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DO NOT PRINT THIS PRESENTATION!! • There are over 100 slides in this presentation • View these animations to illustrate the lecture notes. • anyone caught printing this on lab computers will be severely dealt with by Dr Denyer (oooh scary!) ‘When Good Signals go Bad’ Lecture 18 BCHM2072 2006 Vanessa Gysbers www.toothpastefordinner.com problems with signals • activity – excess ON signal • accelerator stuck down – lack of OFF signal • brakes fail • activity/absence • affect ligands, receptors, downstream transducers, or targets excess G-protein coupled Toxin Cholera receptors Pertussis Intrinsic tyrosine Her+ kinase receptors breast cancer deficient Night blindness (not covered in this lecture) FGFR Achondroplasia Non-receptor TKs Bcr-abl BTK CM Leukaemia Agglobulinemia Cytokine TNFa gc, JAK, ILxR receptors Rheumatoid Severe Combined ImmunoDeficiency arthritis? G-protein Coupled Receptors excess signal: lack of OFF cholera pertussis (whooping cough) Revision: GPCRs normally…. N GPCR G-protein GDP C Inactive G-protein bound to GDP N GPCR G-protein C GDP • • • • Ligand binds adrenaline serotonin glucagon vision N GPCR G-protein C GDP Changes shape of loop N GPCR C G-protein GDP G-protein can bind N GPCR C G-protein GDP GTP Exchanges GDP for GTP N GPCR C G-protein GTP GDP Exchanges GDP for GTP N GPCR C G-protein GTP And becomes activated N GPCR C G-protein GTP a-subunit dissociates from bg N GPCR bg C Gsa GTP stimulatory a-subunit (Gsa) activates adenylate cyclase N a GPCR GTP C a-subunit (Gsa) activates adenylate cyclase N a GPCR GTP C a-subunit (Gsa) activates adenylate cyclase N AC a GPCR GTP C ATP cAMP cAMP = 2nd messenger N a GTP GPCR C ATP activates Protein Kinase A cAMP cAMP cAMP among many others PKA ACTIVE N a GTP GPCR C ATP PKA phosphorylates targets cAMP cAMP cAMP ACTIVE 2 ways to terminate the signal 1. hydrolysis of GTP GDP 2. activation of inhibitory G proteins 1. termination “OFF” a a AC GTP Active G-protein + GTP cAMP Gsa is a GTPase hydrolyses GTPGDP 1. termination “OFF” a a AC a GTP hydrolysis Active G-protein + GTP cAMP GTP GTP GDP GDP Gsa is a GTPase hydrolyses GTPGDP 1. termination “OFF” a a AC a a GTP hydrolysis GTP GDP GDP Active G-protein + GTP cAMP Gsa is a GTPase hydrolyses GTPGDP 1. termination “OFF” a a AC a a GTP hydrolysis GTP Active G-protein + GTP cAMP GDP GDP inactive G-protein + GDP stops activating AC cAMP Gsa is a GTPase hydrolyses GTPGDP 2. signal termination “OFF” N Inhibitory signal GPCR C bg Gia GDP different a subunit inhibitory signal activates Ginhibitory 2. signal termination “OFF” N Inhibitory signal GPCR C bg Gia GTP exchange GDPGTP 2. signal termination “OFF” N Inhibitory signal GPCR bg Gia GTP inhibitory G-protein inhibits AC cAMP Gia inhibits adenylate cyclase 2. signal termination “OFF” N Inhibitory signal GPCR bg Gia GTP inhibitory G-protein inhibits AC cAMP Gia inhibits adenylate cyclase Cholera toxin interferes with termination of GCPR signal • toxin binds to a receptor on enterocyte surface • enters cell by endocytosis Vibrio cholera Gsa GTP C cAMP cholera toxin cleaves inside cell = active enzyme a a Gs GTP C cAMP cholera toxin cleaves inside cell = active enzyme a a Gs cAMP GTP C NAD nicotinamide + ADP ribose NAD in active site of cholera toxin toxin catalyses ADP-ribosylation of active Gsa a a Gs GTP C cAMP ribosylation of Gsa prevents GTPase activity of Gsa a a Gs GTP C cAMP can’t hydrolyse GTPGDP continued activation of AC cAMP ribosylation of Gsa prevents GTPase activity of Gsa Gsa GTP AC cAMP Cholera toxin Gsa GDP can’t hydrolyse GTPGDP cannot stop activating AC cAMP CFTR Gsa GTP AC cAMP cAMP ions and water Cholera toxin Gsa GDP cAMP activate CFTR efflux ions and water diarrhoea pertussis also interferes with termination inhibits an inhibitor! N Inhibitory signal normally... GPCR Gia GTP Gia GDP Gia activated by inhibitory ligands exchanges GDP GTP inhibits AC cAMP N Inhibitory signal normally... GPCR Gia GTP Gia GDP Gia also a GTPase to terminate termination! N Inhibitory signal pertussis toxin... GPCR Gia GTP pertussis toxin Gia GDP ribosylates inactive Gia prevents exchange of GDPGTP N Inhibitory signal pertussis toxin GPCR Gia cAMP pertussis toxin GTP Gia GDP prevents the ACTIVATION of INHIBITOR compare…. Gsa AC Gia GTP GTP Cholera toxin Gsa pertussis toxin GDP Gia GDP cannot stop activating AC cannot inhibit AC accelerator stuck on brake failure Receptor Tyrosone Kinases (RTKs) excess signal: overexpression of receptors Her 2+ breast cancer cancer • Receptor tyrosine kinases are often receptors for growth factors • dysregulated signalling through RTKs dysregulated cell proliferation cancer homodimers or heterodimers GF GF Ligand binds EGF Her-2 chain • Growth factors – Epidermal GF – Platelet derived GF – Nerve GF • Insulin Ligand binds GF dimerisation GF activates Intrinsic tyrosine-kinase domains dimerisation GF P Tyr Tyr P Auto-phosphorylation of tyrosine residue GF P Tyr Tyr P SH2 docking of partners SH2 GF SH2 P Tyr Tyr P SH2 ENZYMES eg PLC-g PIP3 ADAPTORS eg Grb (recruits Ras) docking GF P Tyr SH2 P Tyr Tyr ENZYMES P SH2 Tyr P eg PLC-g PIP3 ADAPTORS eg Grb (recruits Ras) phosphorylation of tyrosines by receptor TK GF Ras P Tyr SH2 P Tyr Tyr P eg Ras mutated in ~15% cancers (60% of melanomas) activation of downstream targets Ras P Tyr MAPKs SH2 P Tyr Tyr P Ras activates downstream kinases eg. mitogen activated protein kinases Ras P Tyr SH2 P Tyr Tyr P MAPKs enter nucleus and activate transcription factors by phosphorylation P Jun Jun Her-2 (=erbB-2) • one type of chain in EGF receptors (=erbB) • overexpressed in ~30% breast Ca spontaneous dimerization of receptor without ligand constitutive receptor activation = pro-proliferative (poor prognosis) Her-2 overexpression P P P P Tyr Tyr P P spontaneous dimerisation WITHOUT LIGAND!!! eventual activation of MAPK MAPKs enter nucleus and activate transcription factors by phosphorylation P Jun Jun ALSO anti-anti proliferative! inhibition of a cdk inhibitor normally………… Cdk2 proliferative signal mitosis normally………… cdk2 inhibitor Cdk2 cdk2 inhibitor anti-proliferative signal arrest Her-2 overexpression cdk2 inhibitor Cdk2 P cdk2 inhibitor anti-proliferative signal cannot arrest Herceptin® (Trastuzumab) • • • • • Herceptin is a monoclonal antibody binds extracellular bit of Her-2 Receptor. disrupts dimerisation signal initiation activation of MAPKs phosphorylation of cdk2-inhibitor (via PI3K) can enter the nucleus inhibit cdk2 activity cell cycle arrest during G1 phase (Kute et al 2004). • proliferation Herceptin monoclonal antibody Tyr binds Her-2 prevents signal initiation cdk2 inhibitor cdk2 inhibitor Jun Her-2 overexpression + Herceptin cdk2 inhibitor Cdk2 P cdk2 inhibitor cdk2 inhibitor can inhibit cdk2 arrest Herceptin® • $$$$$ ($70K/year) • Aug 06 Herceptin® listed on the Pharmaceutical Benefits Scheme (PBS). • Around 2,100 patients are expected to be treated with Herceptin each year. • 46% decrease in reoccurrence not examinable! Receptor Tyrosone Kinases (RTKs) deficient signal: mutation of receptors achondroplasia Achondroplasia: • deficient RTK signal • mutation of Fibroblast Growth Factor Receptor (FGFR3 gene ) proliferation of cartilage at ‘growth plates’ in bone shorter bones, and shorter stature • 98% = Glycine Arginine in TMD • single point mutation of gene • arises spontaneously in 80% of cases!! – neither parent affected • then autosomal dominant inheritance • lethal if homozygous Cytokine receptors deficient signal: underexpression of receptor chains or of associated kinases Severe Combine Immunodeficiency Syndrome (SCID) cytokine revision!!! JAK JAK Typical cytokine receptor = Receptor that interacts with tyrosine kinases cytokine JAK JAK The interacting tyrosine kinase =JAK JAK = Janus kinase Hello my JAK twin!! Let me phosphorylate you, STAT and the receptor Hi JAK! Let me phosphorylate you, STAT and the receptor too! JAK: the two faced kinase Interleukin 3 JAK JAK • • • • Ligand binds Interleukins Interferons Growth hormone Erythropoietin Interleukin 3 JAK JAK dimerisation Interleukin 3 JAK JAK dimerisation Interleukin 3 P JAK JAK P JAKs meet: phosphorylate each other Interleukin 3 P JAK P JAK Tyr Tyr P P JAKs can then ph’late the RECEPTOR: Interleukin 3 P JAK P JAK Tyr Tyr P P JAKS ph’late tyrosine residues Interleukin 3 P Signal Transducer and Activator of Transcription JAK P JAK Tyr Tyr P P SH2 STAT STAT docks to P-tyr on Receptor Interleukin 3 P JAK SH2 STAT P P JAK Tyr Tyr P P SH2 STAT P STAT gets ph’lated by JAK Interleukin 3 P JAK SH2 P STAT P JAK Tyr Tyr P P SH2 STAT P P-STAT then dissociates Interleukin 3 P SH2 P STAT JAK P JAK Tyr Tyr P P SH2 STAT P P-STAT then dissociates P JAK P JAK Tyr Tyr P P SH2 P SH2 STAT STAT P then dimerises P JAK P JAK Tyr Tyr P P SH2 P SH2 STAT STAT P then translocates P JAK P JAK Tyr Tyr P P SH2 P SH2 STAT STAT P then translocates to the nucleus SH2 P SH2 STAT STAT P promoter Where it activates transcription specific ligand specific JAKS and STATS specific genes SH2 P SH2 STAT STAT P promoter eg immune cell development, anti or pro inflammatory genes interleukins (IL) gc ILxR P JAK SH2 P STAT P JAK Tyr Tyr P P SH2 STAT P gc chain shared by receptors of many different interleukins interleukins (IL) gc ILxR JAK SH2 JAK Tyr STAT affects immune cell development gene mutation of g chain X-linked Severe Combined ImmunoDeficiency IL-xR or JAK3 mutation less common specific for particular interleukins gc JAK SH2 Tyr SH2 STAT STAT autosomal recessive (AR)SCID affects fewer receptor types SCID • affects immune cell development • severe infections • g chain mutation worst: shared by many interleukins most common (50%) affects more cell types treatment of SCID • Gene therapy to replace defective gene • • • • gc chain JAK ILxR chain ADA Cytokine receptors excess signal: overexpression of ligand Rheumatoid arthritis normally: negative feedback loop via Supressors of Cytokine Signals TNFa P JAK SH2 STAT P SOCS is induced by same receptor JAK P Tyr Tyr P P SH2 STATP P P P P inhibits JAK target proteins for ubiquitin degradation SOCS normally: negative feedback loop via Supressors of Cytokine Signals TNFa P JAK JAK P SH2 STAT SOCS is induced by same receptor Tyr P ? excess TNFa ligand TNFa P JAK SH2 STAT P JAK P Tyr Tyr P P SH2 STATP P P P P activates transcription eg of inflammatory genes TNFa ? treatments for RhA eg Infliximab binds TNFa what would happen subsequently? P JAK SH2 STAT P JAK P Tyr Tyr P what about other potential targets? P SH2 STATP P P P P SOCS stimulate Non-receptor TKs excess signal: bcr-abl fusion protein chronic myelogenous leukaemia (CML) chronic myelogenous leukaemia bcr abl 22 controlSH3abl Philadelphia chromosome 9 = tyrosine kinase bcr abl bcr-abl fusion protein • fusion change in SH3 domain of abl • bcr-abl constitutively active SH3 domain constitutively active TK abl abl bcrSH3 ATP bcrSH3 ADP kinase domain Ras STAT myc Ras STAT myc uncontrolled proliferation, apoptosis Treatment = imatinib (Glivec ®) competes with ATP at active site of abl ATP abl glivec bcrSH3 Gleevec (purple) in active site of kinase Ras STAT myc Ras STAT myc structure of Gleevec Non-receptor tyrosine kinase deficient signal: mutation in B-cell Tyrosine Kinase (BTK) red spots show mutations that have been located in BTK X-linked agammaglobulinaemia normal btk signal B cell receptor PIP3K BTK phospholipase Cg Inosityl triphosphate (IP3) Ca2+ I certainly don’t expect you to know details of this diagram , but you should know the PIP3K, PLCg, IP3 pathway for Dr E-S, and appreciate how mutation of btk interrupts the pathway. X-linked agammaglobulinemia • mutation in btk (B-cell Tyrosine Kinase) prevents binding to upstream and downstream partners usually signals via phospholipase Cg Ca2+ X-linked agammaglobulinaemia btk (B-cell Tyrosine Kinase) needed for: • production of antibodies • B-cell maturation • deficiency humoral immunity • susceptible to BACTERIAL infections
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