Biol 2056 2024 L4 Lecture Notes PDF

Glucagon and Insulin antagonistically regulate glucose release in the Biol 2056 liver 2024 L4 Fed state Amino acids glu...

Glucagon and Insulin antagonistically regulate glucose release in the Biol 2056 liver 2024 L4 Fed state Amino acids glucose (GT Ga P) glucagon Insulin cAMP Liver + Glycogen - breakdown glucose  Biol 2056 Insulin Receptor is a member of the tyrosine kinase receptor superfamily 2024 L4 Approx 50 RTK In human genome Involved in cell proliferation, differentiation, migration. Critical role in development and tissue homeostasis. Examples of growth factors that function through tyrosine kinase Biol 2056 receptors 2024 L4 Factor Principal Primary Activity Comments Source PDGF platelets, promotes proliferation of two different protein chains form 3 distinct endothelial connective tissue, glial and smooth dimer forms; AA, AB and BB cells, placenta muscle cells EGF submaxillary promotes proliferation of gland, Brunners mesenchymal, glial and epithelial gland cells TGF- common in may be important for normal wound related to EGF transformed healing cells FGF wide range of promotes proliferation of many at least 19 family members, 4 distinct cells; protein is cells; inhibits some stem cells; receptors associated with induces mesoderm to form in early the ECM embryos NGF promotes neurite outgrowth and several related proteins first identified as neural cell survival proto-oncogenes; trkA (trackA), trkB, trkC IGF-I primarily liver promotes proliferation of many cell related to IGF-II and proinsulin, also called types Somatomedin C IGF-II variety of cells promotes proliferation of many cell related to IGF-I and proinsulin types primarily of fetal origin 3  Characteristics of the Tyrosine kinase receptor family. Biol 2056 2024 L4 Transmembrane proteins Catalytic receptors Contain a ligand binding site in a portion of the receptor located outside the cell An intracellular portion of the receptor contains a protein kinase active site The protein kinase is specific for phosphorylating tyrosine residues Ligand binding activates the protein kinase This results in autophosphorylation (self-phosphorylation) of tyrosine residues in the intracellular part of the receptor  Tyrosine Phosphorylation: serendipity but only when you are Biol 2056 very very smart: Tony Hunter. 2024 L4 Middle T antigen drives transformation https:// www.youtube.com/ watch?v=IU1JKUhjVgg How? Looking for PK. Tony hunter Eckhart W, Hutchinson MA, Dimerisation of ligand bound receptor tyrosine kinases is a commonBiol 2056 mechanism of transducing signals to the interior of the cell 2024 L4 Tyrosine GPCR kinase Ligand binding Tyrosine Kinase domain P DIMERISATI Ligand binding in the Difficult for ligand extracellular domains can binding ON mediate long range To induce long range conformational changes conformational Activation of which induce intracellular change in single receptor  Ligand induced dimerisation of tyrosine kinase receptors induces Biol 2056 cross phosphorylation, increased kinase activity and further 2024 L4 receptor tail phosphorylation Point of dimerization: 1. Consider catalytic domains flip between Inactive and active state but will be predominantly inactive due to inhibitory action of the activation loop. 2. Dimerisation enables cross phosphorylation and activation 3. Dimerisation leads to the phosphorylation of other intracellular tyrosine des of dimerization residues Ligand dependent no external repceptor interaction (TRKA ) Receptor dependent but not through ligand interactions (EGF) 4. Essentially conversion of Bi-ligand interaction across receptor interaction interface (KIT) ligand binding into a Ligand, receptor and accessory protein (FGFR heparan sulphate) conformational and chemical change (phosphorylation).  Activation-loop phosphorylation stabilizes the active Biol 2056 conformation of the insulin receptor tyrosine kinase (IRK). 2024 L4 Inactive kinase Active kinase The activation loop occludes the active site. Tyr1162, is bound in the active site but cannot be phosphorylated (in cis) because part of the A-loop interferes with the ATP binding site and the catalytic residue Asp1150 is improperly positioned to co-ordinate MgATP. Tyrosine phosphorylation of T1162 and 1163 and 1158, in trans, disrupts existing interaction and induces new interactions with arg1155 and arg1164 which displaces the activation loop by about 30A and enables ATP and substrate to bind. This movement facilitates a functional spatial arrangement of Lys1030 and Glu1047, SH2 domain binding to tyrosine phosphorylated residues generates Biol 2056 a simple output system describing tyrosine kinase signalling: 2024 L4 1. SH2 domain (Src homology) 2. PTB(Phosphotyrosine binding domain) SH2 domain present in about 100 proteins and thought to mainly interact with phosphorylated tyrosine residues. Approx 100aa and folds into a compact structure with a central b-sheet with two binding pockets. 27 PTB containing proteins in the human genome. Many PTB domains interact with non-phosphorylated tyrosine residues. 1 SH2 domain in yeast. Underpins the paucity of tyrosine kinase signalling Different SH2 domains interact with different tyrosine Biol 2056 phosphorylated peptides 2024 L4 Specificity is determined by two binding pockets 1. Tyrosine Phosphorylation of the intracellular receptor domains Biol 2056 drives the direct interaction and regulation of downstream 2024 L4 signalling proteins. 1. P85 is a regulatory subunit of PI3K. It contains a SH2 domain. When bound to the receptor PI3K becomes active and generates a new signalling molecule called PIP3. 2. PLCg is recruited by its SH2 domain to the phosphorylated receptor and in turn is activated and generates DAG and IP3. 3. The SH2 domain of Grb2 mediates recruitment and subsequent activation of the RAS/MAPK pathway. Direct phosphorylation of receptor tyrosine residues 2. Receptor mediated Tyrosine Phosphorylation of adaptor proteins Biol 2056 also drives downstream intracellular signalling 2024 L4 Some receptors use the recruitment of an adaptor protein to the receptor induced by by phospho-tyrosine binding. The adaptor is then multiply phosphorylated and post--translationally modified and initiates downstream signalling Examples FGFRS1 (fibroblast growth factor) receptor substrate: (FGF and NGF) GAB1 (EGF and others) sine phosphorylation induces receptor ubiquination and internalisation:Biol 2056 pling reader modules to drive signalling pathways 2024 L4 P-tyr SH2 UB UIM (HRS1) Recycled P UB - Internalisation pathway Tyrosine Phosphorylation is coupled to Ubiquitination through the SH2 of the Cbl protein ( Casitas B-lineage Lymphoma) endosome Lysosomal Degradation  Biol 2056 RTK signalling to PI3K 2024 L4 P PI3K PLC RAS migration Proliferation metabolism  Middle T antigen of the polymoma transforming virus interacts Biol 2056 with and activates a PI-kinase 2024 L4 Middle T transforming proteins Induced upon infection with polyoma virus. It is required for transformation of cells. 1. Middle T associates with a tyrosine kinase (pp60cSrc the Polyoma virus cellular homologue of the Src Large T antigen : pRB, p53 oncogene) Middle T antigen: Src, ? Small T antigen: pp2A 2. A PtdIns-Kinase activity was found associated with middle T immunoprecipitates. PtdIns PtdIns4P PtdIns(4,5)P2 PtdIns3P  Biol 2056 PI3K pathway is regulated by RTK and GPCR receptor signalling 2024 L4 Middle T Middle T, RTK and GPCR all regulate the activity of PI3K to generate the second messenger PtdIns(3,4,5)P3 P PI3K How do RTKS P PtdIns(4,5)P2 PtdIns(3,4,5)P2specifically regulate P PI3K to stimulate P 1. PtdIns(3,4,5)P is PtdIns(3,4,5)P3 the Agonist sensitive synthesis ? lipid second messenger Controlling the PtdIns(3,4,5)P3 signal Biol 2056 2024 L4 PTEN: human tumour PI3-Kinase suppressor often deleted writer or mutated in human tumours (40%) PTEN removes the 3 phosphate from PtdIns(3,4,5)P3 (3- PtdIns(4,5)P2 PtdIns(3,4,5)P3 PtdIns(3,4)P2 phosphatase) SHIP1/2 is a 5 P P phosphatase which P P P removes the 5 phosphate from PtdIns(3,4,5)P3. Ship1/2 Although SHIP degrades PtdIns(3,4,5)P3 PtdIns(3,4,5)P3 it also 5-phosphatase generates a new lipid PTEN PtdIns(3,4)P2 which is eraser through to have messenger functions The balance of these enzymes dictates the Class 1A PI3K are directly regulated by RTK activation Biol 2056 2024 L4 1. Class1 A p110 catalytic domains interact with the p85 regulatory domains. 2. Interaction of p110 and p85 : A. suppresses the activity of P110 subunit; B. prevents proteolytic degradation of P110 and C. enable recruitment of the holo enzyme to an activated RTK receptor. Tyrosine kinase receptors couple to the class 1A PI3K through the Biol 2056 interaction of the SH2 domain of p85 with a specific tyrosine 2024 L4 phosphorylated receptor peptide. Class 1A PI3K are recruited to activated tyrosine kinase receptors. The SH2 domain of the p85 subunit mediates the interaction with the YxxM motif on the receptor or an adaptor protein Binding of the SH2 domain to the receptor tyrosine phosphorylated peptide: 1. Brings the enzyme to the membrane where its substrate PtdIns(4,5)P2 resides 2. Relieves the inhibition of the p85 subunit upon the catalytic activity of p110 3. Stimulates interaction of the kinase with the membrane. PtdIns(3,4,5)P3 controls downstream signalling by recruiting proteinsBiol 2056 with specific PtdIns(3,4,5)P3 binding domains 2024 L4 1. Dramatic shift in phosphoinositide signalling paradigm when it was realised that some PH domains interact with phosphoinositides. 2. The chemical synthesis of specific phosphoinositides enabled the generation of tool compounds to be able to be used to rapidly purify proteins that interact with specific PI species. Mass spec Wash unbound Proteins Over 400 proteins have been identified that interact with phosphoinositides. Of these about 50 appear to interact specifically with PtdIns(3,4,5)P3 These proteins are implicated in: 1. Cytoskeletal dynamics 2. Vesicle trafficking 3. Nuclear functions e PH domain is a phosphoinositide sensing protein module. Biol 2056 2024 L4 PH domain: First found in the platelet protein pleckstrin and shown to be a module that binds PtdIns(4,5)P2. PH domain also mediate protein/protein interactions e.g bg subunit of heterotrimeric G proteins. Also involved in generating dimerization interfaces. PH domains are present in many proteins including protein kinases, guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs) for small GTP-binding proteins, lipid transport proteins and phospholipases It is a domain of approximately 120 amino acids. A common structural feature is that they contain two nearly orthogonal β sheets formed by seven β strands arranged in groups of four and three, flanked by a C-terminal helix that `holds' the structure together. The inositide-binding site is almost always formed by the β1-β2 and β3-β4 strands and the variable loops connecting them  SH2 domain mediated activation of PI3-Kinase drives the synthesis Biol 2056 of PIP3 which induces new output signalling through 2024 L4 PtdIns(3,4,5)P3 “readers” PI3-Kinase SH2 interaction writer p85 PI3K IRS1 PtdIns(4,5)P2 PtdIns(3,4,5)P3 Reader Phosphotyrosine binding OUTPUT? Domain interaction P P P P PH i a dom PtdIns(3,4,5)P3 initiates n downstream signalling by Interacting and recruiting proteins PTEN to the membrane that contain eraser specific PIP3 interaction motifs such as the Pleckstrin homology Activation of the PtdIns(3,4,5)P3 reader PKB/AKT protein kinase Biol 2056 couples insulin protein phosphorylation and downstream target 2024 L4 pathway regulation P-tyr SH2 PtdIns(3,4,5)P3 PH (PKB) PtdIns(3,4,5)P3 P P PDK1 IRS1 p85 PI3K P P t308 B / AK / AK PK s473 PKB T mTORC2 T a se ase kin Glut4 ki n AS16 Glucose transporter 0 PDE GSK1 RAB TCS1/2 GAP SREBP1 mTORC1 Decrease Glycogen Fatty Protein Glut4 vesicle cAMP synthesis acid and synthesis Translocation triglyceri increased de glucose uptake synthesis and utilisation  Biol 2056 Abbreviations for the previous slide. 2024 L4 PDE: phosphodiesterase (remember cAMP0 GSK: glycogen synthase kinase (cAMP switches this pathway off) SREBP1: Sterol regulatory element-binding proteins. Transcription factor that regulates fatty acid production. TCS1/2: tuberous sclerosis complex. TSC1–TSC2 (hamartin– tuberin). This complex controls the RHEB small molecular weight G protein which controls MTORC1 complex protein kinase activity mTORC1: mechanistic target of rapamycin complex 1. this is a protein kinase complex that essentially controls protein synthesis. It has multiple inputs into it that enable it to act as an integrator of signalling. mTORC2: mechanistic target of rapamycin complex 2. this is a protein kinase complex that shares the same kinase subunit as mTORC1 but it is involved in the selective regulation of substrates. Its main substrate is the AKT/PKB kinase. PDK1: phosphoinositide dependent kinase 1. a protein kinase that regulates PKB. It is also an important kinase that regulates the maturation of a number of other protein kinases AS180:ATK substrate 160. this is a rab gap that control the GTP loading of the smg RAB which in turn controls vesicle trafficking. Receptor coupled PtdIns(3,4,5)P3 synthesis drives specific Biol 2056 downstream signalling pathways in different tissues 2024 L4 INSULIN PDGF P P P P P P Adipos Fibroblast Liver s e Inhibits glucose Stimulates Stimulates Release and glucose proliferation stimulates uptake and Glycogen Stimulates migration synthesis Triacylglycer Decreases ol synthesis gluconeogenesi and inhibits  Biol 2056 Many different types of receptors drive PIP3 synthesis and downstream signalling 2024 L4 GPCR Tyrosine Immune TGF integrin Cytokine kinase receptors receptors Receptor input Common Protein Protein GTPase Cytoskeletal and kinases phosphatases Regulating proteins Proteins Cell specific downstream PtdIns(3,4,5) proliferation apoptosis Membrane migration metabolism P3Cellular binding trafficking targets outputs Deregulation of the PI3K/PtdIns(3,4,5)P3 pathway is frequent event Biol 2056 in tumour development 2024 L4 Oncogenic Tyrosine kinase receptorPI3K mutations RAS Overexpresion or mutations activating PtdIns(4,5)P2 ATP Tumour oncogenesis suppression PTEN PI3K PtdIns(3,4,5)P3 ADP PTEN mutations Deletions: inactivating + + Tumour development  Biol 2056 RTK mediated PLC activation 2024 L4 P PI3K PLC RAS migration Proliferation metabolism The phospholipase C family Biol 2056 2024 L4 Core enzyme composed of the PH domain, four tandem EF domains, a split tim barrel (X Y) and a C2 domain. The active site is in the TIM, which is interupted by an auto-inhibitory loop insert. In PLCzeta the loop is activatory and its removal inhibits the enzyme. The PH domain can interact with phosphoinositides, rac, bg. The C2 domain interacts with calcium and with the membrane surface and contributes to Gaq binding together with the C-terminal. SH2 mediated recruitment of PLCg induces its tyrosine Biol 2056 phosphorylation of PLCg required for its activation. 2024 L4 SH2 domain of PLCg RTK RTK enables its recruitment to the RTK receptor. Tyrosine Phosphorylation at Y783 and y1253 are important for PLC activation. P P SH 2 P PLCg1 P Receptor dimerization and cross Non-receptor phosphorylation and tyrosine kinases auto phosphorylation Itk lead to the generation BTK of a new tyrosine phosphorylation surface that acts as a  Biol 2056 RTK activation of RAS signalling 2024 L4 P PI3K PLC RAS migration Proliferation metabolism The small molecular weight (monomeric) G proteins of the Ras Biol 2056 family are molecular switches that control diverse cellular outputs 2024 L4 150 members. They are normally lipid Modified at c-terminus WRITER (farneyslation palmitoylation) Ras proliferation Rac Cytoskeletal Rho dynamics CDC42 ARF Membrane dynamics RAB ERASER Many GEFS and GAPS also have phosphoinositide interacting motifs (PH coupling activation to PIP3 pathway) as well as SH2  Biol 2056 RTK mediated RAS activation 2024 L4 GRB2 is an adaptor protein that contains SH2 and SH3 domains. The SH2 domains of the adaptor protein GRB2 interact with specific tyrosine phosphorylated residues on an RTK. GRB2 recruits the SOS (son of seven less) protein through the interaction of its SH3 domain with a proline rich motif on SOS. SOS is a guanine nucleotide exchange factor for RAS RAS regulates two important pathways that coordinate proliferative Biol 2056 responses downstream of many different types of receptors 2024 L4 SH3 domain WRITER SOS PI3K:PIP3/AKT (GEF) RAS GDP READER RA domain( ras association RAS GTP Domains) RAF/MAPK NF1 Many different GAPS NF2 (GAP) ERASER  Biol 2056 change leads to conformational changes in RAS that enable reader interaction 2024 L4 RAS bound to GTP shows conformational changes in switch1 (SW1) and switch 2 (SW2) loops. The changes in the conformation of these loops enables the interaction with downstream reader proteins. Mutations in the SW regions can ablate specific interaction with reader proteins. Mutations in Ras switch I residues T35S, E37G and D38E eliminate PI3K binding. However, T35S and D38E do T35S E37G D38E Y40C Y64G not affect Raf binding. The E37G mutation also abolishes (S2) binding to Raf but not to PI3K X X X Y X RalGEF. The Y40C mutation does not affect PI3K binding, RAF Y X Y X Y but it abrogates Raf and RalGEF binding. RALG Y Y X Y EF In the switch II region, the Y64G ation of downstream pathways by RAS switch loop mutation mutantseliminates PI3K and The binary switch action of RAS converts GTP binding into the Biol 2056 regulation of protein phosphorylation driven by the RAF/MAP 2024 L4 kinase pathway Mutated in melanoma V600E activation loop RAS GTP R RAF-Kinase MAPKKK A MEK MAPKK (example of a MEK Dual S/T and Y-kinase MAPK MAPK MAPK MAPK FOS, JUN ELK1 The binary switch action of RAS converts GTP binding into the Biol 2056 activation of PI3K and PtdIns(3,4,5)P3 signalling 2024 L4 PtdIns(4,5)P2PtdIns(3,4,5)P3 P P Reader RAS GTP R PI3-Kinase P OUTPUT? A P RAS signalling in health and cancers Biol 2056 2024 L4 There are Three isoforms of RAS : H-RAS. N-RAS and K-RAS. HRAS transcripts are highly expressed in brain, muscle and skin and lowest in liver KRAS transcripts are readily detected in gut, lung and thymus. NRAS transcripts are primarily expressed in the testis and thymus Show high degree of homology with main differences in the C terminal hypervariable region. They are post-translationally modified with lipid chains that target them to membranes. They are often mutated in cancer (95% of pancreatic tumour have mutated K-RAS) with different isoforms showing tumour specificity: KRAS primarily in colon and pancreatic cancers,: NRAS in myeloid leukemia and HRAS in bladder carcinomas The G12, G13 and Q61 mutations essentially stop RAS from hydrolysing Deregulation of RAS signalling in human tumours drives cell Biol 2056 proliferation 2024 L4 Deregulated Tyrosine kinase receptor Overexpresion or mutations Mutated in tumours G12, G13 and Q61 Lock RAS in the active state WRITER SOS PI3K:PIP3/AKT (GEF) Activation mutations in PI3K or deletions and inactivating mutants in PTEN RAS GDP RAS GTP READER drive PtdIns(3,4,5)P3 signalling Mutated in melanoma V600E activation loop NF1 RAF/MAPK Mutated or NF2 Deleted in tumours (GAP) Loss of function ERASER  Structural analysis reveals why mutations in Gln61 and Gly12 lead Biol 2056 to RAS GTP accumulation 2024 L4 Glycine-12 of the P-loop (binds the GDP/GTP) is critical for oncogenic activation of Ras because any mutation of this residue [except Gly12Pro (G12P)] activates the oncogenic potential of Ras. Any mutation including Gly12Ala would lead to a clash with Arg789 and with Gln61. Gln61 is essential for GTP hydrolysis and forms contacts with the ARG789 as well as with the gamma phosphate of GTP. Mutation of Gln61 to any other residue d chain is a RAS GAP and shows the arginine finger (789) beside glutamate leads to a loss of low strand is RAS.. GTP hydrolysis and oncogenic here show the van der Waals radii activation. cogenic mutants of RAS prevent GTP hydrolysis leading to the accumulation of RA  Biol 2056 2024 L4 Supplementary slides Examples of SH2 domain containing proteins Biol 2056 2024 L4 1. 120 proteins with SH2 domains 2. Contact many aspects of cell regulation. 3. Many appear to be adaptor proteins 4. RTK signalling is dependent on the repertoire of SH2 binding proteins present in a cell and on the specific tyrosine phosphorylation site. Cross talk with the PI3K pathway drives full activation of PLCg and Biol 2056 PtdIns(4,5)P2 hydrolysis 2024 L4 PtdIns(3,4,5)P3 SH2+PH (PLCg1) DAG C2 (PKC) SH2(PI3K) P-tyr