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FIG 9.9 structure of PKC. The conserved domains C1-C4 are functional modules. C1 binds diacylglycerol (or phorbol ester), C2 is involved in the attachment to phospholipid, which is reinforced by the binding of Ca2+, and C3 + C4 constitute the kinase domain which is linked to C2 by a hinge region. Regulation of activity and interaction with the upstream kinase PDK1 occurs through the turn-and hydrophobic motif (HM) (variable segment V5). The structures shown are a compilation of conventional and novel PKC isoforms: C1 (A+B) domain of PKCdelta, C2 domain of PKCalpha and C3+4 domains of PKCtheta (1ptr, 1dsy, 1xjd).
FIG 9.13 InaD scaffold protein and signalling complexes. (a) the formation of a complex that operates in Drosophila phototransduction. Two copies of InaD gather a number of signalling molecules to form a signalling circuit. Light falls on rhodopsin (1) which activates Galpha-q, which acts on NorpA, a phospholipase C (2). This causes the generation of diacylglycerol, which opens a Trp Ca2+-channel (3), causing membrane depolarization. This rapid event is followed by an almost instantaneous desensitization of Trp due to phosphorylation by eye-PKC ((4). PDZ domains are numbered 1 – 5. (b) A loss of InaD causes a retardation of the return to the resting state and hence a loss of visual resolution.
FIG 11.1 Stained blood smear form a leukaemic fowl. In December 1905, there came into my hands a Buff Cochin Bantam hen showing signs of illness in the way of indisposition to move about and a general weakness of a progressive character. No symptoms of ordinary fowl iseases were present . . . Examination of blood smears showed, however, a great increase of white cells of the large lymphocyte type . . . A diagnosis of leukemia was therefore made . . . A great variety of staining methods were used, including the most recent methods for the staining of spirochetes and protozoan parasites . . . No evidence of the existence of any infective agent could be obtained. Note that avian red blood cells (yellow stain) are nucleated.
FIG 12.4 Activation of the EGF receptor. The EGF receptor is composed of four extracellular domains (I – IV) of which I and III (also called L1 and L2) are leucine-rich repeats that function in ligand binding. II and IV (also called CR1 and CR2) are furin-like, cysteine-rich domains. Binding of EGF causes the receptor to dimerize. The receptor unfolds, a dimerization arm of domain II binds to a docking site at the base of domain II of the partner, allowing close approach and activation of the two kinase domains. The fi rst substrates are tyrosine residues in the C-terminal of the receptor itself (transphosphorylation). The asymmetrically dimerized, phosphorylated molecule constitutes the catalytically active receptor. (2gs6, 39 EGFR 1nql, 40 1ivo 41 ). Adapted from Zhang et al. 39 and Ferguson et al. 40
FIG 12.11 Vulval development in C. elegans. Because it is a relative simple structure, formed from just a few cells, the vulva is well suited for the genetic analysis of cell differentiation during embryological development. It is the product of just three cell lineages, the descendents of cells p5.p, p6.p, and p7.p. Development is initiated by a signal from the anchor cell that lies adjacent to p6.p. The ligand, lin-3 (homologue of EGF), produced by the anchor cell, binds its receptor Let-23 (homologous to the EGF-R) on the surface of cell p6.p. Cell p6.p in turn releases signals to its neighbours, p5.p and p7.p, and initiates a sequence of events involving the MAP kinase pathway, which determines the fate of these cells as components of vulval tissue. For more information, consult http://www.wormbook.org/
FIG 12.20 Dual effector interaction of RasGTP that leads to eff ective signalling to ERK. (a) In order to activate the ERK pathway, Ras has not only to recruit C-Raf (1), but also to remove IMP, the inhibitor which prevents formation of the Raf-MEK-ERK signalling cassette. RasGTP binds IMP (2). This initiates a series of ubiquitylations that mark the protein for destruction by the proteasome (3). MEK1 and ERK2, linked to the scaff old protein KSR1, now join C-Raf, enabling the signal to pass from one kinase to another (4). (b) Conserved domains in KSR1. CA2, proline-rich; CA3, cysteine-rich, resembles the C1 domain (PMA/DAG binding site) of PKC, CA4, serine/threonine-rich; CA5, a kinase domain that resembles that of Raf, but lacks an essential lysine and is therefore inactive (adapted from Matheny 160 and Kolch 152). (c) The domain architecture of IMP reveals a RING finger, involved in ubiquitylation.
FIG 13.5 Tight junction proteins: JAM, occludin, and claudin. (a) Diagram illustrating a tight junction as present in endothelial and epithelial tissues. (b) The junctions are composed of three types of protein. JAM is a member of the immunoglobulin family of adhesion molecules, but claudin and occludin are not. Both of these span the membrane four times and have very short extracellular domains. Their interaction ensures close apposition of the two membranes. There are many variants of the claudins. The combination expressed in tight junctions determines the accessibility of the paracellular space. They all bind to ZO-1 (or ZO-2, ZO-3) which connects the junctional proteins to the actin cytoskeleton. (c) Domain architecture of junctional proteins. Cyan indicates PDZ or PDZ binding motif.
FIG 13.7 Domain architecture of integrins. ( a) Integrins, involved in cell– cell and cell– extracellular matrix contact, are composed of two non-covalently bound subunits ( α and β ). Two examples of a subunit are shown. The stalk region of the α -subunit comprises two calf domains and a thigh domain. The protein articulates at the border of calf-1 and thigh, the PSI and hybrid domain of the β -subunit being pushed outward in a switchblade movement. This plays an important role in the activation process. The heads contain various subdomains of which the β -propeller (on the α -subunit) and β 1 -domain (on the β -subunit) assure the association of the two subunits. (b) Molecular structure of α V and β 3 . Depending on the type of integrin, ligand recognition either occurs through the β 1 -domain or through the α I-domain (in α L ). Yellow spots indicate divalent metal ions; PSI, plexin 7 integrin domain; β TD, β -tail domain; EGFD, EGF-like domain (1jv2).
FIG 13.16 A two-step process to create an integrin signalling complex. (a) With the structural components of the focal adhesion site assembled, the focal adhesion kinase FAK associates with the F2 lobe of the FERM domain of talin; (Fak also has a FERM domain). Autophosphorylation FAK then generates a docking site (Y397) for the SH2 domain of Src which phosphorylates at Y925, in the FAT domain). Src and FAK next phosphorylate the FAK-associated docking protein CAS at multiple sites. An integrin-signalling complex is formed that acts in a manner similar to growth factor-receptor signalling complexes, i.e. attachment of adaptors and eff ectors and tyrosine phosphorylation of substrate. (b) Illustration of the main domains and essential phosphorylation sites of FAK and CAS.
FIG 14.1 Loss of cell – cell contact and the dissipation of cell polarity. The polarized state is well illustrated by the segregation of the components of the tight and adherent junctions on lateral surfaces: IGFRs at the basal surface and the Na+/glucose symporter (SGLT1) at the apical surface. With the dissipation of the adherent junctions, this functional polarization is lost. The Na+/glucose symporters and IGF receptors become randomly distributed and keratin is replaced by vimentin. The cells become de-differentiated.
FIG 14.2 Examples of D rosophila phenotypes due to mutations in the Wnt pathway. In the wild-type fl y, actin, which forms a single hair, polymerizes in a defi ned position with uniform orientation within the plane of the epithelial wing cells. Mutations in Fz and Dsh cause a loss of planar polarity of these cells. Polymerization occurs at random points within the plane of the epithelium giving rise to a frizzled (curly) or dishevelled (disorganized) phenotype. Mutations in Arm give rise to a highly segmented embryo resembling the banded shell of an armadillo. Image of hairs from Wong and Adler 1993, originally published in the Journal of Cell Biology 123, 209 – 221. Images of Drosophila larvae Wt and Arm are kindly provided by Dr Martinez-Arias, Cambridge, UK.
FIG 14.11 Parallels between the Wnt and Hedgehog pathways. (a) Wnt and (b) Hh signalling pathways share components including GSK3 β and members of the CK1 and Lrp families. They also have similar mechanisms. In the absence of ligands, transcription factors are phosphorylated (red arrows) and destroyed or partially degraded, resulting in repression of transcription. In the presence of their ligands, the receptors are phosphorylated (green arrows) and this prevents phosphorylation and destruction of the transcription factors, which now enter the nucleus to activate transcription (Smo, Smoothened).
FIG 15.2 Comparison of domain the architectures of receptors, adaptors and eff ectors involved in signalling through the Toll and Toll-like receptors. Toll and TLR4 both have leucine-rich repeats in their extracellular segment (two of which, at the N-terminus, are highlighted in the inset panel) and an intracellular TIR domain. A signalling complex is recruited to the occupied receptor through adaptors containing TIR-domains. Those that possess only a TIR domain (such as TIRAP) recruit other TIR-containing adaptors. Adaptors having an additional death domain (DD) recruit the DD-containing serine/threonine kinase IRAK-4 (or pelle in the case of Toll). Note that the IL-1R also possesses a TIR domain and therefore resembles both Toll and TLR4, but in addition it has three Ig-like domains in the extracellular segment. The N-terminal is indicated at the left, unless otherwise indicated (1ziw, 22 1fyw 23 ).
FIG 15.6 TRAF6-mediated signalling complex formation. Recruitment of IRAK4 brings IRAK1 and TRAF6 to the receptor signalling complex (1). IRAK1 is phosphorylated by IRAK4 (2) and this induces catalytic activity the complex comprising TRAF6 (E3-ubiquitin ligase), Ubc13 and Mms2 (3). Auto-ubiquitylation of TRAF6 follows (4). The K63-linked ubiquitin chain acts as a docking site for two kinase complexes. One of these comprises TAB2 coupled to the serine/threonine kinase TAK1 with its regulatory subunit TAB1 (5). The other comprises NEMO attached to I κ B-kinases- α and - β (IKK- α and - β ). Recruitment of TAB2/TAB1/TAK1 results in autophosphorylation of the kinase (7) and subsequent phosphorylation and activation of IKK- β (8).
FIG 15.11 Negative feedback of the TLR4 signal transduction pathways: resolution of the infl ammatory response. Among the genes regulated by the diff erent transcription factors are proteases that cleave the ubiquitin isopeptide bonds. Their action causes disassembly of the TRAF6-mediated signalling complexes. Activated p38 phosphorylates the ubiquitin binding protein TAB2 as well as the regulatory subunit TAB1. Both phosphorylations cause inactivation of TAK1. ATF3 is also one of the targets of the TLR4 signal pathways, acting to suppress transcription of IL-6 and IL-12 when bound to NF- κ B and ATF2/c-Jun. Finally, IL-10 has a general inhibitory eff ect on expression of pro-infl ammatory cytokines. Collectively, these negative feedback pathways act to resolve the inflammatory response.
FIG 15.15 Ubiquitin-binding proteins involved in substrate presentation to the proteasome. There are numerous proteins that bind ubiquitins and which are involved in the presentation of substrate to the proteasome. They contain ubiquitinassociated (UBA) or ubiquitin-interacting motifs (UIM) (among others). The domain organization of two examples are illustrated. hHR23a is composed of two UBA sites and one ubiquitin-like domain (UBL). It binds ubiquitylated substrate at both UBA sites, and this may be one mechanism by which only poly-ubiquitylated proteins are selected for destruction. With its UBL domain it binds the ubiquitin-interacting motif from S5a, an integral component of the PA700 particle (1oqy, (79) 1aar (70)).
FIG 16.4 TNFR activation. (a) In the absence of ligand, TNF- α receptors are associated through their N-terminal pre-ligand assembly domain (PLAD). The intracellular segment is bound to SODD. PLAD and SODD control the silencing of the unoccupied receptor. Binding of ligand induces trimerization of the receptors and this reveals death-domain docking sites. TRADD and RIP1 associate with the receptors. TRAF2 is recruited to the protein complex through a TRADD N-terminal interaction with the MATH domain of TRAF2. (b) Structure DR5 (member of the TNF receptor family) bound to TRAIL (member of the TNF family). Three receptor molecules bind a trimeric ligand (1du3 (35)).
FIG 16.9 Chemokine-mediated activation of integrins in leucocytes. Binding of the chemokines causes activation of Gi. The βγ - subunits activate PLCβ giving rise to diacylglycerol and IP3 , both of which are involved in the activation of CalDAG-GEF, which activates Rap1. This binds its eff ector RapL responsible for activation of integrins (for instance αLβ2 (LFA1)).
FIG 17.1 Non-receptor protein tyrosine kinase families. These are subdivided into 10 families most of which contain SH2 and SH3 domains (Src-A and Src-B families are shown as one). Several were discovered as transforming genes of viral genomes, hence names like Src or Abl, derived from Rous sarcoma virus or Abelson murine leukaemia virus. Adapted from Robinson et al (1).
FIG 17.10 STAT proteins act as remote sensors. STAT proteins that dissociate from the DNA are dephosphorylated by tyrosine phosphatases (1). They return to the cytoplasm and only re-enter the nucleus if they are rephosphorylated by (active) IFN receptors (2). With a large proportion of receptors occupied, the cycle is rapid and STAT accumulates in the nucleus. With few receptors occupied, the cycle is slow, resulting in an accumulation of STAT in the cytoplasm. Effectively, STAT acts as a remote sensor of receptor occupation.
FIG 17.12 From c-Src to the deregulated kinase v-Src. (a) c-Src exists in two states. In its inactive form its SH2 domain folds back and binds to pY530 in the C-terminal segment. Dephosphorylation of pY530 or displacement by a higher affinity phosphotyrosine releases the SH2 domain, allowing Src to adopt its open form and become phosphorylated in its activation segment (Y419). (b) Ribbon diagram of c-Src in its inactive and active states. (c) v-Src lacks C-terminal residues that include Y530. It cannot adopt the closed conformation and is constitutively active (2src (58), 1y57 (59)).
FIG 18.6 Activation of PKB β. (a) In the inactive PKB structure, the various regions of the kinase domains comprising the α C-helix of the N-lobe and the activation segment are disordered. Substrate and ATP do not bind. The organization of the C-terminal segment with its hydrophobic motif (HM, shown in yellow) is indicated approximately. (1gzk (40) ). (b) Binding of the C-terminal hydrophobic motif (HM, yellow) with the α C-helix is facilitated by phosphorylation of S474. This induces reorganization of the α C-helix (pink) and a second phosphorylation in the activation segment (T309) organizes the activation segment (red). Binding of ATP and substrate ensues (2jdr (41)). (c). Binding of the hydrophobic motif to the α C-helix (1) leads to structural rearrangements in which E200 engages and correctly positions K180 (2) that coordinates the binding of ATP in the catalytic cleft. The H196 of the ordered α C-helix also engages the (PDK) phosphorylated T309 resulting in a reorientation of the activation segment (3). The kinase now binds both ATP and substrate and is fully competent to phosphorylate substrate.(2jdr (41)).
FIG 18.8 Insulin receptor structure. (a) The insulin receptor is a homodimer. In the mature form, each component is present as two chains, α and β . Each monomer possesses seven domains, L1 (leucine rich region-1), CR (cysteine rich region), L2, FnIII-1 (fi bronectin-III-like domain 1), FnIII-2, FNIII-3, and the intracellular tyrosine kinase domain (structure not shown). Both the α and β chains contribute to the FnIII-2 domain. The long insert region (shown as a dotted line) separating FnIII-2a from -2b contains three disulfide bridges (2) that link the monomers. FNIII-1 provides a fourth disulfi de bridge (1). b) A possible arrangement of the two peptides is shown. The insulin-binding space is between the central β -sheet of L1 and the bottom loops of the FnIII-1 domain. (c) Domain architecture showing locations of the positions numbered in (a). From McKern et al. 42 (2dtg 42 ).
FIG 18.14 Signalling through AMP kinase. Hints of ATP depletion are manifested by the leakage of 5 ’ -AMP from mitochondria which binds the regulatory subunit of AMPK. Subsequent phosphorylation by LKB1 leads to its activation. AMPK phosphorylates TSC2 thereby preventing the inhibitory control by PKB. The active TSC1/2 complex maintains Rheb in its GDP-bound state thus preventing activation of mTOR. Protein synthesis is suppressed.
FIG 19.4 Par in C. elegans. (a) The sperm microtubule organizing centre arranges the disassembly of the actin cytoskeleton. This leads to a redistribution of Par proteins. Par-3 and Par-6 remain associated with the cytoskeleton and dominate the future anterior site. Par-1 and Par-2 remain ‘active’ at the future posterior site. Each Par complex has its specific kinase activity, Par-1 at the posterior and PKC-3 at the anterior site. This leads to phosphorylation of other polarity-determining proteins and results in an asymmetric distribution of mRNA and proteins. (b) Then, following replication of DNA and during attachment of the chromosomes to the microtubule cytoskeleton, the Par complexes ensure the right orientation of the mitotic spindle by correctly positioning the astral microtubules.
FIG 19.5 Activation of Par-6/atypical PKC by Cdc42. (a) Activation of Cdc42, by a receptor binding an external polarity cue, leads to binding of Par-6/aPKC. This acts to relieve inhibition and allows phosphorylation and activation of the atypical PKC. The active complex now phosphorylates substrates that operate in the determination or maintenance of cell polarity. (b) Domain architecture of the Par-6 polarity complex. Domain interactions are indicated by red lines. (c) Structure of the domains involved in formation of the Par-6 polarity complex. The switch 1 and 2 regions of GTP-bound Cdc42 interact with the partial CRIB and with the PDZ domains of Par-6. Par-6 and PKCiota are linked through their PB1 domains (phox-bem domains, 1 and 2). Interacting surfaces are highlighted.
FIG 20.1 Big beef. Springhill Wizzard, owned by Martin Brothers, Newtownards, was the best Belgian Blue Junior Bull and Reserve Male Champion at the Balmoral Show 2007. Thomas and James Martin are pictured exhibiting the prizewinner. Photograph by kind courtesy of Columba O’Hara and the British Blue Cattle Society.
FIG 20.5 Schematic view of TGFβ receptor activation. The TGF β type I receptor exists in three different states. Inactive has the GS wedged into the N-lobe (held in place by FKBP12); intermediate allows oscillation between the attached and partly-detached positions of GS; in the active state the phosphorylated GS cannot wedge into the N-lobe. Phosphorylation is induced by the close proximity of the types I and II receptors. The phosphorylated GS, together with the L45 loop, form the binding site of receptor-regulated Smad proteins (R-SMADs) (1), which are subsequently phosphorylated on two serine residues at their C-termini (2). SARA facilitates the interaction between R-Smads and the type I receptor. Once phosphorylated, R-Smad proteins detach from the receptor (3). FYVE, MH1 and MH2 indicate domains (see text).
FIG 20.10 Smad activation and nuclear translocation. (a) On phosphorylation of the C-terminal SxS motif, receptor-regulated Smads complex with each other and then with Smad4. The phosphoserines bind the basic pocket in the MH2 domain. The trimers enter the nucleus to bind DNA at the SBE. The complexes also bind other DNA-binding proteins and transcriptional cofactors (for instance p300). (b) Detail of a Smad2 – Smad4 complex showing the interaction of the phosphoserines in the C-terminus with the basic pocket of the MH2 domain. The Smad proteins are viewed from two diff erent angles to show the presumed orientation of the MH2 domain relative to the DNA-binding MH1 domain (1u7v (51)).
FIG 20.15 Neuroectoderm induction through FGF and the release of the BMP-traps noggin and chordin. Neuroectoderm formation in Xenopus laevis occurs through inhibition of the BMP signal by release of noggin and chordin from the Spemann organizer and through diff usion of FGF from the mesoderm marginal zone into dorsal ectoderm. Neuroectoderm develops into notochord and gives rise to the brain.
FIG 21.4 The arrangement at the active site of PTP1B. (a) The sides of the catalytic cleft of tyrosine phosphatases are characterized by three motifs: the WPD loop, containing an invariant Asp, the Q-loop, containing an invariant Gln, and the pTyr-loop, containing an invariant Tyr (1ptt (10) ). (b) The invariant tyrosine determines the depth of the catalytic cleft, ~10 Å, and allows the target phosphotyrosine to make contact with the catalytic cysteine residue (yellow). (c) The pocket is too deep for phosphoserine or phosphothreonine to contact the catalytic cysteine residue (2hnp (11)).
FIG 21.8 Generation of superoxide (O2- ) by NADPH oxidase and the mitochondrial electron transport chain. Superoxide is produced by NADPH oxidase, a complex comprising NOXA (NOX activator), NOXO (NOX organizer), Rac, and NOX1. It is also released from mitochondria. Superoxide is converted to oxygen and peroxide, to become H2O2, which then transiently converts the active site cysteine thiolate into a catalytically inactive cyclic sulfenamide. Glutathione is required to restore enzyme activity. This reaction scheme applies for cysteines with a low pKa.
FIG 21.20 Dorsal closure in Drosophila and the role of the dual specifi city phosphatase puc. (a) Phenotypes of D rosophila embryos arising as a consequence of altered or enhanced expression of the puc gene. The weak mutant puc Eh yields an almost perfect seal between the two sides of the ectoderm. Loss of function ( puc E69) causes a puckered phenotype, in which the cells of the two borders penetrate each other’s territory. Over-expression prevents the leading edges reaching each other and the embryo reveals dorsal openings. (b) Stages in dorsal closure during Drosophila embryogenesis. The embryos are shown dorsal side up, with the anterior (future head region) to the left. At stage 12, a large part of the embryo is still covered by amnioserosa (pink). At stage 13, the ectoderm cell sheet extends towards the dorsal midline (red arrow). This movement takes about 2 h and is the consequence of the progressive fl attening of cells which extend a leading edge towards the midline. At stage 15, the leading edges of both sides converge. The process of fl attening and migration ceases. (c) The signal transduction pathway that controls dorsal closure. Positive control of fl attening and migration occurs through induction of dpp (decapentaplegic, member of the TGF β family of growth factors, see page 607) by a pathway involving mekk (M1K), hep (M2K), and bsk (M3K). When cells make contact, negative feedback, due to Puc-mediated dephosphorylation and inactivation of Bsk, arrests fl attening and migration. (d) Phylogenetic tree analysis of puc , yeast MSG5, and human-dual specifi city phosphatases. Image courtesy of Dr Martinez-Arias, Cambridge, UK. Embryonic stages according to Campos-Ortega and Hartenstein (109).
FIG 21.26 Members of the PPP family share a common catalytic domain structure. (a) The catalytic domain fold consists of a central β-sandwich surrounded on one side by seven α-helices and on the other by a subdomain comprising three α-helices and a three-stranded β-sheet. (b) The catalytic cleft has a Y-shape, with three branches commonly referred to as hydrophobic, acidic and C-terminal grooves. (c) Crystallographic data provide compelling evidence for the role of two metal ions in the catalytic reaction. Most data are consistent with a single step mechanism, employing a metal-activated nucleophilic water molecule or hydroxide ion. (d) The metal coordinating residues (asparagines, aspartates and histidines) are invariant amongst the PPP family members. The position of some of the amino-acids of the signature sequences are shown. (1jk7 (134)).
FIG 21.29 Regulation of PP1c by MYPT1 and inhibition by okadaic acid. (a) Attachment of the regulatory subunit MYPT1 to PP1c (the PP1 δ variant) creates an extended acidic groove on its surface which forms a perfect fit for the N-terminal sequence of the regulatory light chain of myosin II, which in turn has many basic residues (K or R in orange). At its other end, the groove is hydrophobic enabling it to accommodate a stretch of hydrophobic residues (blue) in the regulatory light chain. This causes it to align so that the phosphoserine lies above the catalytic pocket (indicated by ++). Thus MYPT1 increases the affinity of PP1δ some 10-fold for this particular substrate. Dephosphorylation of myosin regulatory light chain inactivates the ATPase activity of myosin-II and hence prevents movement of its head, resulting in muscle relaxation. (b) Okadaic acid, a causative toxin of diarrhetic shellfish poisoning in Europe, inhibits PP1c by occupying the catalytic cleft, preventing access of substrate.
FIG 22.3 Structure of the Notch regulatory region (NRR). (a) A ribbon representation of the Notch protein. A, B, and C indicate the three Ca2+-binding Lin-12 repeats and the S1 site. Notch is cleaved in the cis-Golgi by a furin-like convertase at the S1-site and arrives at the cell surface as a heterodimer reassembled from the fragments. (b) The Lin-12 repeats mask the S2-cleavage site. The Notch ligand renders this site accessible to the ADAM family of proteases. Cleavage at S1 is ligand-independent, whereas S2 cleaved by ADAM, and S3/S4 cleaved by γ-secretase, are dependent on ligand interaction. LNR, lin-12/Notch repeat; HD, heterodimerization domain. (c) Lin-12 repeats (spheres) covering the S2 site (2oo4).
FIG 22.5 The Notch transcriptional complex. (a) The intracellular Notch segment, Nicd, first binds the β -trefoil domain of CSL and then folds over to attach, by its ankyrin repeats, to the C-terminal domain. (b) This creates a binding site for Mam and a trimeric complex is formed. The C-terminal domain of Mam recruits numerous proteins that cause acetylation of histones, so rendering the DNA amenable for transcription. Among the recruited molecules is CDK8, which later phosphorylates Nicd in both its TAD and PEST regions. Phosphorylated PEST is recognized by the receptor subunit of a nuclear E3-ligase complex, resulting in polyubiquitylation of Nicd, followed by proteasome-mediated destruction. (c) Numerous transcriptional complexes thus formed are readily visible by immunostaining of p300 (d) and (e) Histone acetylation unwinds DNA and transcription follows removal of repressors and recruitment of transcriptional activators. Image (c) from Fryer et al. 34.
FIG 22.11 Imaginal discs of a third-instar larva and the corresponding body parts of the adult fly. (a) The imaginal discs harbour cells that form the diff erent body parts of the adult fly during the process of metamorphosis,. Here, only the eye and the wing imaginal discs are indicated (by double-headed arrows). The abdomen arises from histoblast nests. Note that the adult is much smaller than the larva, most of the cells outside the imaginal discs having been removed by apoptosis. Image adapted with permission from V. Hartenstein, http://flybase.bio.indiana.edu. (b) The imaginal wing disc contains clusters of cells, proneural clusters, which express the neurogenic genes achaete/scute. These clusters later form the mechanosensory organs. In each selected cluster, just one cell that expresses a particularly high level of achaete/scute retains the neural fate. The others develop as epidermal cells. Image of immunochemical staining adapted from Cubas et al. 57.
FIG 23.1 Mode of action of alkylating agents and cisplatin. (a) Binding of cisplatin to two guanine residues in a DNA strand. Binding occurs at the same nitrogen that is bound by the alkylating agents. (b) Nitrogen mustard links to guanosine through methylene groups that have lost a chlorine atom. Two linkages are formed which are either intra- or inter-strand. (c) Doxorubicin, a cytotoxic antibiotic, intercalates between the bases of two DNA strands. In doing so it prevents the action of DNA polymerase (1au5 (4), 2des (5)).
FIG 23.7 Regulation of c-Abl. c-Abl has many different activation states. (a) and (b) represent the inactive and fully active states. Intermediate states, which have limited kinase activity, are represented in panels (c) – (f). (a) Latched conformation, the kinase domain clamped in an inactive state. (b) Displacement of myristate (yellow) from the C-lobe, detachment of the SH3 domain by phosphorylation of Y245 and reorganisation of the activation segment by phosphorylation of Y412 render c-Abl fully competent. (c) Partial activation through displacement of myristate. (d) Partial activation through phosphorylation of the linker region (Y245). (e) Partial activation through displacement of the SH2 domain by a phosphotyrosine-containing protein. (f) Partial activation through displacement of SH3 by a proline rich sequence. (g) The fusion protein Bcr-Abl is also in an intermediate activation state. It lacks the Cap region (together with its myristate chain) and the Bcr moiety binds and holds the SH2 domain away from the kinase domain. Full activation then requires only phosphorylation of Y245 and Y412
FIG 23.8 Optimization of imatinib as a chemotherapeutic agent. The discovery that 2-phenylaminopyrimidine inhibitors of PKC also inhibit the unrelated v-Abl oncogene turned attention to its potential use in the treatment of chronic myelogenous leukaemia. Starting with the 2-phenylaminopyrimidine backbone, addition of the benzamidine group increased activity against tyrosine kinases, the methyl group reduced its activity against PKC (so-called ‘ target hopping ’ ). Addition of a 3’-pyridyl group improved the activity in cellular assays. Subsequent addition of N -methylpiperazine increased water solubility and oral bioavailability, enabling the drug to survive the stomach and to enter the bloodstream.
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