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Receptor Pharmacology (3rd edition)
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Chapter 2 Structure and function of 7-TM G-protein coupled receptors p79

FIG 2.1 The molecular structure of rhodopsin-like 7-TM domain receptors. These receptors are characterized by the presence of seven membrane-spanning domains (TM1 to TM7) with a short extracellular N-terminus and intracellular C-terminus shown in a two-dimensional schematic representation in (a). Agonist binding is predicted to be within the transmembrane domains. The extracellular structure is stabilized by a disulphide bond joining the first and second extracellular loops. The third intracellular loop is the main site of G-protein interaction, while both the third intracellular loop and carboxy tail are targets for phosphorylation by kinases responsible for initiating receptor desensitization and internalization. (b) Analysis of crystal structures has shown that the packing arrangement of the transmembrane domains is more complicated than that implied in (a) with transmembrane domains tilted to form the ligand binding pocket.

FIG 2.3 GRK-mediated desensitization and trafficking of GPCRs. Ligand binding to GPCRs not only promotes G-protein activation but makes the receptor a substrate for phosphorylation by GRKs (G-protein coupled receptor kinases). GRK phosphorylation promotes arrestin binding, which causes G-protein uncoupling and promotes receptor internalization via clathrin-mediated endocytosis.

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Chapter 3 The structure of ligand-gated ion channels p89

FIG 3.3 (a) Model of the 4-TM receptors. The model shows the ligand binding site, the membrane bilayer and the position of the channel gate.
(b) Electron density map of the nAChR in profile at 4.6 Å resolution. The electron density is shown through a cross-section of an α-subunit and the interface between the other α-subunit and the δ-subunit. The asterisk indicates the proposed Ach binding site. (c) Molecular structure of the nAChR receptor resolved at 4 Å. Only the two α- and γ-subunits are shown. The TM2 is shown in dark.

FIG 3.4 (a,b) A model of a single a-subunit. It is base on the 4 Å structure analysis of the Torpedo nAChR (figure 3.3c) where it has been proposed that ligand binding induces a rotation in the binding domain that is transmitted to the transmembrane regions through interactions between the β1-β2 loop and the M2-M3 loop.
(c) The orientation of the TM2 helical segment in the closed and open state of the channel. At the top: A view of two of the five helices from the side where the helical segment is illustrated as two helices (rods) separated by a kink where leucine (ellipse) is located. At the bottom: in the resting state, the five leucine of the five helices, viewed here from the synaptic cleft, block the pore of the channel. The binding of an agonist cause the helical segments to rotate, turning the leucines away from the transition pore, thus allowing cations (Na+ and K+) to cross the channel.

FIG 3.6 Crystal structure of the Aplysia AchBindingProtein. (a,b) The complex, as viewed into the extracellular vestibule that is formed by the subunits that are either in the apop form (a) or bound to epibatidine (b). The complex consists of five identical subunits (pentamonomer) of which one is highlighted. The open arrow indicates the ligand binding site whereas the C loop is indicated by the black arrow. (c) View of the epibatidine (agonist) binding site viewed from the pore (compare with position open arrow in (b)).

FIG 3.10 (a) Molecular structure of a glutamate receptor subunit. (b) Binding of glutamate at the GluR2 binding pocket. Glutamate interacts directly with residues in both domain 1 and domain 2 (R485, P478, T480 and S654, T655, E750 respectively), thereby stabilizing the domain closure. (c) Schematic illustration of the proposed model for receptor subunit activation (only two subunits are shown). Agonist binding stabilizes a closed conformation of the lobes in the binding domain, thereby inducing an opening of the pore. Agonist stabilizing a high degree of domain closure (α) will subsequently induce a twist (β) between the subunits. This results in a conformational changes that reduces the distance between the domains projecting to the pore. (d) Relationship between the degree of domain closure (a) and the distance for the regionsions projecting to the posre (<-> d) for eight agonists. (e) Relationship between efficacy, measured as the relative maximal response (compare with a full response of glutamate) in a nondesensitizing mutant of the receptor domain, and domain closure. (f) Activation of the receptor is proposed to require activation of at least two subunits, and activation of more subunits opens the channels further, resulting in a higher conductance level (from 6 pS, 12 pS to 17 pS when, respectively, 2, 3 or 4 subunits are occupied by agonist.

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Chapter 4 Molecular structure of receptor tyrosine kinases p111

FIG 4.1 Classification of tyrosine protein kinase containing receptors.

FIG 4.2 Receptors employ different dimerization strategies.
a) PDGF forms a ligand dimer of which each growth factor engages one receptor; b) EGF has one binding site and its binding reveals a receptor dimerization motif; c) insulin has two binding sites and its action somehow must change the conformation of an existing receptor dimer; d) FGF has two binding sites but two ligands are needed to bring two receptors together. Stable dimers only form when two heparin sulphate oligosaccharides combine with receptor ligand complexes.

FIG 4.3 Conserved substructures within the consensus kinase fold involved in the phosphoryltransfer from ATP to substrate.
a) Dormant versus active protein-kinases. For clarity reasons only a few molecular connections are shown and one of the two Mg2+ atoms is omitted (situated between β- and γ-phosphate). Note the rather superficial binding of the substrate peptide in the right panel. This explains, in part, why serine and threonine are not good substrates; their hydroxyl group does not get close enough to both the γ-phosphate and the catalytic aspartate residue.
b) Active serine/threonine protein kinase (protein kinase B). Note that the substrate-peptide penetrates deeper into the cleft and that a short serine residue easily reaches both ATP and the catalytic aspartate.

PDB files: 2GS6 and 2GS7
Highlighted residues in the catalytic domain of the EGFR (right panel 3a): Gly695-Val702 (glycine-rich loop); Lys721 (in b3 sheet); Glu738 (in αC helix); Arg812 (in VHRDLA motif); Asp813 (catalytic residue in VHRDLA motif); Asp831 (in DFG motif); Leu833-Gly850 (activation segment); Val852-Met859 (P+1 substrate binding residues in spheres in the right panel of figure 3a).
PDB file: 1O6K
Highlighted residues in the catalytic domain of protein kinase B (PKB) (figure 3b): Gly159-Lys165 (glycine-rich loop); Lys181 (in β3 sheet); Glu200 (in αC Helix); Asp275 (in RDI motif); Asp293 (in DFG motif); Leu296-Phe310 (activation segment); Gly312-Glu320 (P+1 substrate binding residues in spheres in figure 3b).

FIG 4.4 Modelled structures of the dimerization process of the extracellular domain of the EGFR. (a) Domain architecture of the EGF receptor; (b) Interaction between domain II and IV keeps the EGF receptor in a tethered conformation. EGF is bound to domain I but, because of (experimental)acidic conditions, cannot make contact with domain III. (c) EGF binding leads to the adaptation of an extended configuration and liberates the dimerization finger in domain II. This now interacts with another domain II and forms an EGFR dimer.
PDB files:1NQL and 1YY9

FIG 4.5 The kinase domain of the EGF receptor is kept in an inactive state by a leucine wedge that dislocates the αC-helix.
Mutation of these residues leads to constitutive active kinases.
PDB file: 2GS7

FIG 4.6 Allosteric regulation of the EGF receptor kinase domain by the formation of an asymmetric dimer.
In this proposed model of kinase activation, one of the two protein kinases acts as the regulator of the other kinase domain (activator kinase), a mechanism of activation reminiscent of cyclin mediated activation of cylin-dependent kinases.

FIG 4.7 Insulin receptor structure
The insulin receptor is a homodimer. In the mature form, each component is present as two chains, α and β. The protein is organized in seven domains, L1 (leucine rich region-1), CR (cysteine rich region), L2, FnIII-1 (fibronectin-III-like domain 1), FnIII-2, FNIII-3 and the intracellular tyrosine kinase domain. Both the α and β chain contribute to the FnIII-2 domain. The long insert region (shown as a dotted line) separating FnIII-2a from -2b contains three disulphide bridges (position 2) that link the monomers. FNIII-1 provides a fourth disulphide bridge (position 1).
A possible orientation between the two peptides is indicated in the right hand panel (dimer). The insulin binding space is between the central b-sheet of L1 and the bottom loops of the FnIII-1 domain.
PDB file: 2DTG

FIG 4.8 Insulin binding and possible consequence for receptor conformation.
How binding of insulin leads to activation of the kinase domains remains to be elucidated. In this schematic representation we propose that the kinase domains approach each other due to a change in the relative position of the two receptors

FIG 4.9 Molecular mechanism of insulin receptor kinase activation
In the inactive state, the activation segment occupies the site where normally substrate would bind to the kinase. Note that one of the tyrosines of the activation segment is even correctly orientated for phosphorylation. However, other important structures are not in the right configuration for accommodating ATP. Upon ligand binding the kinase domains phosphorylate each other at three tyrosine residues in the activation segment. This leads to a reorganization of the N-terminal lobe and to interdomain closure (compare the relative positions of lys, glu and asp.

FIG 4.10 Inhibition of insulin tyrosine kinase by its substrate IRS-2
One of the phosphorylation sites of the IRS-2 substrate does not occur in the classic YΦXM motif but in a YGDI motif, in a segment of the protein that was originally designated a kinase regulatory loop binding region (KRLB). Because of the unusual motif, the orientation of the target tyrosine (Y628) is slightly distorted thereby reducing the efficiency of phosphorylation. Moreover, a tyrosine seven residues downstream inserts into the ATP pocket and competes with binding. Altogether, these features make the IRS-2 protein not only a substrate but also an inhibitor of the insulin receptor tyrosine kinase.
PDF file: 3BU3

FIG 4.11 ephosphorylation of the insulin receptor activation segment by PTP1B;
PTP1B binds preferentially to the bis- or tris-phosphorylated activation segment of the insulin receptor. Residue pTyr-1162 is most readily recognized by the catalytic cleft of the phosphatase. Dephosphorylation renders the insulin receptor catalytically incompetent. Sequence comparison with other PTPs suggests that the features that confer the specificity of this reaction are unique to PTP1B and its close relative TCPTP.
PDB files: 1PTT and 1IRK

FIG 4.12 Top and side view of the FGF receptor bound to ligand and heparin sulphate oligosaccharides. Note that FGF2 binding to its receptor creates a canyon which nicely fits the heparan sulphate motifs. This surface illustration shows the multiple contacts between receptor, ligand and the heparan sulphate oligosaccharides
PDB file: 1FQ9

FIG 4.13 Multiple interaction sites between FGF2, FGFR1 and heparin sulphate.
FGF interacts with two receptors. Its primary interaction is rather extensive, occurring with both Ig-domain II and III. The secondary interaction site is limited to domain Ig-II. It also has a binding site for heparin. Receptors interact with their ligand, with heparin and with each other. Altogether, these interactions result in high affinity binding, giving rise to stable dimers.
PDB file : 1FQ9

FIG 4.14 The molecular brake of the FGF receptor kinase family
The FGF receptor kinase is kept in a dormant state through a network of hydrogen bonds, in between N549, E565 and K641, which prevents the right orientation of the N-lobe relative to the C-lobe. Phosphorylation of the activation segment breaks this inhibitory spell (right panel).
A classic view of the FGF tyrosine kinase shows the moderate shift of the overall structure between the inactive and active conformation. Compare the position of the essential residues glu, asp and asp* in the left and right panel. This shift suffices to accommodate ATP and leads to phosphorylation of substrate.
PDB file : 2PVF

FIG 4.15 Structure of Epo bound to its receptors
a) The Epo receptor occurs in dimers in unliganded state but the relative orientation of the two receptors (here arbitrarily depicted as 180°) does not allow for kinase activation. Epo binding positions the first FnIII-domains of two receptors in a 120° orientation which is commensurate with kinase activation. b) A side view shows the structural role of the N-terminal sequence, which makes contacts with both the 1st and the 2nd FnIII-domains.
c) Domain architecture of JAK2 and the Epo Receptor The FERM and SH2 are in between quotation marks because they are related domains and do not fully match the signature sequences.
PDP file: 1EER

FIG 4.16 Model for Epo-mediated activation of JAK2
In the resting state, the pseudo kinase (JH2) maintains a strong interaction with the kinase domain (JH1) thus preventing its activity. Epo binding triggers the extracellular domains to adopt a 120° angular orientation and this somehow relieves the inhibitory constraint of JH2. Phosphorylation in trans of the activation segments (on two tyrosine residues) leads to kinase activation and subsequent phosphorylation of tyrosine residues on the cytosolic segment of the Epo receptor.
(Image adapted from Lu et al J Biol Chem 2008 ;283 :5258-5266)

FIG 4.17 Overview of antibodies binding to EGFR or ERBB2
Trastuzumab binds domain IV and as shown from two perspectives (top and side view) it does not really hinder EGF binding. The dimerization arm is coloured in green
The structural data show only the Fab fragment of the antibody but realise that these are much bigger structures with an Fc segment that can interact with Fc-receptors carried by blood-borne cells.
Cetuximab binds domain III and does compete with EGF for binding. Notice that the receptor is in the tethered conformation, with its dimerization arm buried in domain IV.
Pertuzumab binds the dimerization arm (in green) and prevents its association with another receptor.
PDB files 1N8Z (trastuzumab), 1S78 (pertuzumab), 1VY9 (cetuximab)

FIG 4.18 Optimization of Gleevec as a chemotherapeutic agent
The discovery 2-phenylaminopyrimidine inhibitors of PKC also inhibit the unrelated v-Abl oncogene turned the attention to its potential use in the treatment of chronic myelogenous leukemia. Starting with the 2-phenylaminopyrimidine backbone, addition of the benzamidine group increased activity against tyrosine kinases, the methyl group reduced its activity against PKC. Addition of a 3'-pyridyl group improved the activity in cellular assays (bioavailability). Subsequent addition of N-methylpiperazine increased water solubility and oral bioavailability (surviving the stomach and entering the bloodstream).

FIG 4.19 Binding of Imatinib to the tyrosine kinase Abl.
Only the catalytic domain of the tyrosine protein kinase Abl is shown. Note that imatinib binds the ATP binding pocket of the kinase in its inactive conformation. The activation segment is not phosphorylated and covers the substrate binding site. In fact, imatinib prevents outward movement of the activation segment. Note also that Asp and Glu (in the αC-helix) are not in the correct position to coordinate ATP binding.
PDB file:1OPJ

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Chapter 7 G-proteins p199

FIG 7.2 G-protein schematic realistic

FIG 7.2 G-protein receptor interaction

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Chapter 8 Signal transduction through protein tyrosine kinases p225

FIG 8.1 Receptor dimerization and phosphorylation
(a) On occupation by its ligand, the EGF receptor takes on an extended conformation through which one receptor recognizes the other. Receptor dimerisation brings together the tyrosine protein kinase domains that form an asymmetric dimer, leading to a conformational change that renders the kinases catalytically competent. This is followed by phophosphorylation in trans (one kinase phosphorylates the other) of the C-terminal segments of the receptor. The dimerized, phosphorylated molecule constitutes the active receptor, able to attract adaptors and effectors and able to phosphorylate other substrates.
(b) The insulin receptor is already dimerized and yet requires ligand to reveal its tyrosine kinase activity. Here it is postulated that receptor occupation causes the correct orientation of the two kinase domains so that they can phosphorylate each others' activation segment. This leads to their activation and is followed by phosphorylation of other receptor-residues and then phosphorylate of other substrates. The phosphotyrosines act to recruit adaptors and effectors.

FIG 8.2 Recruitment of SH2- and PTB-containing proteins to activated tyrosine kinase receptors
(a) receptor signalling complex formation through the binding of SH2- and PTB-containing proteins with the EGF receptor (adaptor proteins Grb2 and Shc respectively). (b) Molecular detail of an SH2-binding sequence interacting with a peptide of the EGF receptor. The phosphotyrosine and asparagine bury themselves into the SH2 domain and constitute the key determinants of the interaction between the receptor and Grb2. The loss of phosphate weakens the interaction and causes separation of the two proteins.
(c) Recruitment of Grb2/Sos results in activation of the GTPase Ras. Sos is a guanine exchange factor which facilitates the exchange of GDP for GTP. Ras is active in its GTP-bound state. Hydrolysis brings Ras back in its inactive GDP-bound state.

FIG 8.3 Domain organization of proteins that associate with phosphorylated tyrosine kinase-containing receptors.
Proteins that associate with tyrosine-phosphorylated receptors contain either SH2 or PTB domains. Unlike the enzymes, the adaptors and docking proteins lack intrinsic catalytic activity but serve to link phosphorylated receptors with other effector proteins. Some of the proteins presented in this figure are discussed in this chapter.

FIG 8.4 ERBB receptor family, their ligands and some of their adaptors and effectors
EGF receptors, ERBB1 to -4, form different receptor combinations when bound to ligand. The different receptor dimers, in turn, recruit different sets of effectors and adaptors. Note that ERBB2 does not bind ligand, it naturally occurs in an extended confirmation but cannot form homodimers (ERBB2/ERBB2). It is the favourite partner of ligand-bound EGFR (ERBB1) and of ERBB3. Note also that ERBB3 lacks kinase activity. However, it can nevertheless act as an "active receptor" by recruiting adaptors and effectors after being phosphorylated by ERBB2.

FIG 8.5 Branching of the signal transduction pathways.
Following activation of receptor tyrosine kinases, several signal transduction pathways can be activated. Five of these are indicated. Further details feature in the following paragraphs and figures.

FIG 8.6 Regulation of the Ras-ERK pathway by receptor protein tyrosine kinases.
The adaptor protein Grb2, in association with the guanine exchange factor Sos, attaches to the tyrosine-phosphorylated receptor through its SH2 domains. This brings the Grb2/hSos complex into the vicinity of the membrane where it catalyses guanine nucleotide exchange on Ras. The activated Ras associates with the serine/threonine protein kinase C-Raf. Its localization at the membrane results in activation and subsequent phosphorylation of the dual specificity kinase MEK1 (at S217 and S221), which in turn phosphorylates ERK2 on a threonine residue (T183) and on a tyrosine (Y185) residue. Dimerization allows ERK2 to enter the nucleus (by an as of yet unknown mechanism). The insert shows a ribbon presentation of the kinase domain of ERK2 with its two phosphorylated residues (pT183 and pY185). Inside the nucleus ERK2 phosphorylates Elk-1 which then associates with SRF to form an active transcription factor complex. This binds to DNA at the serum response element (SRE). ERK2 also phosphorylates and stabilizes c-Fos, which, in complex with c-Jun, binds to DNA at the AP-1 sequence. Both SRE and AP-1 induce a strong expression of c-Fos as well as genes involved in the onset of cell proliferation (e.g. cyclin D).
Insert : PDF file: 2ERK

FIG 8.7 Central role of the serine/threonine phosphatase PP2A in the formation of a productive Ras-MAP kinase signalling cassette.
The serine/threonine phosphatase PP2A plays two important roles in the onset of the Ras-ERK pathway. Both effect the relief of inhibitory constraints imposed by the phosphoserine-binding scaffold protein 14-3-3.
(a) C-Raf and the MEK/ERK-signalling cassette are blocked through their association with the scaffold protein 14-3-3. With respect to the signalling cassette, this attachment is made possible through phosphorylation of KSR1 on serine residues by the kinase C-TAK1. Growth factor signalling causes the release of a regulatory subunit of PP2A (PP2A-B) which binds the catalytic domain, leading to its activation. (b) Activated PP2A dephosphorylates C-Raf on S259 causing detachment of 14-3-3 from the C-terminus. This allows C-Raf to associate with RasGTP. PP2A also dephosphorylates two residues in KSR1 (S297 and S392). (c) Dephosphorylation of KSR1 causes detachment of 14-3-3 and translocation of the signalling cassette to the membrane, there to bind to RasGTP.

FIG 8.8 ual effector interaction of RasGTP that leads to effective 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) and this initiates a series of ubiquitylations that mark the protein for destruction by the proteasome (3). MEK1 and ERK2, linked to the scaffold protein KSR1, are now able to join C-Raf, enabling the signal to pass from one kinase to another. (b) KSR1 has a number of conserved domains, CA2, proline rich domain, CA3, cysteine rich domain which resembles the PMA/DAG binding site (C1 domain) of PKC, CA4, Ser/Thr rich domain and CA5, a kinase domain that resembles that of Raf, but lacks an essential lysine and is therefore inactive. The domain architecture of IMP reveals a Zn-(RING)-finger, a motif known to be involved in the ubiquitylation of protein. IMP qualifies as an E3-ligase and binds E2-ubiquitin. The E3-ligase activity is activated by binding to RasGTP and this results in the auto-ubiquitylation of IMP, followed by its destruction.

FIG 8.9 Parallel MAP kinase pathways.
The MAP kinases can be classified in three groups, based on the identity of the intermediate residue in their dual phosphorylation motifs (TEY, TGY or TPY) in the activation segment. This classification also defines three distinct signal transduction pathways indicated as the ERK, the P38and the JNK kinase pathway, each having unique upstream activators. The ERK pathway is said to act in response to mitogens, whereas the p38 and JNK pathways are reserved for the response to stress and inflammatory cytokines.

FIG 8.10 Phosphorylation of the β-adrenergic receptor by GRK2 activates the Ras-ERK pathway.
Phosphorylation of the β adrenergic receptor by GRK2 allows recruitment of the adaptor β arrestin2 and terminates communication with the G-protein. It also activates the Ras ERK pathway by recruitment of Src (via its SH3-domain) and MEK1/ERK2. β arrestin2 also directs the receptor to clathrin-coated pits, there to be removed from the cell surface by endocytosis and degraded in the lysosomal pathway.

FIG 8.11 Phosphoinositide 3-kinases and the generation of 3-phosphorylated lipids.
(a) The PI 3-kinases phosphorylate the 3-OH-position in the inositol ring of the phosphatidylinositol lipids. The 3-OH phosphorylated inositol lipids are not substrates for PLC. The phosphatases PTEN and SHIP reverse the reaction. (b) composition of inositol lipids before and after phosphorylation by PI 3-kinase. The PH domain of PKB interacts preferentially with the PI-3,4,5-P3 product. (c) domain architecture of the type 1A PI 3-kinase family. The catalytic subunits (a, b and d) all possess a p85- and Ras-binding site. They also have a PI-C2 domain with which they interact with phospholipids. The PI 3K accessory domain serves as a spine on which the other domains are fastened. The regulatory subunits, p85a is particularly versatile, its SH3 domain interacts with proline rich sequences, its BCR/GAP domain interacts with monomeric GTPases of the Rho family (Cdc42 and Rac), whereas its SH2 domain interacts with phosphotyrosines.

FIG 8.12 Activation of PI 3-kinase by the insulin receptor.
Insulin binding to the insulin receptor dimer (1) induces a conformational change (2) (as of yet to be determined how) that causes trans-phosphorylation of the activation segments at three tyrosine residues (3). Further phosphorylation follows both upstream and downstream of the catalytic domain. The IRS-1 (insulin receptor substrate-1) binds phosphotyrosine-972 (also numbered Y960, situated in the NPEY motif) with its PTB domain (phosphotyrosine binding domain) (4). Phosphorylation of a number of tyrosine residues follows (5) which then serves as a docking site for the SH2-domains of the p85 regulatory subunit of PI 3-kinase (6) leading to the generation of phosphatidylinositol-3,4,5-trisphosphate.

FIG 8.13 Mechanism of activation of protein kinase PKB.
Left panel. Generation of phosphatidylinositol 3,4,5-trisphosphate (1) serves as a membrane recruitment signal for PKB and PDK1. (2). A first phosphorylation of PKB occurs in the C-terminal hydrophobic motif (3). The identity of the kinase, tentatively named PDK2, remains elusive. Possible candidates are the complex mTOR/Rictor and PKB itself (low level of autophosphorylation). Close apposition of PKD1 causes binding of the phosphorylated hydrophobic motif to the αC-helix of PDK1 (4). The kinase now fully competent and phosphorylates the activation segment of PKB. Full kinase activity is achieved (5). Detachment of activated PKB from the membrane may occur after dephosphorylation of PI-3,4,5-phosphate by PTEN (or SHIP) (6).
The sequences in the lower right hand corner box shows the position of the phosphorylated threonine (T*) and serine(S*) residues (the hydrophobic motif is FPQFS).
Right panel. Domain architecture of AGC kinases whose activity are controlled by two phosphorylation sites, one in an hydrophobic motif, which is sometimes part of a bigger autoregulatory C-terminal domain, and one in the activation segment. For these protein kinases, PDK1 acts as the "master switch" by phosphorylating the activation segment.

FIG 8.14 Regulation of mTOR by PKB-mediated phosphorylation of TSC2.
(a) One of the substrates of PKB is the protein TSC2. This, in complex with TSC1, acts as a GTPase activing protein (GAP) of the monomeric GTPase Rheb. TSC1/TSC2 keeps Rheb in its inactive state and this holds protein synthesis in check. Insulin augments the rate of protein synthesis by activation of Rheb. This occurs through phosphorylation and inactivation of TSC2 by PKB. Rheb gradually accumulates in its GTP bound state (by an as yet unidentified guanine exchange factor) and interacts with the protein kinase mTOR, complexed to Raptor (and GaL, which is not shown).
(b) Binding of Rheb to mTOR/Raptor somehow unveils its kinase activity and causes the complex to associate with eIF3. The first action of mTOR/Raptor is to phosphorylate S6K1 (1), in its C-terminal segment, which causes its detachment from eIF-3 (2). This enables the initiation factor to join the mRNA (3) and to start to assemble other initiation factors that eventually form the protein translation initiation complex.

FIG 8.15 A series of ordered phosphorylation events facilitate the assembly of the translation initiation complex on the mRNA.
(a) Activation and association of mTOR with the initiation complex causes the phosphorylation of 4E-BP (indicated as "BP") (1). Phosphorylated 4E-BP detaches from eIF-4E.
(b) The dissociation of 4E-BP (2) permits the association of a big initiation factor eIF-4G that interacts with eIF-4E, eIF-3 and eIF-4A (3). Partly phosphorylated S6K1 is now fully activated by PDK1 through phosphorylation of its activation segment (4). S6K1 phosphorylates the ribosomal S6 protein (rbS6) and phosphorylates eIF-4B, a regulatory component of the RNA helicase that next joins the subunit eIF-4A (5).
(c) The conditions are now favourable for binding of PABP (poly-A binding protein) which is attached to the 3'-poly A tail of the mRNA (6). The initiation complex now moves towards the start AUG codon (7) where it will be joined with the 60S ribosomal particle.

FIG 8.16 Regulation of translation and transcription by insulin and growth factor receptors
Both the Ras/MAPK and the PI 3-kinase/PKB pathway affect protein synthesis and gene transcription. This is an effective way to alter the cellular proteome necessary to respond to the signals of insulin and growth factors.

FIG 8.17 Direct phosphorylation of STAT transcription factors.
Through their SH2 domains, STAT1a and STAT1b bind to the tyrosine-phosphorylated receptor and become phosphorylated. They then form a dimer, (called a Sis-inducible factor, SIF) which translocates to the nucleus, where it binds to a Sis-inducible element (SIE) within the fos promoter.

FIG 8.18 Non-receptor protein tyrosine kinase families.
These protein kinases are subdivided in 10 families (Src-A and Src-B are shown as one "Src" family). Most of them contain SH2 and SH3 domains which plays an important role in the regulation of kinase activity. Several were originally discovered as transforming genes of a viral genome, hence names like Src or Abl, derived from Rous sarcoma virus or Abelson murine leukaemia virus. The acronym in between brackets indicates the family member of which the domain architecture is illustrated.

FIG 8.19 Regulation of Src kinase activity.
Phosphorylation of the C-carboxy terminal tyrosine of Src causes binding of its own SH2 domain. This event places the SH3 domain adjacent to the N-terminal lobe of the kinase domain which affects the coordination of ATP (orange) through a change in the position glutamate-310 in the C-helix. Detachment of the SH2 domain, through dephosphorhylation of the carboxy-terminal tyrosine (or through binding of the SH2 domain to tyrosine phosphates of other proteins) removes this restraint (1). Subsequent phosphorylation of tyrosine-419 in the activation segment liberates the entry path for substrate (2); the protein kinase is now catalytically competent.

FIG 8.20 Signalling complex formation at the TCR complex.
The TCR bound to antigen/MHC activates Lck that phosphorylates the two CD3 ζ-chains in the ITAM motif (1). The phosphotyrosine residues form a docking site for the SH2 domains of ZAP70 (2), another cytosolic PTK. ZAP70 is phosphorylated by Lck in the linker region between the SH2 domains and the catalytic domain (3). Phosphorylated and activated ZAP70, in turn, phosphorylates several (maximally nine) tyrosine residues on the transmembrane adaptor protein LAT (4). Various proteins attach onto LAT. These include PLC-γ (5), which upon attachment gets phosphorylated and activated by Lck (6). Other proteins are the guanine exchange factor Vav, the adaptor proteins Grb2 and SLP76 and the p85 regulatory subunit p85 of PI 3-kinase. All of these play important roles in the activation of the IL-2 gene. Production of diacylglycerol and IP3 (7) by PLC-γ is an important starting point for the two signalling pathways described in more detail in this section.

FIG 8.21 Two signalling pathways downstream of PLC-γ.
Activation of PLC-g results in the production of diacylglycerol and IP3. These second messengers activate two signalling pathways. One involves IP3-mediated release of Ca2+ from intracellular stores and result in the activation of the serine/threonine phosphatase calcineurin. This leads to activation of NFAT. The other involves the diacylglycerol-mediated activation of PKCΘ which phosphorylates the adaptor protein CARMA1. The unfolded protein acts as a docking site for the assembly of a TRAF6-ubiquitin ligase complex that results in the activation of IKKβ and subsequent nuclear translocation of NFκB. The genes whose transcription is regulated by NFAT and NFκB are involved in the regulation of the immune response. Expression of IL-2 is particulary important for the clonal expansion of the activated T-cells (Th1 subtype).

FIG 8.22 Calcineurin-mediated activation of NFAT1.
Activation of PLC-g results in the liberation of IP3 into the cytosol. This binds to its receptor and causes release of Ca2+ from intracellular stores. Ca2+ binds to calmodulin (CaM) and calcineurin B (CnB), leading to the activation of the serine/threonine phosphatase PP2B. The phosphatase dephosphorylates numerous phosphoserine residues in the N-terminal segment of the transcription factor NFAT1 and this causes exposure of the nuclear localisation signal (NLS) together with a masking of the nuclear export signal (NES). NFAT enters the nucleus and associates with c-Jun and c-Fos (AP-1 complex) to drive gene expression.

FIG 8.23 PLC-γ-mediated activation of PKCΘ causes activation of the TRAF6 E3-ubiquitin ligase complex The production of diacylglycerol by PLC-γ causes membrane attachment, followed by multiple phosphorylations and activation of PKCΘ (theta), a member of the nPKC subfamily which lacks a functional C2-domain and therefore does not require Ca2+ for its activation. PKCΘ phosphorylates the adaptor protein Carma1 resulting in its unfolding. The CARD domain of CARMA1 recruits another adaptor, Cbl-10, via its CARD domain. This brings Malt1, TRAF6 (E3-ligase) and Ubc13/Mms2 (E2-conjugating enzyme) into the complex. As a result, TRAF6 is poly-ubiquitylated. The K63-type ubiquitin chain acts as a docking site for the NEMO/IKKα/IKKβ as well as the TAB1, -2/TAK1 protein kinase complex (not shown).

FIG 8.24 IKK-β-mediated activation of the RelA/NFκB complex Phosphorylated/activated IKK-β phosphorylates IκB on two serine residues in its N-terminal domain, allowing its recognition by the SCF/βTrcp ubiquitin-ligase complex. Following ubiquitylation (K48-type), IκB is marked for destruction by the S26 proteasome, thus liberating RelA (p65) and NFκB1 (p50) and exposing their nuclear localisation signal (NLS). They enter the nucleus, there to bind DNA at the κB-element, driving expression of inflammatory cytokines.

FIG 8.26 Adhesion-mediated survival.
The focal adhesion site promotes cell proliferation signals through activation of Ras. The Src phosphorylated Tyr925 acts as a binding site for the SH2 domain of Grb2. This interaction recruits the Ras guanine exchange factor Sos to the membrane, leading to activation of Ras. Ras-GTP initiates the activation of the Raf-ERK pathway, necessary for initiation of the cell cycle. The focal adhesion site promotes cell survival signals through activation of serine/threonine protein kinase-B (PKB). The phosphorylated Tyr397 residue of focal adhesion kinase (FAK) provides a binding site for the SH2 domain of the regulatory subunit (p85) of the lipid kinase phosphatidyl inositol 3-kinase (PI 3-kinase). Subsequent production of PI-3,4,5-P3 provides a binding site for the PH domain of PKB (and PDK1). After its activation (through phosphorylation of a serine (in hydrophobic motif) and a threonine (in activation segment) residue, PKB phosphorylates a large number of proteins that directly or indirectly deal with cell death (see text for further detail).

FIG 8.27 Adhesion-mediated cell cycle control.
(a) In epithelial cells, intergrin α6β4, attached to the extracellular matrix, forms a special adhesion complex named hemi-desmosome. These complexes are linked to intermediate filaments via plectin (a protein that resembles plakoglobin). ERBB2/3 receptors are recruited into these complexes leading to phosphorylation of ERBB2 onTyr869 by Src bound to FAK (1). This phosphorylation promotes the catalytic activity of the tyrosine kinase domain of ERBB2 and enhances growth factor receptor signalling output. (Note that ERBB3 is kinase dead and cannot phosphorylate ERBB2). Src also phosphorylates STAT3 and this signal is enforced by a second phosphorylation on serine through ERK2 (3). Both phosphorylations enhance its transcriptional activity. In the case of breast tumor cells, this pathway promotes cellular invasion. Finally, the α4-integrin subunit is also a target of Src and this may affects its interaction with plectin (4).
(b) Two exemples of FAK signaling via the intermediate of CAS. The phosphotyrosines of CAS bind the SH2 domain of Crk. The proline rich region (pro) of the guanine exchange factors C3G and 180DOCK bind the SH3 domain of Crk. Their recruitment to the focal adhesion complex causes the activation of Rab1 and Rac1. Rab1 signals to B-Raf, which then phosphorylates MEK1, thus enforcing the growth factor receptor-stimulated Ras-ERK pathway, whereas Rac1 stimuates PAK1, which signals to JNK1. Both ERK and JNK stimulate expression of genes that initiate progression into the G1 phase of the cell cycle (cyclinD, c-myc etc).

FIG 8.28 Removal of the EGFR from the cell surface and sorting into the lysosome pathway.
Activated EGF receptors are recognized by Cbl which either binds directly through a phosphotyrosine-binding motif or by interaction with the SH3 domain of Grb2. Cbl causes mono-ubiquitylation of the EGFR and this acts as a sorting signal directing the receptor into the lysosomal pathway for degradation. The receptor-Cbl complex is recognized by CIN85 and endophilin which couple the receptor to a complex of proteins that includes the key endocytic adaptor AP-2. The complex then recruits clathrin monomers. As a result, active EGFRs accumulate in clathrin-coated membrane pits which then pinch off from the plasma membrane as endocytic vesicles. Within the intracellular network of vesicular transport pathways, the receptors are sorted into a pathway that takes them via the early and late endosomes towards the lysosome. They are thus destroyed.

FIG 8.29 Attenuating growth factor signalling.
(a) the Ras-ERK1 pathway has a number of feed back controls. The activated ERK1 or its downstream kinase RSK1 negatively feed back on Sos (1) and on C-Raf (2). ERK1 also induces expression of the dual specificity phosphatase MKP-1 (3) which leads to its deactivation. ERK1 also induces expression of the Epha2 receptor which, if neighbouring cells express its ligand ephrin-A1, causes attenuation of the Ras signal.
(b) The PI 3-kinase/PKB pathway also exerts negative control, in part by S6K1-mediated phosphorylation of IRS1 and IRS2 (serine/threonine phosphorylations that block the SH2/PTB docking function). mTOR reduces expression of the PDGF receptor.
(c) C-Raf is regulated by numerous phosphorylations. With respect to negative regulation by ERK1, phosphorylation of serine residues 29, 289, 296, 301 and 642 all excert an inhibitory action.
(d) The tyrosine protein phosphatase PTP1B plays a key role in the regulation of the insulin signal. Its presence reduces phosphorylation of the receptor (removal of phosphatases in the activation segment) and of the IRS-1 and -2 docking proteins. The consequences are a lack of cell membrane expression of the glucose transporter Glut-4 and a low level of glycogen synthesis (symptoms of diabetes).

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Last Updated January 17, 2011 10:00 PM | admin news