Signaling Pathways That Control Gene Expression

Signaling Pathways That Control Gene Expression
Wailap Victor Ng National Yang Ming University Lodish  Berk  Kaiser  Krieger  scott  Bretscher  Ploegh  Matsudaira MOLECULAR CELL BIOLOGY SEVENTH EDITION Copyright © 2013 by W. H. Freeman and Company

General properties of signaling pathways
Many cells respond to multiple types of hormones and other signaling molecules Some mammalian cells express ~100 different types of cell-surface receptors, each of which binds a different ligand Eukaryotes have about a dozen classes of highly conserved cell-surface receptors, which activate several types of highly conserved intracellular signaling pathways

Extracellular signals can have short-term and/or long-term effects in responding cells
Short-term effects are usually triggered by modification of existing proteins or enzymes which lead to changes in cell function such as metabolism or movement Many extracellular signals also affect gene expression and thus induce long-term changes in cell function, and alterations in cell division and differentiation, which occur during development and cell fate determination Changes in gene expression also enables differentiated cells to respond to their environment by changing their shape, metabolism, or movement The pathways discussed in this chapter have been conserved throughout evolution and operate in much the same manner in flies, worm, and human.

Properties of genes affected by
signaling pathways Expression of any gene can be regulated by a single or multiple extracellular signals Many genes are regulated by multiple transcription factors which are activated or repressed by different intracellular signaling pathways

Signal transduction pathways can be grouped into several basic types based on the sequence of intracellular events

Several common cell-surface receptors and
PK1 PK2 PK3 PK Several common cell-surface receptors and signal transduction pathways that affect gene expression Figure 16.1 Several common cell-surface receptors and signal transduction pathways 6

There are 3 representative classes of receptors that activate receptor-associated protein kinases
Receptor tyrosine kinases (RTKs) TGF-β receptors Cytokine receptors

Receptor tyrosine kinases (RTKs) and cytokine receptors are two large classes of receptors that activate protein tyrosine kinases Human genome encodes ~90 protein tyrosine kinases which phosphorylate specific tyrosine residues on target proteins The phosphorylated targets can then activate one or more signal pathways The activated pathways regulate most aspects of cell proliferation, differentiation, survival, and metabolism

Properties of the two broad categories of receptors
that activate tyrosine kinases Receptor tyrosine kinases (RTKs) are those in which the tyrosine kinase enzyme is an intrinsic part of the receptor’s polypeptide chain Cytokine receptors are those in which the receptor and kinase (JAK) are proteins encoded by different genes yet bound tightly together Both classes of receptors activate similar intracellular signal transduction pathways (See next slide) The JAK-STAT signaling pathway transmits information from chemical signals outside the cell, through the cell membrane, and into gene promoters on the DNA in the cell nucleus, which causes DNA transcription and activity in the cell. The JAK-STAT system is a major signaling alternative to the second messenger system. The JAK-STAT system consists of three main components: (1) a receptor (2) Janus kinase (JAK) and (3) Signal Transducer and Activator of Transcription (STAT). Many JAK-STAT pathways are expressed in white blood cells, and are therefore involved in regulation of the immune system. The receptor is activated by a signal from interferon, interleukin, growth factors, or other chemical messengers.

Overview of signal transduction pathways triggered by receptors that activate protein tyrosine kinases Figure Overview of signal transduction pathways triggered by receptors that activate protein tyrosine kinases Both RTKs and cytokine receptors activate multiple signal transduction pathways that ultimately regulate transcription of genes. (a) In the most direct pathway, mainly employed by cytokine receptors, a STAT transcription factor binds to the activated receptor, becomes phosphorylated, moves to the nucleus, and directly activates transcription. (b) Binding of one type of adapter protein (GRB2 or SHC) to an activated receptor leads to activation of the Ras/MAP kinase pathway. (c, d) Two phosphoinositol pathways are triggered by recruitment of phospholipase Cγ and PI-3 kinase to the membrane. Elevated levels of Ca2+ and activated protein kinase B modulate the activity of transcription factors as well as of cytosolic proteins that are involved in metabolic pathways or cell movement or shape. e.g. Signal transducer and activator of transcription 3 (STAT3) 10

Receptor tyrosine kinases (RTKs)

Numerous factors that regulate cell division
and metabolism are ligands for RTK The signaling molecules are soluble or membrane-bound peptide or protein hormones Many of which were initially identified as growth factors such as nerve growth factor (NGF), platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), and epidermal growth factor (EGF) that control the division of specific types of cells Others, such as insulin regulates multiple genes that control sugar and lipid metabolism in liver, muscle, and adipose cells

How did RTKs and ligands identify in the past?
Many RTKs and ligands were identified in studies of human cancers associated with mutant forms of growth- factor receptors that stimulate proliferation even in the absence of growth factor Others had been identified through analysis of gene mutations that affect differentiation of certain cell types in C. elegans, Drosophila, and mouse

Structure of receptor tyrosine kinases
All RTKs have three essential components: an extracellular domain containing a ligand binding site a single hydrophobic transmembrane α helix a cytosolic segment with protein tyrosine kinase activity Kinase

Activation of receptor tyrosine kinases
Resting state – activation lip is unphosphorylated and assumes a conformation that blocks the kinase activity Ligand binding causes a conformation change that promotes formation of a functional dimeric receptor that leads to activation of its intrinsic kinase

Activation of receptor tyrosine kinases
Figure 16.3 General structure and activation of receptor tyrosine kinases (RTKs) The cytosolic domain of RTKs contains an intrinsic protein tyrosine kinase catalytic site. In the absence of ligand [1], RTKs generally exist as monomers with poorly active kinases. Ligand binding causes a conformational change that promotes formation of a functional dimeric receptor, bringing together two poorly active kinases that then phosphorylate each other on a tyrosine residue in the activation lip [2]. Phosphorylation causes the lip to move out of the kinase catalytic site, thus increasing the ability of ATP and the protein substrate to bind. The activated kinase then phosphorylates several tyrosine residues in the receptor’s cytosolic domain [3]. The resulting phosphotyrosines function as docking sites for various signal transduction proteins. * The resulting phosphotyrosines function as docking sites for various signal transduction proteins 16

Functional dimers can be formed in multiple ways
Monomeric ligands binding to two receptor monomers e.g., Binding of epidermal growth factor (EGF) to HER1 receptor (Next slide) Dimeric ligand binding bring two monomeric receptors together (Slide 16) Monomeric ligands bind to two receptor, which have already been linked together (e.g., disulfide bonds) in resting state. The binding leads to conformation change and activation of receptor kinase, e.g., insulin receptor, epidermal growth factor receptors

Ligand-induced dimerization of HER1, a human receptor for epidermal growth factor (EGF)
TGF-α (Transforming growth factor α; a member of EGF) HER1 Monomeric ligands binding to two receptor monomers Figure 16.4 Ligand-induced dimerization of HER1, a human receptor for epidermal growth factor (EGF) Schematic depiction of the extracellular and transmembrane domains of HER1, which is a tyrosine receptor kinase. Binding of one EGF molecule to a monomeric receptor causes an alteration in the structure of a loop between the two EGF-binding domains. Dimerization of two identical ligand-bound receptor monomers in the plane of the membrane occurs primarily through interactions between the two “activated” loop segments. Structure of the dimeric HER1 protein bound to transforming growth factor α (TGF-α), a member of the EGF family. The receptor’s extracellular domains are shown in blue; the transmembrane domain is shown in red as an alpha helix, but its structure is not known in detail. The two smaller TGF-α molecules are colored green. Note the interaction between the “activated” loop segments in the two receptor monomers. * Binding of EGF to its RTK triggers a conformation change in the receptor extracellular domain so that it “clamps” down on the ligand. This action pushes out a loop located between the two EGF-binding domains, and interactions between the two extended loop segments allow the formation of the functional receptor dimer. 18

Structure of EPO, a dimeric ligand, bound to the extracellular domain of an EPO receptor
Dimeric ligand binding to two receptor monomers Figure 16.9 EPO contains of 4 long conserved alpha helices folded together EPO receptor is a dimer of identical subunits Each EPO receptor extracellular domain consists of 2 subdomain, each with 7 conserved beta strands folded in a characteristic fashion Side chains of residues on 2 of the alpha helices in EPO (site 1) contact loops on one EpoR monomer, while residues on the two other Epo alpha helices (site 2) bind to the same loop segments in a second monomer, thereby stabilizing the dimeric receptor in a specific conformation The structures of other cytokines and their receptors are similar to Epo and EpoR 19

tyrosine kinase becomes activated
Once a receptor tyrosine kinase (RTK) is locked into a functional dimeric state, its associated tyrosine kinase becomes activated In the inactive monomeric state, the activation lip is localized in the active site of the kinase, blocking its activity so the kinase is in “off” state Ligand binding leads to conformational change, causing the activation lip to move out of the kinase active site and allowing the kinase to function

Activation of EGF receptor by EGF results in the formation of an asymmetric kinase domain dimer
Figure 16.6 Activation of EGF receptor by EGF results in the formation of an asymmetric kinase domain dimer In the inactive, monomeric state [1] the unstructured segment of the juxtamembrane domain (JM-B; green) binds to the upper, or N lobe of the kinase domain, causing a conformational change that positions the activation lips in the kinase active site and thus inhibits kinase activation. Receptor dimerization generates an asymmetric kinase dimer [2] such that the activator kinase binds the juxtamembrane segment of the receiver kinase, causing a conformation change that removes the activation lip from the kinase site of the receiver kinase, activating its kinase activity. [3] The active kinase then phosphorylates tyrosine residues (yellow circles) in the C-terminal segment of the receptor cytosolic domain. EGF (Epidermal growth factor) JM (Justamembrane domain) 21

Human epidermal growth factor receptors (EGFR or HER) are RTKs that bind members of the epidermal growth factor superfamily In human, the four members of the HER (human epidermal growth factor receptor) family are: HER1 – binds EGF, HB-EGF (heparin-binding EGF), and TGF-α HER2 – does not directly bind a ligand *** HER3 – binds neuregulins 1 and 2 (NRG1 and NRG2) HER4 – binds NRG1 and NRG2 and HB-EGF These receptors form homo- or heterodimeric receptors – HER1/HER1, HER2/HER1, HER2/HER3, and HER2/HER4 TGF-α (Tumor-derived or transforming growth factor alpha) Change: hetero-oligomers to heterodimers

The HER family of receptors and their ligands
EGF (Epidermal growth factor) HB-EGF (Heparin-binding EGF) TGF-α (Tumor-derived or transforming growth factor α) NRG1 and NRG2 (Neuregulins 1 and 2) Figure 16.7 HER3 has a very poor active kinase domain and can signal only when complexed with HER2 23

HER2 receptor and breast cancer
Understanding of the HERs has helped explain why a particular form of breast cancer is so dangerous Amplification of the HER2 gene occurs in ~25% of breast cancers – poor prognosis Overexpression of HER2 makes the tumor cells sensitive to growth stimulation by low levels of any member of the EGF family of growth factor Therapeutics: Monoclonal antibodies specific for HER2 reduce recurrence by ~50% in these patients, e.g., Trastuzumab (Herceptin) and Pertuzumab (Perjeta) Trastuzumab (Herceptin) and Pertuzumab (Perjeta) of Genentech mab: Monoclonal antibody

Cytokine receptors

Cytokine receptors – Cytokines influence development of many cell types
Cytokines form a family of relatively small (~160 a.a.), secreted signaling molecules that control growth and differentiation of specific types of cells. Examples: Prolactin – induces epithelial cells in mammary gland to differentiate into acinar cells that produce milk proteins and secrete them into the ducts Interleukins – essential for proliferation and functioning of T and B cells Interferons – produced and secreted by certain cell types following virus infection and act on nearby cells to induce enzymes that render them more resistant to virus infection Erythropoietin – induces proliferation and differentiation of erythroid progenitors to RBC (Next slide)

Erythropoietin and formation of red blood cells
In the absence of EPO, CFU-E undergo apoptosis Binding of EPO to its receptors on CFU-E induces the expression of several proteins which prevent programmed cell death Other EPO-induced proteins trigger the developmental program of 3~5 terminal cell divisions (Each CFU-E produces 30~100 RBC) Figure 16.8 CFU-E (Colony-forming units erythroid) are derived from hematopoietic stem cells, which also give rise to progenitors of other blood cells. In the absence of EPO, CFU-E undergo apoptosis Bind of EPO to its receptor 27

Binding of a cytokine to its receptor activates a tightly bound JAK protein tyrosine kinase
All cytokines are evolved from a common ancestral protein and have a similar tertiary structure consisting of four long conserved α helices folded together Likewise, the various cytokine receptors undoubtedly evolved from a single common ancestor since they have similar structure Cytokine receptors do not possess intrinsic enzymatic activity. JAK (Janus kinase) is tightly bound to the cytosolic domain of all cytokine receptors JAK family has 4 members (JAK1~3 and TYK2), each contains: an N-terminal receptor-binding domain an C-terminal kinase domain a middle domain that regulates kinase activity by an unknown mechanism The four JAK family members are: Janus kinase 1 (JAK1) Janus kinase 2 (JAK2) Janus kinase 3 (JAK3) Tyrosine kinase 2 (TYK2) 28

General structure and activation of cytokine receptors
Homodimer Figure General structure and activation of cytokine receptors The cytosolic domain of cytokine receptors binds tightly and irreversibly to a JAK protein kinase. In the absence of ligand [1], the receptors form a homodimer but the JAK kinase are poorly active. Ligand binding causes a conformation change that brings together the associated JAK kinase domains, which them phosphorylate each other on a tyrosine residue in the activation lip [2]. Downstream signaling [3] then proceeds in a manner similar to that from receptor tyrosine kinases. Ligand binding brought the associated JAKs close enough together so that one can phosphorylate the other on a critical tyrosine in the activation lip 29

Phosphotyrosine residues are the binding surfaces for multiple proteins with conserved domains
Once the RTK kinases or JAK kinases become activated, they first phosphorylated several tyrosine residues on the cytosolic domain of the receptor Several of these phosphorylated residues then served as binding sites for signal transduction proteins containing conserved phosphotyrosine-binding domain. Examples: SH2 domain PTB domain (Phosphotyrosine binding domain) SH2 (Src homology 2 domain which is homologous to a region of Src cytosolic tyrosine kinase encoded by the src gene)

residues in receptors or receptor-associated proteins
Recruitment of intracellular signal transduction proteins to the cell membrane by binding to phosphotyrosine residues in receptors or receptor-associated proteins Figure 16.12 PTB domain are often found on so called multidocking proteins, which serve as binding site for other signal transduction proteins IRS is a multidocking protein of several RTKs (e.g., insulin receptor) and cytokine receptors (e.g. IL-4 receptor) Multidocking proteins expand the number of intracellular signaling pathways that can be activated by the receptor 31

SH2 domains in action: JAK kinase activates STAT transcription factors
The JAK/STAT pathway operates downstream from all cytokine receptors and some RTKs All STAT proteins (STAT1, 2, 3, 4, 5A, 5B , and 6) contain: N-terminal DNA-binding domain C-terminal domain with a critical tyrosine residue SH2 domain that binds to one or more specific phosphotyrosine in the cytosolic domain of the receptors or the phosphorylated “critical tyrosine residue” on another STAT molecule Some examples of the molecules that use the JAK/STAT signaling pathway are colony-stimulating factor, prolactin, growth hormone, and many cytokines.

Activation and structure of STAT proteins
Inactive STAT monomers bound to phosphotyrosines on receptors are phosphorylated by receptor associated JAKs (or some RTK kinases) Then two STATs dimerize and translocate to the nucleus to activate transcription of genes with specific enhancers Figure (a) 33

Activation of the same intermediate signaling molecule (e. g
Activation of the same intermediate signaling molecule (e.g., STAT) by different cytokine receptors in different cells leads to the activation of different genes - Why? Since different cell types have unique complements of transcription factors and unique epigenetic modifications on their chromatin, the genes that are available to be activated by any STAT are also different

Multiple mechanisms down-regulate signaling from RTKs and cytokine receptors
Receptor-mediate endocytosis – Endocytosis of ligand- receptor complexes and their degradation in lysosomes is a principal way of reducing RTK and cytokine receptors on the cell surface, thus decreasing the sensitivity of cells to many peptide hormones Dephosphorylation of activated receptors – Signaling from cytokine receptors is terminated by phosphotyrosine phosphatase SHP1 (Short-term regulation) Signal blocking and protein degradation by several SOCS proteins (Long-term regulation)

Two mechanisms for terminating erythropoietin (EPO) receptor signal transduction
Figure 16.14 (a) Short-term regulation Binding of an SH2 domain in SHP1 to a particular phosphotyrosine in the activated receptor unmasks its phosphatase catalytic site and position it near the phosphotyrosine in the lip region of JAK2 Removal of the phosphate from this tyrosine inactivates the JAK kinase (b) Long-term regulation SOCS protein, whose expression is induced by STAT in Epo stimulated erythroid cells Binding of SOCS to phosphotyrosine residues on EpoR or JAK2 blocks binding of other signaling proteins The SOCS box can also target proteins such as JAK2 for degradation by ubiquitin-proteasome pathway * Similar mechanisms regulate signaling from other cytokine receptors * SOCS (suppressor of cytokine signaling proteins) refers to a family of genes involved in inhibiting the JAK-STAT signaling pathway. SOCS (Suppressor of cytokine signaling proteins) is a family of proteins involved in inhibiting the JAK-STAT signaling pathway 36

The Ras/MAP kinase pathway

The Ras/MAP kinase pathway
Almost all RTKs and cytokine receptors activate this pathway Ras protein, a monomeric (small) G protein belongs to the GTPase superfamily of intracellular switch proteins Activated Ras promotes formation, at membrane, of signal transduction complexes containing 3 sequentially acting protein kinases; e.g., Raf → MEK → MAPK → nucleus Among the targets for MAPK are transcription factors that regulate expression of proteins with important roles in the cell cycle and in differentiation

Ras is a GTPase switch protein that operates downstream of most RTKs and cytosine receptors
Unlike trimeric G protein, Ras is not directly linked to cell- surface receptors Activation of Ras requires adaptor proteins to transduce the signals from the activated receptors to Ras Active “on” state with a bound GTP and an inactive “off” state with a bound “GDP” Ras mutant: e.g., RasD contains a mutation at position 12, blocks the functional binding of GTPase accelerating protein (GAP) and thus “locks” Ras in the active GTP- bound state

Receptor tyrosine kinases and JAK kinases are linked to Ras by adapters proteins GRB and SOS
Human GRB proteins: GRB2, 7, 10 ,14, GRB: Growth factor receptor-bound protein (GRB2, 7, 10, 14) SOS: Son of sevenless homolog (SOS1 and SOS2) - Human

Activation of Ras following ligand binding to receptor tyrosine kinases (RTKs) [or cytokine receptors] Figure Activation of Ras following ligand binding to receptor tyrosine kinases (RTKs) or cytokine receptors The receptor for epidermal growth factor (EGF) and many other growth factors are RTKs. The cytosolic adapter proteins GRB2 binds to a specific phosphotyrosine on an activated, ligand-bound receptor and to the cytosolic Sos protein, bringing it near the plasma membrane and to its substrate, the inactive Ras·GDP. The guanine nucleotide exchange factor (GEF) of Sos then promotes formation of active Ras·GTP. Note that Ras is tethered to the cytosolic surface of the plasma membrane by a hydrophobic farnesyl anchor (a lipid group). GRB has SH2 and SH3 domains 41

change that triggers an exchange of GTP for GDP
Structures of Ras bound to GDP, Sos protein, and GTP – Binding of Sos to inactive Ras causes a conformation change that triggers an exchange of GTP for GDP Figure Structures of Ras bound to GDP, Sos protein, and GTP In Ras·GDP, the Swicth I (green) and Switch II (blue) segments do not interact directly with GDP. One alpha helix (brown) in Sos binds to both switch regions of Ras·GDP, leading to a massive conformation change in Ras. In effect, Sos pries Ras open by displacing the Switch I region, thereby allowing GDP to diffuse out. (c) GTP is thought to bind to the Ras-Sos complex first through its base (guanine); subsequently binding of GTP phosphates completes the interaction. The resulting conformation change in Switch I and Switch II segments of Ras, allowing both to bind to the GTP gamma phosphate, displaces Sos and promotes interaction Ras·GTP with its effectors. Ras 42

Ras and each kinase has multiple isoforms
The Ras/MAPK pathway Activation of Ras induces a cascade that includes three kinases: Raf → MEK (MAPKK) → MAP kinase (MAPK) Raf and MAPK are serine/threonine kinase MEK is a dual specific protein kinase that phosphorylates its target proteins on both tyrosine and serine/threonine Ras and each kinase has multiple isoforms Human has 3 RAS, 3 Raf, 2 MEK, 12 MAPK proteins, each has overlapping but also nonredundant functions MAPK (Mitogen-activated protein kinase) MAPKK (Mitogen-activated protein kinase kinase; MEK, or MAP2K) The acronym MEK comes from MAP and ERK kinase Mitogen-activated protein kinase kinase (also known as MAP2K, MEK, MAPKK) is a kinase enzyme which phosphorylates mitogen-activated protein kinase (MAPK). ERK was a microtubule-associated protein (MAP) kinase Extracellular-signal-regulated kinases (ERKs) Raf/Mek/Erk pathway Mitogen-activated protein kinase (MAPK)

Ras/MAP kinase pathway - Signals pass from activated Ras to a cascade of protein kinases, ending with MAPK Active MAPK translocates to nucleus; activates many transcription factors Figure 16.20 In unstimulated cells, binding of a dimer of the protein to Raf stabilizes it in an inactive conformation Interaction of the Raf N-terminal regulatory domain with Ras·GTP results in dephosphorylation of one of the serines that bind Raf to , phosphorylation of other residues, and activation of Raf kinase activity Dissociation of activated Raf → MEK MEK phosphorylate one threonine and one tyrosine on the activation lip of MAP kinase (MAPK), thereby activating its catalytic activity

Structures of inactive, unphosphorylated MAP kinase (MAPK) and the active, phosphorylated form –
Phosphorylation of activation lip results in a conformation change that enhances its catalytic activity and promote kinase dimerization Figure 16.21 Binding of MEK to MAP kinase destabilize the lip structure, resulting in exposure of tyrosine-185, which is buried in the inactive conformation. Following phosphorylation of this critical tyrosine, MEK phosphorylates the neighboring threonine-183 Both phosphorylated residues interact with additional amino acids, thereby conferring an altered conformation to the lip region, which in turn permit binding of ATP at the catalytic site (groove region) 45

MAP kinase (MAPK) regulates the activity of many transcription factors controlling early response genes Addition of a growth factor (e.g., EGF or PDGF) to quiescent culture mammalian cells causes a rapid increase in the expression of as many as 100 different genes These are called early response genes because they are induced before cells enter the S phase and replicate their DNA One important early response gene encodes the transcription factor c-Fos Together with other transcription factors, such as c-Jun, c-Fos induces expression of many protein genes necessary for cells to progress through the cell cycle Most RTKs that bind growth factors utilize the MAP kinase pathway to activate genes such as c-Fos

Induction of c-fos transcription by MAP kinase
SRF (Serum response factor) SRE (serum response element) TCF (Ternary complex factor) Figure Induction of gene transcription by MAP kinase Steps [1-3]: In the cytosol, MAP kinase phosphorylates and activates the kinase p90RSK, which then moves into the nucleus and phosphorylates the SRF (serum response factor) transcription factor. Steps [4] and [5]: After translocating into the nucleus, MAP kinase directly phosphorylates the transcription factor TCF (ternary complex factor) that is already bound to the promoter of the c-fos gene. Steps [6]: Phosphorylated TCF and SRF act together to stimulate transcription of genes (e.g., c-fos) that contain an SRE sequence in their promoter. Association of the phosphorylated TCF with two molecules of phosphorylated SRF forms an active trimeric factor that activate gene transcription. p90RSK: Ribosomal protein S6 kinase family 47

Multicellular animals MAP kinase (MAPK) is often activated by RTKs or cytokine receptors, but signaling from other receptors can also activate MAP kinase in different eukaryotic cells. Example: G protein-coupled receptors (GPCR) transmit signals to MAP kinase in yeast mating pathways Yeast has six MAP kinases, different receptors activate different MAP kinase pathways The mating in S. cerevisiae, a well-studied example of a MAP kinase cascade linked to GPCRs are induced by two secreted peptide pheromones (a and α factors)

Pheromone-induced mating of
haploid yeast cells The a and α type haploids cells synthesize and secrete the a and α mating factors, respectively Activation of the MAP kinase by either the a or α receptors (GPCRs) induce transcription of genes that inhibit progression of the cell cycle and others that enable the cells of opposite mating type to fuse together and ultimately form a diploid cell Figure Pheromone-induced mating of haploid yeast cells The α cells produce α mating factor and a-factor receptor; the a cells produce a factor and α-factor receptor. Both receptors are G protein-coupled receptors. Binding of the mating factors to their cognate receptors on cells of the opposite type leads to gene activation, resulting in mating and production of diploid cells. In the presence of sufficient nutrients, these cells will grow as disploids. Without sufficient nutrients, the cells will undergo meiosis and form four haploid spores. 49

Yeast MAP kinase cascades in the mating and osmoregulatory pathways are mediated by two different scaffold proteins Raf analog DIG 1 and 2 are inactivated via phosphorylation by MAPK Figure 16.24 (a) Activation of the MAP kinase by either the a or α receptors induce transcription of genes that inhibit progression of the cell cycle and others that enable the cells of opposite mating type to fuse together and ultimately form a diploid cell (b) Hog1 phosphorylates specific protein targets, including ion channels; after translocating to the nucleus, Hog1 phosphorylates several transcription factors and chromatin-modifying enzymes Guanine nucleotide exchange factor (GEF)

Scaffold proteins separate multiple
MAP kinase pathways in eukaryotic cells Different extracellular signals induce activation of different kinase pathways, which regulates diverse cellular processes by phosphorylating different sets of transcription factors The kinase components of each MAP kinase cascade assemble into a large pathway-specific complex stabilized by a scaffold protein (e.g., yeast Ste5 and Pbs2) Formation of such complex ensures that activation of one MAP kinase pathway by a particular extracellular signal does not lead to activation of other pathways containing shared components * Human has 3 RAS, 3 Raf, 2 MEK, and 12 MAPK proteins Human has 3 RAS, 3 Raf, 2 MEK, 2 ERK proteins, each has overlapping but also nonredundant functions

Phosphoinositide signaling pathways
Some RTKs and cytokine receptors initiate signaling pathways that involve special phosphorylated phospholipids derived from phosphatidyl inositol (PI): IP3/DAG pathway [involves activation of phospholipase Cγ (PLCγ) ] PI-3 kinase (PI3K) pathway These pathways end with a variety of kinases, including protein kinases C and B (PKC and PKB) that play key roles in cell growth and metabolism The phospholipase that is activated by GPCR is the beta isoform (PLCβ).

IP3/DAG pathway - Phospholipase Cγ (PLCγ) is activated by some RTKs and cytokine receptors to synthesize inositol 1,4,5- triphosphate (IP3) and DAG The SH2 domain of PLCγ binds to specific phosphotyrosines on the hormone activated receptors, that position the enzyme close to its membrane bound substrates The activated receptors phosphorylate PLCγ to promote its enzyme activity IP3 and DAG produced by PLCγ cleavage initiate the IP3/DAG pathway  PKC Figure 16.25 The phophoslipase C activated by GPCRs are the beta isoform of this enzyme 53

(Also known as PI3K/AKT or PI3K/AKT/mTOR pathway)
PI-3 kinase pathway (Also known as PI3K/AKT or PI3K/AKT/mTOR pathway) RTKs or cytokine receptors  PI-3Kinase  PKB IP3/DAG pathway Figure 16.25 The phophoslipase C activated by GPCRs are the beta isoform of this enzyme PI3K/AKT pathway 54

(Also known as PI3K/AKT or PI3K/AKT/mTOR pathway)
PI-3 kinase pathway (Also known as PI3K/AKT or PI3K/AKT/mTOR pathway) Many activated RTKs and cytokine receptors recruit PI-3 kinase to the membrane and activate the enzyme to generate PI 3,4-bisphosphate (PIP2) and PI 3,4,5-triphosphate (PIP3) * PI: phosphatidylinositol Figure 16.25 The phophoslipase C activated by GPCRs are the beta isoform of this enzyme 55

Accumulation of PI 3-phosphates in the plasma membrane leads to activation of several kinases
The 3-phosphate groups in PI 3,4-bisphosphate (PIP2) and PI 3,4,5-triphosphate (PIP3) provide the docking sites and activate many protein kinases with a PH domain One important kinase that binds to PI 3-phosphates is protein kinase B (PKB; a serine/threonine kinase; also known as Akt) The activated kinases in turn affect the activity of many cellular proteins PI-3 kinase pathway (PI3K/AKT pathway or PI3K/AKT/mTOR pathway) Pleckstrin homology domain (PH domain)

Recruitment and activation of protein kinase B (PKB)
PI-3 kinase pathway (PI3K/AKT or PI3K/AKT/mTOR pathway) - Recruitment and activation of protein kinase B (PKB) RTKs or cytokine receptors  PI-3Kinase  PKB T S Figure Recruitment and activation of protein kinase B (PKB) in PI-3 kinase pathway In unstimulated cells [1], PKB is in the cytosol with its PH domain bound to the catalytic kinase domain, inhibiting its activity. Hormone stimulation leads to activation of PI-3 kinase and subsequent formation of phosphatidylinositol (PI) 3-phosphate. The 3-phosphate group serves as docking sites on the plasma membrane for the PH domain of PKB [2] and another kinase, PDK1. Full activation of PKB requires phosphorylation both in the activation lip by PDK1 and at the C-terminus by a second kinase, PDK2 Activation by hormone stimulation ted PKB and PDK1 diffuse randomly in thPDK1 and PDK2 are recruited to phosphorylate PKB for maximal activation PDK1 is recruited to the plasma membrane via binding of its own PH domain to PI-3 phosphates. Both membrane-associate plane of the membrane, eventually bringing them close enough together so that PDK1 can phosphorylate PKB on a critical threonine residue in its activation lip. Phosphorylation of a second serine, in the C-terminus of PKB by PDK2 is necessary for maximum PKB activity. Similar to the regulation of Raf activity, release of an inhibitory domain and phosphorylation by other kinases regulate the activity of PKB. 57

Activated PKB ------| Bad & FOXO3a (pro-apoptotic)
PI-3 kinase pathway - Activated protein kinase B induces many cellular responses Once fully activated, PKB can dissociate from the plasma membrane and phosphorylate its many target proteins, which have a wide range of effects on cell behavior In many cells, activated PKB directly phosphorylates and inactivates pro-apoptotic proteins such as Bad, a short-term effect that prevents activation of apoptotic pathway It also promotes survival of many cultured cells by phosphorylating the Forkhead transcription factor FOXO3a, thereby reducing its ability to induce expression of several pro-apoptotic genes Anti-apoptosis Activated PKB | Bad & FOXO3a (pro-apoptotic) [Anti-apoptosis]

Negatively regulation of PI-3 kinase pathway
by PTEN phosphatase The PI-3 kinase pathway is negatively regulated by PTEN phosphatase which hydrolyzes the 3-phosphate in PI 3-phosphates Loss of PTEN, a common occurrence in human tumors, promotes cell survival and proliferation

TGF-β receptors (Receptor serine kinases)
TGF-β/Smad signaling pathway

TGF-β/Smad signaling pathway
The transforming growth factor β (TGF-β) superfamily includes a number of related extracellular signaling molecules that play widespread roles in regulating development TGF-β monomers are stored in an inactive form on the cell surface or in the extracellular matrix; release of active monomers (e.g., by protease digestion) leads to formation of functional homodimers or heterodimers TGF-β bind to conserved family of receptor serine kinases (i.e., the TGF-β receptor superfamily) The ligand-activated receptors phosphorylate and thus trigger the activation of one conserved class of transcription factors (Smads) TGF-β + TGF-β receptor  Smads

The principal function of all 3 human TGF-β isoforms: TGF-β1, 2, and 3, on most normal cells is to potently prevent their proliferation by inducing synthesis of proteins that inhibit the cell cycle via autocrine or paracrine signaling TGF-β proteins also promote expression of cell-adhesion molecules and extracellular-matrix molecules, which play important roles in tissue organization Loss of TGF-β receptors or any of the several intracellular signal transduction proteins in the TGF-β pathway occurs frequently in early development of human tumors TGF-β + TGF-β receptor  Smads  Protein synthesis --| Cell cycle (Autocrine/paracrine signaling)

Structure of TGF-β superfamily of signaling molecules
Figure Structure of TGF-β superfamily of signaling molecules In this ribbon diagram of a mature TGF-β, the two subunits are shown in green and blue. Disulfide-linked cysteine residues (yellow and red) are shown in ball-and –stick form. The three intrachain disulfide linkages (red) in each monomer form a cystine-knot domain, which is resistant to degradation. Red and yellow ball-and-stick form indicate the three intrachain and one interchain disulfide linkages, respectively 63

Three separate TGF-β receptor proteins (RI, RII, RIII)
participate in binding TGF-β and activating signal transduction In some cells, RIII, a transmembrane proteoglycan, binds and concentrate mature TGF-β molecules near the cell surface, facilitating their binding to RII In some cells, TGF-β binds directly to RII, a constitutively phosphorylated and active kinase Binding of TGF-β induces formation of complexes containing two copies each of RI and RII, which consist serine/threonine kinases as part of their cytosolic domains Proteoglycans are proteins that are heavily glycosylated. Proteoglycans represent a special class of glycoproteins where, because they are so heavily glycosylated, the balance has shifted toward carbohydrate in the nomenclature. The protein does play an important role as the core of the structure, with covalently attached carbohydrate polymers. The carbohydrate chains are negatively charged at neutral pH due to sulphate and uronic acid groups in proteoglycans.

TGF-β/Smad signaling pathway
3 types of Smad proteins function in the TGF-β signaling pathway: R-Smads (Receptor-regulated Smads: Smads 2 and 3) co-Smads (Smad 4) I-Smad (Inhibitory Smads) Ran GTPase PAI-1 (plasminogen activator inhibitor-1) NLS: Nuclear-localization signal Figure TGF-β/Smad signaling pathway Step [1a]: In some cells, TGF-β binds to type III TGF-β receptor (RIII), which increase the concentration of TGF-β near the cell surface and also presents TGF-β to the type II receptor (RII). Step [1b]: In other cells, TGF-β binds directly to RII, a constitutively phosphorylated and active kinase. Step [2]: Ligand bound RII recruits and phosphorylates the juxtamembrane segment of the type I receptor (RI), which does not directly bind TGF-β. This releases the inhibition of RI kinase activity that otherwise is imposed by the segment of RI between the membrane and its kinase domain. Step [3]: Activated RI then phosphorylates Smad2 or Smad3 (shown here as Smad2/3), causing a conformational change that unmasks its nuclear-localization signal (NLS). Step [4]: Two phosphorylated molecules of Smad2/3 bind to a co-Smad (Smad4) molecule, which is not phosphorylated, and with an importin, forming a large cytosolic complex. Step [5 & 6]: After the entire complex translocates into the nucleus, Ran·GTP causes dissociation of importin. Step [7]: A nuclear TF (e.g., TFE3) then associates with the Smad2/3/Smad4 complex, forming an activation complex that cooperatively binds in a precise geometry to regulatory sequences of a target gene. Step [8]: This complex then recruits transcriptional co-activators and induces gene transcription. Smad2/3 is dephosphorylated by a nuclear phosphatase (Step [9]) and recycles through a nuclear pore to the cytosol (Step [10]) , where it can be reactivated by another TGF-β receptor complex. Shown at bottom is the activation complex for the gene encoding plasminogen activator inhibitor (PAI-1), and similar transcriptional complexes activates expression of genes encoding other extracellular matrix proteins such as fibronectin. NLS (nuclear-localization signal) PAI-1 (plasminogen activator inhibitor-1) An RII then phosphorylates serine and threonine residues in a highly conserved sequence of the RI subunit, thereby activating the RI kinase activity Activated RI receptors phosphorylate Smad2 or 3, causing a conformation change to unmask its NLS ….. 65

Negative feedback loops regulate TGF-β/Smad signaling
Among the proteins induced by TGF-β stimulation are the I-Smad, especially Smad 7, blocks the ability of activated RI to phosphorylate Smad2/3, and it may target TGF-β receptor for degradation Oncoproteins (e.g., Ski and SnoN) act as negative regulators of the TGF-β signaling by inhibiting transcription mediated by the Sma2/3/Smad4 complex (next slide)

Model of Ski-mediated down-regulation of Smad transcription-activating function
TGF-β/Smad signaling pathway Figure Model of Ski-mediated down-regulation of Smad transcription-activating function Ski represses Smad function by binding directly to Smad4. Since the Ski-binding domain on Smad4 significantly overlaps with the Smad4 MH2 domain required for binding the phosphorylated tail of Smad3, binding of Ski to Smad4 disrupts the normal interactions between Smad3 and Sma4 necessary for transcription activation. In addition, Ski recruits the protein N-Cor, which binds directly to mSin3A; in turn, mSin3A interacts with histone deacetylase (HDAC), an enzyme that promotes histone deacetylation on nearby pomoters, repressing gene expressing. As a result of both processes, transcription activation induced by TGF-β and mediated by Smad complexes is shut down. The related protein SnoN functions similarly to Ski in repressing TGF-β signaling. Binding of Ski to Smad4 disrupts the normal interactions between Smad3 and Sma4 necessary for transcription activation and indirectly recruits histone deacetylase (HDAC) which deacetylates histones on nearby promoters SnoN functions similarly to Ski in repressing TGF-β signaling

Signaling pathways controlled by ubiquitination

Signaling pathways controlled by ubiquitination:
Wnt, Hedgehog, and NF-κB PK1 PK2 PK3 PK

Wnt and hedgehog signaling pathways
Wnt and hedgehog bind to receptors that are similar to seven-spanning GPCRs but do not activate G proteins Activation of each pathway involves disassembly of large cytosolic protein complexes, inhibition of ubiquitination, and release of the active transcription factor They play key roles in many developmental pathways and often induce expression of genes Resting state: Key transcription factor in both pathways are ubiquitinated and targeted for proteolytic cleavage Kinases including glycogen synthase kinase 3 (GSK3) play key roles in both signaling pathways Activation of the Wnt pathway controls numerous critical development events, such as brain development, limb patterning, and organogenesis. (Slide-72)

Wnt signaling pathways
Canonical Wnt signaling pathway (Wnt/β-catenin signaling pathway) Non-canonical Wnt signaling pathway (Atypical WNT- Frizzled signaling pathway or Wnt/Ca2+ pathway) Humans make at least 19 distinct Wnt species and 10 Fizzled receptors

Wnt signaling molecules
Wnts are secreted extracellular proteins with a palmitate group near their N-termini to tether Wnt lipoproteins to the plasma membrane of the Wnt-secreting cells, thus limiting their range of action to adjacent cells Wnts bind to the receptor Frizzled (Fz) & co-receptor LRP (Lipoprotein receptor-related protein) The central player in intracellular Wnt signal transduction is β-catenin (vertebrates) and Armadillo (Drosophila) β-catenin functions as a transcription activator and as a membrane-cytoskeleton linker protein Palmitate contains palmitic acid, a 16-carbon saturated fatty acid. lipoprotein receptor-related protein (LRP)

\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\
Wnt/β-catennin signaling pathway – Wnt signaling triggers release of a transcription factor (β-catennin) from a cytosolic protein complex \\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\ Axin: Scaffold protein CK1: Casein kinase 1 GSK3: Glycogen synthase kinase 3 APC: Adenomatous polyposis coli protein Gro: Groucher (represses TCF) Figure Wnt signaling pathway In the absence of WNT signal (Resting state) The transcription factor TCF bound to the enhancer is repressed by Groucher (Gro). The β-catenin that is not attached to cell-adhesion molecules in the membrane or cytoskeleton is bound to a complex based of the scaffold protein Axin and containing the adenomatous polyposis coli (APC) protein. Casein kinase 1 (CK1) and Glycogen synthase kinase 3 (GSK-3) sequentially phosphorylate β-catenin on multiple serine and threonine residues. Some of the phosphorylated residues serve as the binding sites for a TrcP. β-catenin is then uniquitinated and rapidly degraded by the 26S proteasome. In the presence of Wnt Complete pathway has not been identified. Wnt binding to both Fz and LRP leads to phosphorylation of the LRP cytosolic domain, probably by free GSK3 and CK1 This enable Axin to bind to the cytosolic domain of the LPR co-receptor This shift in Axin localization disrupt the interactions that stabilize the cytosolic complex containing Axin, GSK3, CK1, APC and β-catenin and thus prevent the phosphorylation of β-catenin by CK1 and GSK3, which in turn prevent ubiquitination and degradation of β-catenin. The process requires the dishevelled (Dsh) protein binding to the cytosolic domain of Fz. The free β-catenin translocate to the nucleus, where it associates with a transcription factor (TCF) and functions as a co-activator to induce the expression of particular target genes. Inappropriate activation of this pathway plays a critical role in cancer pathogenesis

Non-canonical Wnt signaling via different subsets of Wnt signals and Fizzled receptors
This pathway control various cancer cell phenotypes, including motility, invasiveness, and maintenance of self-renewal potential

Activation of Wnt signaling pathway controls numerous critical developmental events
in human including: Control formation of osteoblasts (bone-forming cells) and stem cells Brain development, limb patterning, and organogenesis Inappropriate activation of Wnt pathway is a characteristic of many human cancers Delta is one of the ligands for Notch

Hedgehog (Hh) pathway In mammals Hh signaling is restricted to the primary cilium that protrudes from the cell surface (see Slide 79) Similar to Wnt signaling, two membrane receptors [Patched (Ptc) and Smoothened (Smo)] are required to receive & transduce a signal Hh pathway also involves disassembly of an intracellular complex containing a transcription factor, which relieves repression of target genes (Unlike Wnt, the Hh protein undergoes distinctive post-translation modification, and the two membrane receptors move between the plasma membrane and intracellular vesicle membrane) Inappropriate activation of Hh signaling is the cause of several types of human tumors

Hedgehog (Hh) is a morphogen
Signals that induce different cell fates depending on their concentration at their target cells are referred to as morphogens Although Hh is a secreted protein, it moves only a short distance from a signaling cell, on the order of 1 to 20 cells, and is bound by receptor on receiving cells As Hh diffuses farther and farther away from secreting cells, its concentration decreases, and different Hh concentrations induce different fates in target cells

Processing of Hedgehog (Hh) precursor protein
25 kDa The two attached hydrophobic groups may cause secreted Hedgehog to bind nonspecifically and reversibly to cell plasma membranes, thereby limiting its diffusion and so its range of action in tissues Figure 16.31 Cell synthesize a 45-kDa Hh precursor, which undergoes a nucleophillic attach by the thiol side chain of C-258 on the carbonyl carbon of G-257, forming a high-energy thioester intermediate. The enzymatic activity in the C-terminal domain catalyzes the formation of an ester bond between the beta-3 hydroxyl group of cholesterol and glycine-257, CLEAVING the precursor into two fragments. The N-terminal fragment retains the cholesterol moiety and is also modified by addition of a palmitoyl group to the N-terminus The two attached hydrophobic groups may cause secreted Hedgehog to bind nonspecifically and reversibly to cell plasma membranes, thereby limiting its diffusion and so its range of action in tissues. 78

Hedgehog (Hh) signaling in vertebrates
This involves primary cilia, long plasma membrane- enveloped structures that protrude from the cell surface Mammalian genomes contain 3 hh genes and 2 ptc genes, which expressed differentially among various tissues Mammals express three Gli transcription factors 2 ptc genes: Ptch1 and Ptch2 receptors

Primary cilia Most cells have a single immotile cilium called the primary cilium Primary cilium plays crucial roles in vertebrate development and human genetic diseases Different intraflagellar transport (IFT) proteins move proteins and particles from the base of the cilium to the tip and in the opposite direction

Hedgehog signaling in vertebrates occurs in primary cilia
-Hh KIF7: Kinesin KIF7 motor protein +Hh IFT (Intraflagellar transport proteins) Figure Hedgehog signaling in vertebrates Hedgehog (Hh) signaling occurs in primary cilia, but otherwise the overall process is similar to that in flies. In the absence of Hh, Patched is localized to the ciliary membrane and in an unknown manner blocks the entry of Smoothened into cilia; Smo is present mainly in the membrane of internal vesicles. The kinesin KIF7 (the Cos2 homolog) binds to microtubules at the cilium base, where it may form a complex with the transcription factor Gli (the vertebrate homology of Ci), SuFu, and kinases. KIF7 prevents Gli enrichment within the cilium and promotes proteolytic processing of Gli to the Gli repressor GliR. Hh binding triggers movement of Smo to the ciliary membrane and movement of the KIF7 motor protein up to the microtubule to the ciliary tip, where Gli accumulates and gets activated by an as yet unknown mechanism. Activated Gli is then transported down the cilium and is released into the cytosol. IFT (Intraflagellar transport proteins)

NF-κB signaling pathways
Unlike the Wnt and hedgehog signaling pathways, an inhibitor of a key transcription factor (NF-κB), is deactivated by ubiquitination when the pathway is activated In the resting state, NF-κB is sequestered in the cytosol bound to an inhibitor (I-κBα) Several stress-inducing conditions cause ubiquitination and immediate degradation of the inhibitor, allowing cells to respond immediately and vigorously by activating gene expression NF-κB (nuclear-factor kappa-light-chain enhancer of activated B cells)

NF-κB Pathway - Degradation of an inhibitor
protein (I-κBα) activates the NF-κB transcription NF-κB is the master transcriptional regulator of the immune system in mammals and is rapidly activated in immune-system cells in response to infections and inflammation In many immune cells, NF-κB stimulates transcription of >150 genes, including those encoding cytokines and chemokines The heterodimeric NF-κB (p65 and p50) subunits share a region of homology at their N-termini that is required for their dimerization and binding to DNA In resting state, I-κBα inhibitor binding to p65 and p50 masks their nuclear-localizing signals (NLS) and sequesters NF-κB in the cytosol

NF-κB signaling pathway – resting state
NF-κB (P50 and P65) Figure Activation of the NF-κB signaling pathway In resting cell, the dimeric transcription factor NF-κB (p50 and p65) is sequestered in the cytosol, bound to the inhibitor I-κBα. Step [1]: Activation of the trimeric I-κB kinase is stimulated by many agents, including virus infection, ionizing radiation, binding of the pro-inflammatory cytokines TNFα or IL-1 to their respective receptors, or activation of any of several Toll-like receptors by components of invading bacteria or fungi. Step [2]: The β subunit of the I-κB kinases then phosphorylates the inhibitor I-κBα, which then binds to an E3 ubiquitin ligase. Step [3 & 4]: Subsequent lysine 48-linked polyubiquitination of I-κBα targets it for degradation by proteasomes. Step [5]: The removal of I-κBα unmasks the nuclear localization signals (NLS) in both subunits of NF-κB , allowing their translocation to the nucleus. Step [6]: In the nucleus, NF-κB activates transcription of numerous target genes, including the gene encoding I-κBα, which acts to terminate signaling, and gene encoding various inflammatory cytokines. Turn off by a negative loop since one of the genes induced by NF-κB is I-κBα (inhibitor) 78

Activation of the NF-κB signaling pathway (1/2)
Figure Activation of the NF-κB signaling pathway In resting cell, the dimeric transcription factor NF-κB (p50 and p65) is sequestered in the cytosol, bound to the inhibitor I-κBα. Step [1]: Activation of the trimeric I-κB kinase is stimulated by many agents, including virus infection, ionizing radiation, binding of the pro-inflammatory cytokines TNFα or IL-1to their respective receptors, or activation of any of several Toll-like receptors by components of invading bacteria or fungi. Step [2]: The β subunit of the I-κB kinases then phosphorylates the inhibitor I-κBα, which then binds to an E3 ubiquitin ligase. Step [3 & 4]: Subsequent lysine 48-linked polyubiquitination of I-κBα targets it for degradation by proteasomes. Step [5]: The removal of I-κBα unmasks the nuclear localization signals (NLS) in both subunits of NF-κB, allowing their translocation to the nucleus. Step [6]: In the nucleus, NF-κB activates transcription of numerous target genes, including the gene encoding I-κBα, which acts to terminate signaling, and gene encoding various inflammatory cytokines. Turn off by a negative loop since one of the genes induced by NF-κB is I-κBα (inhibitor) Ubiquitin protein itself consists of 76 amino acids 79

Activation of the NF-κB signaling pathway (2/2)
Polyubiquitin chains serve as scaffolds linking IL-1 or Toll receptors to downstream proteins in the pathway IL-1 and Toll receptors lack enzymatic activity in their cytosolic domain to phosphorylate the I-κB kinase subunit I-κBβ TRAF, an E3 ubiquitin ligase, links the carboxyl terminus of one ubiquitin to lysine 63 (K63) on another to form the poly-K63- linked ubiquitin chains The ubiquitins act as scaffolds that bind proteins with a K63-binding domain, i.e., TAK1 and NEMO I-κBβ is then phosphorylated by TAK1, which in turn phosphorylate the inhibitor I- κBα Figure Activation of the NF-κB signaling pathway (b) Binding of IL-1β to IL-1 receptor triggers receptor oligomerization and recruitment of several proteins to the receptor cytosolic domain, including TRAF6, and E3 ubiquitin ligase, which catalyze synthesis of long lysine-63-linked polyubiquitin chains linked to TRAF6 and other proteins in the complex. The polyubiquitin chains function as a scaffold to recruit the kinase TAK1 and the NEMO subunit of the trimeric I-κB kinase complex. TAK1 then phosphorylates itself and the β subunit of the trimeric I-κB kinase complex, activating its kinase activity and enabling it to phosphorylate I-κBα. * TRAF6 (tumor necrosis-associated factor 6, E3 ubiquitin protein ligase) 86

Signaling pathways controlled by protein cleavage: Notch/Delta
A transcription factor is released by proteolytic cleavage after the activation of the pathway Delta is one of the ligands for Notch Notch also has other ligands, utilizing the same activation mechanism

Notch/Delta signaling pathway
Both the receptor (Notch) on responding and its ligand (Delta) on signaling cell surface are single-spanning transmembrane proteins Notch consists of two subunits derived from proteolytic cleavage of a precursor protein in Golgi complex In the absence of Delta, the extracellular subunit of Notch is noncovalently associated with it transmembrane-cytosolic subunit Delta is one of the ligands for Notch Notch also has other ligands, utilizing the same activation mechanism

On binding Delta, Notch receptor is sequentially cleaved by the ADAM10 (matrix metalloprotease) and the presenilin 1 (a subunit of a tetrameric γ-secretase complex), releasing a component transcription factor The location of Notch and Delta in different, adjacent cells is essential for the highly conserved and important cell differentiation process in both invertebrates and vertebrates, called lateral inhibition Delta is one of the ligands for Notch Notch also has other ligands, utilizing the same activation mechanism

Activation of Notch/Delta signaling pathway – two
sequential proteolytic cleavages release the TF Inside nucleus, the Notch segment interacts with several transcription factors to affect the expression of genes that in turn influence the determination of cell fate during development. Figure Notch/Delta signaling pathway In the absence of Delta, the extracellular subunit of Notch on a responding cell is noncovalently associated with it transmembrane-cytosolic subunit. When Notch binds to its ligand Delta on an adjacent signaling cell (Step [1]), Notch is first cleaved by the, matrix metalloprotease ADAM10, which is bound to the membrane, releasing the extracellular Notch segment (Step [2]). Next the nicastrin subunit of the four protein γ-secretase complex binds to the stump generated by ADAM 10, and the presumed protease, presenilin 1, catalyzes an intramembrane cleavage that releases the cytosolic segment of Notch (Step [3]). Following translocation to the nucleus, this Notch segment interacts with several transcription factors to affect the expression of genes that in turn influence the determination of cell fate during development (Step [4]). Notch and most of its ligands are transmembrane proteins, so the cells expressing the ligands typically must be adjacent to the notch expressing cell for signaling to occur.[citation needed] The notch ligands are also single-pass transmembrane proteins and are members of the DSL (Delta/Serrate/LAG-2) family of proteins. In Drosophila melanogaster (the fruit fly), there are two ligands named Delta and Serrate. In mammals, the corresponding names are Delta-like and Jagged. In mammals there are multiple Delta-like and Jagged ligands, as well as possibly a variety of other ligands, such as F3/contactin.[36] 90

Integration of cellular responses to multiple signaling pathways
Example: Multiple signal transduction pathways interact to regulate adipocyte differentiation through PPARγ, the master transcription regulator

Multiple signal transduction pathways interact to regulate adipocyte differentiation
PPARγ is the master transcription regulator of adipocyte differentiation Together with C/EBPα, PPARγ induces expression of all genes required for differentiation of preadipocyte into mature fat cells Figure Multiple signal transduction pathways interact to regulate adipocyte differentiation The transcription factor PPARγ is the master transcription regulator of adipocyte differentiation; together with C/EBPα, it induces expression of all genes required for differentiation of preadipocyte into mature fat cells Both PPARγ and C/EBPα are induced early in adipogenesis; each of them enhances the transcription of the other’s genes; leading to their rapid increase during the first 2 days of differentiation Signals from hormones such as insulin and growth factors such as Wnt and TGF-β that activate or repress adipogenesis in the nucleus by transcription factors that regulate - directly or directly – the expression of PPARγ and C/EBPα genes IGF1: A receptor tyrosine kinase (Insulin-like growth factor 1 receptor) 92

Prospectives for the future
Abnormalities in signal transduction underlie many different diseases, including the majority of cancers and many inflammatory conditions Detailed knowledge of the signal pathways involved and the structure of their protein components will continue to provide important molecules for the design of specific therapies Gefitinib ( Iressa) is a drug used for certain breast, lung and other cancers. REMICADE® | Home Details about this drug treatment for Crohn's Disease and rheumatoid arthritis, by Centocor Pharmaceuticals. Enbrel (R) (etanercept) is a treatment for moderate to severe plaque psoriasis and rheumatoid arthritis, and psoriatic arthritis. Enbrel, Amgen (Immunex)

Prospectives for the future - Examples
A small-molecule drug (Iressa) specifically inhibits the kinase activity of the mutant EGF receptor (EGFR) but not on the wild-type or other receptors Monoclonal antibodies or decoy receptors that prevent pro- inflammatory cytokines such as IL-1 and TNF-α from binding to their cognate receptors are now being used in treatment of several inflammatory diseases such as arthritis and Crohn’s disease; e.g., Infliximab (Remicade, Centocor; mAB against TNF-α) Etanercept (Enbrel, Amgen; TNF receptor fused with a IgG1 constant region) Canakinumab (Ilaris, Novartis; mAB against IL-1β) Gefitinib ( Iressa) is a drug used for certain breast, lung and other cancers. REMICADE® | Home Details about this drug treatment for Crohn's Disease and rheumatoid arthritis, by Centocor Pharmaceuticals. Enbrel (R) (etanercept) is a treatment for moderate to severe plaque psoriasis and rheumatoid arthritis, and psoriatic arthritis. Enbrel, Amgen (Immunex)

Top-ten-selling biologic drugs of 2013
Humira – anti-TNF alpha mAb Enbrel – anti-TNF mAB Rituxan - chimeric anti-human CD20 mAB Glatiramer acetate, the active ingredient of COPAXONE, consists of the acetate salts of synthetic polypeptides, containing four naturally occurring amino acids: L-glutamic acid, L-alanine, L-tyrosine, and L-lysine with an average molar fraction of 0.141, 0.427, 0.095, and 0.338, respectively.

Table 5.2 Tyrosine kinase growth factor receptors altered in human tumors

Table 5.2 Examples of human tumors making autocrine growth factors

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Signaling Pathways That Control Gene Expression

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Signaling Pathways That Control Gene Expression

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