Structure and Function in Cell Signalling

Structure and Function in Cell Signalling......Page 1
Contents......Page 7
Acknowledgments......Page 19
Preface......Page 21
1 The components and foundations of signalling......Page 23
1.1.2 Glands and types of secretion......Page 24
1.1.4 Agonists......Page 26
1.1.5 Antagonists......Page 27
1.1.6 Receptors for first messengers......Page 28
1.1.7 Second messengers......Page 30
1.1.9 Membrane-bound second messengers......Page 31
1.2.1 When did the discipline of cell signalling begin?......Page 34
1.2.2 The discovery of hormones  Bayliss and Starling, 1902......Page 35
1.2.4 Peptide sequencing  Fred Sanger, 1951......Page 36
1.2.6 AcrasincAMP  the ancient hunger signal......Page 37
1.3.2 Fluoride  a stimulator of G proteins......Page 38
1.3.4 Heat-stable factor  cAMP......Page 39
1.3.6 The discovery of hormonally regulated protein kinases  phosphorylase kinase, serine phosphorylation and Ca2 Krebs and Fischer, 19581968......Page 40
1.3.8 cAMP-dependent protein kinase......Page 41
1.4.2 Oestrogen receptor directly detected by radioligand binding assays  Jensen and Gorski, 1962......Page 42
1.4.5 Magnesium......Page 43
1.4.7 GTP (contaminant of ATP) lowers 7-pass receptor affinity......Page 44
1.4.8 GTP analogues and adenylyl cyclase activation......Page 45
1.4.9 cAMP toxicity and clonal mutants of S49 cells......Page 46
1.4.11 Use of bacterial toxins......Page 47
1.5.1 A simple mammalian signalling pathway  F-2,6-bisP as a second messenger......Page 48
1.5.2 PFK-1 and FBP-1......Page 50
1.5.4 Control of PFK-2/FBP-2 by phosphorylation  liver......Page 51
1.5.5 Control of PFK-2/FBP-2 by phosphorylation  heart......Page 52
1.6.1 Slime moulds......Page 54
1.6.2 cAMP andE. Coli......Page 56
References......Page 57
2.1 Enzyme steady state assays  Michaelian enzymes......Page 61
2.1.2 Steady state......Page 62
2.1.4 Vmaxis reached when the enzyme becomes saturated......Page 64
2.1.5 What does theKM mean?......Page 65
2.2.1 Equilibrium......Page 67
2.2.3 Bmax the maximum binding capacity is a count of the receptors in a sample......Page 69
2.2.4 The meaning ofKD......Page 71
2.2.5 Displacement assays......Page 72
2.3 The receptors environment......Page 73
2.3.1 Heterogeneity of binding sites  positive cooperativity......Page 74
2.3.2 Binding site heterogeneity  two site models versus negative cooperativity......Page 75
2.3.3 Negative cooperativity of the insulin receptor or two site model......Page 77
2.3.4 Site heterogeneity of the EGF receptor  independent two site model?......Page 79
2.4 Guanine nucleotides and the agonist affinity-shift of 7-pass receptors......Page 81
2.4.1 The ternary complex equilibrium model......Page 82
2.4.3 The thermodynamic catalytic or kinetic model......Page 84
2.4.4 Constitutive signalling in the absence of ligand......Page 86
2.4.6 Agonist binding in membrane preparations where the cognate G protein is in unlimited supply......Page 87
2.4.7 In vivoGTP versus GDP concentrations......Page 88
References......Page 89
3 Modules and motifs in transduction......Page 93
3.1.1 Src-homology-1 (SH1) region represents the tyrosine kinase domain......Page 94
3.1.2 Src-homology-2 (SH2) modules are phosphotyrosine- binding domains......Page 95
3.1.3 Src-homology-3 (SH3) modules are polyproline-binding domains......Page 97
3.1.4 Src-homology-4 (SH4) motif and Src unique domain......Page 100
3.1.5 The C-terminal Src regulatory motif and Src family autoinhibition......Page 104
3.2 PH superfold modules: PH-, PTB- and PDZ-domains......Page 107
3.2.2 PTB domains  phosphotyrosine binding modules......Page 108
3.3 Bcr-homology (BcrH) domains......Page 110
3.4 Dbl homology (DH) domains  partners of PH domains......Page 111
3.6 Ras binding domains......Page 112
3.7 Phosphoserine/phosphothreonine-binding domains......Page 114
3.7.1 14-3-3 proteins......Page 115
3.8 EF-hands  calcium-sensing modules......Page 117
3.9 C1 and C2 domains  a Ca2 -activated, lipid-binding, module......Page 118
References......Page 119
4 Protein kinase enzymes  activation and auto-inhibition......Page 123
4.1.1 Invariant residues......Page 124
4.1.2 The phosphate-binding loop or p-loop......Page 126
4.1.3 Critical differences between serine/threonine kinases and tyrosine kinases......Page 129
4.1.4 Closed and open conformations......Page 130
4.1.5 The catalytic loop or C-loop......Page 131
4.1.6 The activation segment/loop or A-loop......Page 134
4.2 Protein kinases activated by A-loop phosphorylation......Page 135
4.2.1 Phosphorylation of A-loop residues and assembly of active site......Page 136
4.2.3 ATP binding......Page 137
4.3 The insulin receptor kinase (IRK)  a gated kinase......Page 138
4.4 Cyclin dependent kinases......Page 141
4.4.1 Monomeric Cdk2 structures......Page 142
4.4.3 Cyclin-bound phosphorylated Cdk......Page 145
4.5 Secondary inhibition mechanisms  PKA......Page 146
4.5.2 The extended binding surface of RSUB......Page 148
4.5.3 Effects on cAMP binding at CBD-A......Page 151
References......Page 153
5 7-pass receptors and the catabolic response......Page 155
5.2 Functional mechanisms of 7-pass receptors......Page 156
5.2.2 Gaq-coupling receptors  bombesin- anda1-adrenergic receptors  stimulation of calcium release from the endoplasmic reticulum......Page 157
5.2.3 Gai-coupling receptors  somatostatin anda2-adrenergic receptors  inhibition of adenylyl cyclase, activation of K ion channels, inhibition of Ca2channels, and activation of phospholipase Cb2......Page 158
5.3 Amplification......Page 159
5.3.4 How much cAMP is needed?......Page 161
5.4.1 Adenylyl cyclase  signal termination and PDE isoforms......Page 162
5.5.1 Transmembrane isoforms of adenylyl cyclase......Page 163
5.6 G proteins and the adenylyl cyclase effector isoforms......Page 165
5.6.4 a1-adrenergic receptor stimulation of glycogenolysis......Page 170
5.7 Regulatory subunits of PKA and A-Kinase Anchoring Proteins......Page 171
5.7.1 RII regulatory subunits  reversible phosphorylation and scaffolding......Page 172
5.7.2 Segregation of pathways......Page 175
5.7.3 PKA and its inhibitors......Page 176
5.8.1 PhK structure......Page 177
5.8.3 Regulatory subunits......Page 178
5.8.5 Possible mechanisms of activation and holoenzyme conformation......Page 179
5.9.1 Glycogen phosphorylase isoforms......Page 182
5.9.2 Glycogen phosphorylase allosteric sites......Page 183
5.9.3 Control by hormones or metabolite effectors  functional differences between muscle and liver isoforms......Page 184
5.9.4 How do these properties of GP isoforms fit with metabolic necessities?......Page 185
5.9.7 Activation by phosphorylation......Page 187
5.9.8 Activation by 50-AMP......Page 190
5.10 Glycogen synthase......Page 191
5.10.1 GSK-3  a multi-tasking enzyme......Page 192
5.11.1 Protein kinase C......Page 193
5.11.3 Alternative DAG/phorbol ester receptors......Page 194
5.11.5 What does PKC actually do?......Page 195
5.12 G protein coupled receptor kinases  downregulators, signal integrators......Page 196
References......Page 197
6.1 Receptor tyrosine kinases  ligands and signal transduction......Page 201
6.1.1 RTK ligands and receptors......Page 202
6.2 The PDGFR family  signal transduction......Page 203
6.2.1 PDGFR signal transduction particle......Page 204
6.2.2 MAP kinases and MAPK kinases......Page 206
6.2.3 PDGFR kinase insert tyrosines  PI-3-kinase, and RasversusRac......Page 208
6.2.5 PDGFR, PI-3-kinase, Rac and motility......Page 209
6.2.6 PDGFR insert phosphotyrosines and Ras regulators......Page 210
6.2.7 Sos-1  a bi-functionalguanine nucleotideexchange factor (GEF)......Page 211
6.2.8 Sos  the switch from RasGEF to RacGEF......Page 212
6.2.11 Shc......Page 214
6.2.13 PDGFR Juxtamembrane tyrosines......Page 215
6.3.1 PDGFR juxtamembrane and A-loop tyrosines! phenylalanines......Page 216
6.3.4 PDGFR A-loop (Y!F) mutant cannot bind exogenous substrate polypeptides......Page 217
6.3.6 PDGFR Y579/581A is constitutively active......Page 218
6.4.1 Flt-3 juxtamembrane interactions and autoinhibition/activation......Page 219
6.5.1 EGFR family members and ligands......Page 222
6.6 ErbB-type receptor signal transduction particles......Page 224
6.6.1 The epidermal growth factor receptor kinase  a pre-assembled active site......Page 226
6.7 Autoinhibition of EGFR and activation......Page 227
6.7.1 Ligand binding, dimerisation and activation......Page 228
6.7.2 The EGFR juxtamembrane domain  a nexus for crosstalk......Page 229
6.7.3 EGFR activation and calcium......Page 231
References......Page 233
7 G proteins (I)  monomeric G proteins......Page 237
7.1 Classification......Page 238
7.2 ON and OFF states of Ras-like proteins......Page 239
7.3 Raf  a multi-domain serine/threonine kinase family of Ras effectors......Page 240
7.3.1 Raf-Ras binding  translocation of Raf from cytosol to membrane......Page 241
7.3.3 Raf activation by translocation......Page 243
7.3.4 B-Raf is less stringently inhibited than C-Raf......Page 244
7.3.7 MAPK scaffolds......Page 245
7.3.8 Signal termination......Page 246
7.4 Ras protein structure and function......Page 247
7.4.2 The P-loop (G-1)......Page 248
7.4.3 Switch I (G-2)......Page 250
7.4.4 Switch II (G-3)......Page 252
7.5 The switch mechanism: hydrolysis-driven conformational change in Ras......Page 253
7.6 GTP hydrolysis......Page 254
7.7.1 RasGAP......Page 256
7.7.2 RasGEFs......Page 258
7.7.3 The Ras effector region and Raf binding......Page 261
7.7.4 Rap1 and cAMP effects......Page 262
References......Page 263
8 G proteins (II)  heterotrimeric G proteins......Page 267
8.1 Classification and structural relationship with Ras......Page 268
8.2 Ga-subunits: the Ras-like core, G-boxes and switch regions......Page 271
8.2.2 Switch I/G-2......Page 272
8.2.4 Switch II/G-3......Page 275
8.3 GTP exchange, hydrolysis and switch movements......Page 276
8.3.1 GTP conformations......Page 277
8.4.1 Theb/gbinding site of GDP-occupieda......Page 278
8.4.2 The receptor-binding interface of GDP-occupied G proteins......Page 279
8.4.4 Switch II helix rotation......Page 281
8.4.5 Switch II  the primary effector-binding surface of a-subunits......Page 284
8.5.1 RGS proteins and GTPase activation......Page 285
8.5.2 RGS proteins: inhibition of nucleotide exchange;crosstalk with other pathways......Page 286
8.5.4 GRKs  RGS domain-containing S/T-kinases......Page 287
8.6 Signal transduction byb/csubunits......Page 288
References......Page 290
9.1 The insulin receptor  a pre-dimerised RTK with a unique substrate......Page 293
9.1.2 Three clusters of autophosphorylated tyrosines in the InsR intracellular region......Page 295
9.2 InsR and IGF-IR: differentiation leads differential tissue effects......Page 297
9.3 Features of metabolic control in key tissues......Page 298
9.4.1 MAPK/p90Rsk pathway only mediates growth effects......Page 299
9.5 The insulin receptor substrate  a surrogate signal transduction particle......Page 300
9.5.1 IRS protein targetting......Page 301
9.6 IRS-1/2 phosphorylation and PI-3-kinase activation......Page 302
9.7.1 Glycogen granule targetting of PP-1......Page 303
9.7.2 p70Rsk  inducer of GS dephosphorylation?......Page 304
9.8.2 Insulins reversal of adrenaline- and glucoagon-induced glycogenolysis in liver......Page 305
9.9 PIP3 downstream effects  glycogen synthesis......Page 307
9.9.1 PKB and GSK-3 inactivation......Page 308
9.9.3 PIP3 downstream effects  GLUT4 mobilisation......Page 309
9.10.1 Insulin activates the Erk1/2 MAPK pathway  why, then, is the insulin receptor not as mitogenic as the PDGF receptor......Page 311
9.10.3 The insulin receptor and the IGF-I receptor are homologues  why is one anabolic and the other mitogenic?......Page 312
9.10.4 Do differingC-terminal tails cause differing regulation of growth responses in InsRversusIGF-IR?......Page 313
9.10.5 IFG-II, insulin receptor-A and half receptors......Page 314
References......Page 315
10 Mitogens and cell cycle progression......Page 319
10.1.2 The cyclin model......Page 320
10.1.4 Mammalian cyclin cycle model......Page 323
10.1.5 Embryonic cell cycle has no gaps......Page 324
10.2.1 What is G0?......Page 325
10.2.3 Growth factors and the fibroblast cell cycle......Page 326
10.3 Oncogene products derived from growth factor pathway components......Page 327
10.4 Transcription and cyclins......Page 328
10.5.2 Inactivating phosphorylations of Cdks......Page 329
10.6.1 APC/cyclosome (APC/C) and SCF  E3 ubiquitin ligase complexes......Page 331
10.6.3 SCF......Page 332
10.8.2 Myc......Page 333
10.8.5 The serum response factor  MADS box-containing transcrition factors......Page 334
10.8.7 Activation of Jun (and Myc) by inactivation of glycogen synthase kinase-3 (GSK-3)......Page 335
10.8.8 AP-1 complexes  bZip transcription factors......Page 337
10.8.9 AP-1 response elements on DNA......Page 340
10.10 Retinoblastoma-related pocket proteins  negative modulators of E2F......Page 341
10.10.1 Phosphorylation/inactivation mechanism......Page 343
10.10.2 The E2F family of transcription factors  the targets of the pocket proteins......Page 345
10.10.4 E2F targets  genes for DNA replication and licensing, delayed early response genes (cyclin E and A), and NPAT......Page 346
10.11.1 Cyclin E/Cdk-2 substrates  RB , NPAT, nucleophosmin......Page 347
10.11.2 Cyclin E  licensing and loading of helicase......Page 348
10.12.2 Terminating S-phase  cyclin A effects......Page 349
10.13.3 Pre-replicative complex formation begins in G1......Page 350
10.13.4 Helicase loading......Page 352
10.13.6 Origin firing  Ddk and Cdc45......Page 353
10.14.1 What triggers mitosis?......Page 354
10.14.2 POLO  the ultimate mitotic trigger?......Page 355
10.15.1 The INK proteins......Page 356
10.15.2 The Cip/WAF family......Page 357
10.16.1 p53 and Cip/WAF......Page 358
10.16.2 Mdm2 and p19Arf control of p53......Page 359
10.16.4 Apoptosis or cell cycle arrest  majority verdict by a jury of Cdk inhibitors, survival factors, and pro-apoptotic factors......Page 360
10.16.6 p53 and apoptosis......Page 361
10.17.1 The Gsp oncogene......Page 362
10.17.2 Wnt/b-catenin......Page 363
10.18 Concluding remarks and caveats......Page 367
References......Page 369
A.1.1 An alkaline phosphatase assay......Page 377
Appendix 2: RasMol: installation and use......Page 387
Index......Page 399