly, if extracellular conditions are not appropriate for cell proliferation, the control system blocks progression through Start, thereby preventing cell division until conditions become favorable

The Components of The Cell Cycle Control System

• The central components of the cell-cycle control system are members of a family of protein kinases known as cyclin-dependent kinases (Cdks)

• The activities of these kinases rise and fall as the cell progresses through the cycle, leading to cyclical changes in the phosphorylation of intracellular proteins that initiate or regulate the major events of the cell cycle

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• An increase in Cdk activity at the G2/M checkpoint, for example, increases the phosphorylation of proteins that control chromosome condensation, nuclear envelope breakdown, spindle assembly, and other events that occur at the onset of mitosis

• Cyclical changes in Cdk activity are controlled by a complex array of enzymes and other proteins that regulate these kinases

• The most important of these Cdk regulators are proteins known as cyclins

• Cdks, as their name implies, are dependent on cyclins for their activity: unless they are tightly bound to a cyclin, they have no protein kinase activity

• Cyclins were originally named because they undergo a cycle of synthesis and degradation in each cell cycle

• The levels of the Cdk , by contrast, are relatively constant

• There are four classes of cyclins, each defined by the stage of the cell cycle at which they bind Cdks and function

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• All eukaryotic cells require three of these classes:1. G1/S-cyclins activate Cdks in late G1and thereby

help trigger progression through Start, resulting in a commitment to cell-cycle entry. Their levels fall in S phase

2. S-cyclins bind Cdks soon after progression through Start and help stimulate chromosome duplication. S-cyclin levels remain elevated until mitosis, and these cyclins also contribute to the control of some early mitotic events

3. M-cyclins activate Cdks that stimulate entry into mitosis at the G2/M checkpoint. M-cyclins are destroyed in mid-mitosis

• In most cells, a fourth class of cyclins, the G1-cyclins, helps govern the activities of the G1/S cyclins

• There are four Cdks. Two interact with G1 cyclins, one with G1/S- and S-cyclins, and one with M-cyclins

• In the absence of cyclin, the active site in the Cdk protein is partly obscured by a slab of protein, like a stone blocking the entrance to a cave

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• Cyclin binding causes the slab to move away from the active site, resulting in partial activation of the Cdk

• Full activation of the cyclin-Cdk complex then occurs when a separate kinase, the Cdk-activating kinase (CAK), phosphorylates an amino acid near the entrance of the Cdk active site

• This causes a small conformational change that further increases the activity of the Cdk, allowing the kinase to phosphorylate its target proteins effectively and thereby induce specific cell-cycle events

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• Several additional mechanisms fine-tune Cdk activity at specific stages of the cycle

• Phosphorylation at a pair of amino acids in the roof of the kinase active site inhibits the activity of a cyclin-Cdk complex while dephosphorylation of these sites increases the activity

• The binding of Cdk inhibitor proteins (CKIs) also regulates cyclin-Cdk complexes

• CKI binding stimulates a large rearrangement in the structure of the Cdk active site, rendering it inactive

The Inhibition of Cdk

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Summary of The Cell Cycle Progression G1 progression • Following exit from mitosis, cells can enter a

quiescent state or they can continue in G1, which requires the presence of mitogenic signals in the form of growth hormones

• Signaling by growth hormones increases the level of D-type cyclins because of increased transcription

• The increase in D-type cyclins and the formation of cyclin D-CDK4/6 complexes has at least a twofold effect

• The metabolism and growth of the cells are stimulated and the cells are able to reach the critical size required for crossing of the restriction point

• Furthermore, the pRb (retinoblastoma protein) becomes initially phosphorylated by the cyclin D-CDK4/6 complexes, and cells are thus prepared to cross the restriction point

90

• In quiescent cells, Rb is complexed with E2F (a class of transcription factors), resulting in inhibition of these transcription factors

• Phosphorylation of Rb releases it from E2F, and E2F is then free to activate the transcription of genes required for entry into S

Activation of cyclin E/CDK2 and restriction point crossing

• As a consequence of the increased formation of cyclin D-CDK4/6 complexes, the inhibitor p27 is sequestered from complex formation with (and inhibition of) cyclin E-CDK2

• The now active cyclin E-CDK2 continues phosphorylation of pRb and thereby initiates transcription of E2F-responsive genes, among which is the gene for cyclin E

• Activation of cyclin E-CDK2 also requires dephosphorylation

• Now the requirements for restriction point crossing are fulfilled and the continued action of the E2F transcription factors provides for the enzymes that are necessary for entry into and progress through S phase

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Control of the G1/S Transition in the Cell Cycle

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S phase progression• Among the target genes of the E2F transcription

factors is the gene for cyclin A, which increases at the beginning of S phase

• The cyclin A-CDK2 and the cyclin E-CDK2 complexes are thought to phosphorylate important components of initiation complexes of DNA replication and thereby induce the transition of pre-replication complexes to the post-replicative state

• Shortly after entry into S phase, the cyclin E is targeted for degradation in the ubiquitin-proteasome pathway, and the activity of the cyclin E-CDK2 is shut off

• Further progress through S phase requires the continued action of cyclin A-CDK2 complexes

Cyclins and Corresponding Phases of the Cell Cycle

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G2/M transition and progress through M Phase• During S phase and G2 phase, the cyclin B-CDK1

complex (also known as the MPF, mitosis-promoting factor) accumulates in an inhibited phosphorylated state and is activated by the action of phosphatases at the G2/M transition

• The active cyclin B-CDK1 complex phosphorylates numerous substrates and is inactivated by proteolysis only at the end of M phase and during G1 phase

The Cell Cycle and the DNA Damage Response • Progression through the cell cycle, and thus the

rate of cell proliferation, is controlled not only by extracellular mitogens but also by other extracellular and intracellular mechanisms

• One of the most important influences is DNA damage, which can occur as a result of spontaneous chemical reactions in DNA, errors in DNA replication, or exposure to radiation or certain chemicals

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• It is essential that the cell repair damaged chromosomes before attempting to duplicate or segregate them

• The cell-cycle control system can readily detect DNA damage and arrest the cycle at either of two checkpoints-one at Start in late G1, which prevents entry into the cell cycle and into S phase, and one at the G2/M checkpoint, which prevents entry into mitosis

• DNA damage initiates a signaling pathway by activating one of a pair of related protein kinases called ATM and ATR, which associate with the site of damage and phosphorylate various target proteins, including two other protein kinases

• Together these various kinases phosphorylate other target proteins that lead to cell-cycle arrest

• A major target is the gene regulatory protein p53, which stimulates transcription of the gene encoding a Cdk inhibitory protein called p21

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• p21 binds to G1/S-Cdk and S-Cdk complexes and inhibits their activities, thereby helping to block entry into the cell cycle

• DNA damage activates p53 by an indirect mechanism

• In undamaged cells, p53 is highly unstable and is present at very low concentrations

• This is largely because it interacts with another protein, Mdm2, which acts as a ubiquitin ligase that targets p53 for destruction by proteasomes

• Phosphorylation of p53 after DNA damage reduces its binding to Mdm2

• This decreases p53 degradation, which results in a marked increase in p53 concentration in the cell

• In addition, the decreased binding to Mdm2 enhances the ability of p53 to stimulate gene transcription

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How DNA damage arreststhe Cell Cycle in G1

97

• A low level of DNA damage occurs in the normal life of any cell and this damage accumulates in the cell’s progeny if the damage response is not functioning

• Over the long term, the accumulation of genetic damage in cells lacking the DNA damage response leads to an increase in the frequency of cancer-promoting mutations

• Indeed, mutations in the p53 gene occur in at least half of all human cancers

• This loss of p53 function allows the cancer cell to accumulate mutations more readily

o What happens if DNA damage is so severe that it cannot be repaired?

• Animal cells with severe DNA damage do not attempt to continue division, but instead commit suicide by undergoing apoptosis

• Thus, unless the DNA damage is repaired, the DNA damage response can lead to either cell-cycle arrest or cell death

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• Many of the components of mitogenic signaling pathways are encoded by genes that were originally identified as cancer-promoting genes, or oncogenes because mutations in them contribute to the development of cancer

• The mutation of a single amino acid in the small GTPase Ras, for example, causes the protein to become permanently overactive, leading to constant stimulation of Ras-dependent signaling pathways, even in the absence of mitogenic stimulation

• However, when a hyperactivated form of Ras or Myc is experimentally overproduced in most normal cells, the result is not excessive proliferation but the opposite: the cells undergo either cell-cycle arrest or apoptosis.

• The normal cell seems able to detect abnormal mitogenic stimulation, and it responds by preventing further division

• Excessive mitogenic stimulation, often leads to the production of a cell-cycle inhibitor protein called Arf

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• Arf binds and inhibits Mdm2 –p53 will be activated • With this protective mechanisms in place, it seems

hard for cancer cells to arise• But the protective system is often inactivated in

cancer cells by mutations in the genes that encode essential components of the checkpoint responses, such as Arf or p53 or the proteins that help activate them

Excessive Mitogenic Stimulation and Cell Cycle

Arrest

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Apoptosis• Apoptosis depends on a family of proteases that

have a cysteine at their active site and cleave their target proteins at specific aspartic acids

• They are therefore called caspases (c for cysteine and asp for aspartic acid)

• Caspases are synthesized in the cell as inactive precursors, or procaspases, which are typically activated by proteolytic cleavage

• Procaspase cleavage is catalyzed by other already active caspases

• Once activated, caspases cleave, and thereby activate, other procaspases, resulting in an amplifying proteolytic cascade

• Some of the procaspases that operate in apoptosis act at the start of the proteolytic cascade and are called initiator procaspases; when activated, they cleave and activate downstream executioner procaspases

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• Executioner procaspases then cleave and activate other executioner procaspases as well as specific target proteins in the cell

• Among the many target proteins cleaved by executioner caspases are the nuclear lamins, the cleavage of which causes the irreversible breakdown of the nuclear lamina

• Another target is a protein that normally holds the DNA-degrading enzyme , endonuclease in an inactive form; its cleavage frees the endonuclease to cut up the DNA in the cell nucleus

• Other target proteins include components of the cytoskeleton and cell-cell adhesion proteins that attach cells to their neighbors

• The cleavage of these proteins helps the apoptotic cell to round up and detach from its neighbors, making it easier for a healthy neighboring cell to engulf it, or, in the case of an epithelial cell, for the neighbors to extrude it from the cell sheet

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Activation of Procaspases During Apoptosis

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• The two best understood signaling pathways that can activate a caspase cascade leading to apoptosis in mammalian cells are called the extrinsic pathway and the intrinsic pathway

The Intrinsic Pathway of Apoptosis• Cells can activate their apoptosis program from

inside the cell, usually in response to injury or other stresses, such as DNA damage or lack of oxygen, nutrients, or extracellular survival signals

• This intrinsic pathway depends on the release into the cytosol of mitochondrial proteins that normally reside in the intermembrane space of these organelles

• A crucial protein released from mitochondria in the intrinsic pathway is cytochrome c, a water-soluble component of the mitochondrial electron-transport chain

• When released into the cytosol, it has an entirely different function: it binds to a procaspase-activating adaptor protein called Apaf1 (apoptotic protease activating factor-1)

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• Apaf1 oligomerizes into a wheel-like heptamer called an apoptosome

• The Apaf1 proteins in the apoptosome then recruit initiator procaspase proteins (procaspase-9) which are activated by proximity in the apoptosome

• The activated caspase-9 molecules then activate downstream executioner procaspases to induce apoptosis

• A major class of intracellular regulators of apoptosis is the Bcl2 family of proteins

• Bcl2 proteins regulate the intrinsic pathway of apoptosis mainly by controlling the release of cytochrome c and other intermembrane mitochondrial proteins into the cytosol

• Some Bcl2 proteins are pro-apoptotic and promote apoptosis by enhancing the release, whereas others are anti-apoptotic and inhibit apoptosis by blocking the release

• p53 accumulated in response to irreparable DNA damage activates the transcription of genes for pro-apoptotic Bcl2

105The Intrinsic Pathway of Apoptosis

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The Extrinsic Pathway of Apoptosis• Extracellular signal proteins binding to cell-surface

death receptors trigger the extrinsic pathway of apoptosis

• Death receptors are transmembrane proteins containing an extracellular ligand-binding domain, a single transmembrane domain, and an intracellular death domain, which is required by the receptors to activate the apoptotic program

• The receptors are homotrimers and belong to the tumor necrosis factor (TNF) receptor family, which includes a receptor for TNF itself and the Fas death receptor

• The ligands that activate the death receptors are also homotrimers; they are structurally related to one another and belong to the TNF family of signal proteins

• A well-understood example of how death receptors trigger the extrinsic pathway of apoptosis is the activation of Fas on the surface of a target cell by Fas ligand on the surface of a killer (cytotoxic) lymphocyte

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• Fas has a central role in the physiological regulation of programmed cell death in the immune system, where it is mainly used to instruct lymphocytes to die during immune responses

• When activated by the binding of Fas ligand, the death domains on the cytosolic tails of the Fas death receptors recruit intracellular adaptor proteins, which in turn recruit initiator procaspases (procaspase-8, procaspase-10, or both), forming a death-inducing signaling complex (DISC)

• Once activated in the DISC, the initiator caspases activate downstream executioner procaspases to induce apoptosis

• In some circumstances, death receptors activate other intracellular signaling pathways that do not lead to apoptosis.

• TNF receptors, for example, can also activate the NFκ B pathway, which can promote cell survival and activate genes involved in inflammatory responses. Which responses dominate depends on the type of the cell and other signals acting on it

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The Extrinsic Pathway of Apoptosis

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The Molecular Basis of Cancer• Tumor cells have special features compared to

normal cells• The phenotype of a tumor cell is characterized by

the following characteristics: increased rate of cell division, loss of normal

growth control loss of ability to differentiate loss of contact inhibition increased capability for invasion of neighboring

tissue (metastasis) immortalization • The cells of a fully grown, aggressive tumor have

acquired these properties in a slow, multi-step process with the characteristics of cellular evolution

• This development is associated with a selection process, in the course of which, cells that have lost their growth-regulating mechanisms predominate

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• Tumor cells differ from their progenitor cells in having acquired a large number of genetic changes

• It is estimated that tumor cells accumulate several thousand to several hundred thousand changes in DNA sequence

• Two types of gene are associated with development of a tumor cell: oncogenes (onkos Greek for mass or bulk) and tumor suppressor genes

• Oncogenes encode proteins involved in the process of proliferation; that is, those involved in the cell cycle

• These proteins are synthesized in amounts or with an activity that accelerates progression through the cycle

• In contrast, tumor suppressor genes encode proteins that can decelerate or arrest progression through the cycle

• An oncogene has been compared with a car with a stuck accelerator: the car moves even if you take your foot off the accelerator. However, the car will stop if the brake is pressed (this is the effect of the tumor suppressor gene)

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• A mutation in or the loss of a tumor suppressor gene is analogous to a faulty brake, i.e. it no longer halts proliferation of the abnormal cell

Tumor Suppressors • There are two classes of tumor suppressor genes:

gatekeeper genes and caretaker genes• Gatekeeper genes directly prevent growth of

tumors by inhibiting cell division or promoting apoptosis. Examples are pRb and p53

• Caretakers have an indirect influence on tumor formation. These are susceptibility genes that indirectly suppress tumor formation by maintaining the integrity of the genome

• Impairment of the caretaker function will enhance the accumulation of mutations

• An important class of caretakers includes DNA repair enzymes

• Loss of tumour suppressor activity leads to production of a tumour in a homozygous recessive manner

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• This means that both alleles must be mutated for loss of control of the cell cycle to be lost and uncontrolled proliferation to occur

• At least 30 different tumour suppressor genes have been identified

• The mutations that affect tumor suppressor genes are known as “loss-of-function” mutations

Oncogenes• Several genes are involved in the stimulation of

proliferation in a normal cell; that is, they express proteins that directly or indirectly regulate this process

• These proteins include growth factors, growth factor receptors, signaling proteins and transcription factors, which are organized into a sequence that links a growth factor to the process of proliferation

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• If a mutation occurs in any one of these genes which results in an increase in the activity of one of the proteins in this sequence, the risk of development of a tumour increases

• Such a mutated gene is termed an oncogene. The precursor gene for the oncogene is termed a proto-oncogene

• An oncogene is defined by its ability to transform cells in culture

• The proteins expressed by oncogenes are similar to those expressed by proto-oncogenes but are more active or less well controlled than the normal protein

• This means, the mutation in oncogenes is a “gain-of-function” mutation

• Oncogenes generally have dominant character• The mutation of a proto-oncogene to an oncogene

is phenotypically visible when only one of the two copies of the gene in a diploid chromosome set is affected by the mutation

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• Oncogenes were not originally discovered in tumour cells but, as an integral part of the genome of some retroviruses. When RNA, isolated from a retrovirus, was transfected into cells in culture, the cells were transformed

• During the process of transcription and production of viral genomic RNA, a portion of the host DNA sequence is sometimes incorporated into the viral genome

• This host genetic sequence may contain a proto-oncogene (c-onc), which is then subject to a mutation that accompanies viral replication

• During a subsequent round of viral infection, the mutated proto-oncogene sequence (now an oncogene, v-onc) will be inserted into the DNA of the host cell

• Proto-oncogenes can also be converted, via activating mutations, into oncogenes, without the involvement of viruses

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• Radiation and chemical carcinogens act by causing a mutation in the regulatory region of a

gene, increasing the rate of production of the proto-oncogene protein, or

producing a mutation in the coding portion of the proto-oncogene that results in the synthesis of a protein of slightly different amino acid composition capable of transforming the cell

• The entire proto-oncogene or a portion of it may be transposed or translocated, that is, moved from one position in the genome to another

• In its new location, the proto-oncogene may be controlled by a more active promoter and, therefore, overexpressed (increased amounts of the protein product may be produced)

• If only a portion of the proto-oncogene is translocated, it may be expressed as a truncated protein with altered properties, or it may fuse with another gene and produce a fusion protein

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Transforming Mutations in Proto-Oncogenes

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• The truncated or fusion protein would be hyperactive and cause inappropriate cell growth

• The proto-oncogene may be amplified, so that multiple copies of the gene are produced in a single cell

• If more genes are active, more proto-oncogene protein will be produced, increasing the growth rate of the cells

Groups of Proto-Oncogenes Growth factors and growth factor receptors• If too much of a growth factor or a growth factor

receptor is produced, the target cells may respond by proliferating inappropriately

• Mutations may also lead to a receptor being stuck in the “on” position even in the absence of ligand

Signal transduction proteins • A common example is the gene for the GTPase

switch protein Ras. Ras is involved in the MAPK pathway

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• If the GTPase activity of Ras is blocked, downstream mitogenic signaling would continue unabated

Transcription factors • The MAPK pathway leads to the transcription of

Myc, Fos, Jun, that in turn activate the transcription of many proteins needed for cell division

• The ways in which transcription factors could be converted to oncogenes include stimulation of transcription of the gene encoding the transcription factor; increasing the rate of its translocation from the cytosol to the nucleus; activation of the transcription factor by phosphorylation or by increasing its affinity for DNA

Cyclins • Oncogenic activation of cyclins is mostly observed

for the D-type cyclins, which play a central role in the transition from Go to G1 and for G1 progression. Increased levels of D-type cyclins and of CDK4/6 activity are frequently found in tumors

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