Phospholipids, Phosphoinositols & EicosanoidsSlide 2Slide 3Slide 4Slide 5Slide 6Arachidonic Acid MetabolismSlide 8Slide 9Slide 10Growth Factor ReceptorsSlide 12Slide 13Slide 14Slide 15Cytokine ReceptorsSlide 17Slide 18Slide 19Ser/Thr-kinase ReceptorsSlide 21NFkBBIMM118Phospholipids, Phosphoinositols & EicosanoidsBIMM118Phospholipids, Phosphoinositols & EicosanoidsCommon types ofPhospholipids:BIMM118Phospholipids, Phosphoinositols & EicosanoidsSecond messenger –released through hydrolysis by phospholipases and/or –generated through the actions of lipid kinasesPhospholipases: Phosphatidylinositol-kinases:BIMM118Phospholipids, Phosphoinositols & EicosanoidsPhospholipases:•PLA2:–Cytoplasmic form (90 kDa) is regulated through nM Ca++ (Annexins) and phosphorylation; AA specific => signaling function–Secreted form (pancreas, 14 kDa) is also Ca++ dependent (mM range)=> digestive function•PLC: coupled to a variety of (growth factor) receptors:–PLC is activated through GPCRs (Gq) => binding enhances its catalytic activity and in return the GTPase activity of Gq(similar to GAP function in ras signaling) –PLC couples with its SH2 domains directly to growth factor receptors (EGFR, PDGFR) or the TCR, where it is activated through tyrosine phosphorylationBIMM118Phospholipids, Phosphoinositols & EicosanoidsBoth phospholipases yield finally arachidonic acid (see below), in addition, PLC activity also produces DAG and IP3: •DAG: remains membrane bound; diacylglycerol kinase phosphorylates DAG to generate phosphatidic acid which functions as a substrate for PLA2.Phosphatidyl-serine (PS), Ca++ and DAG activate PKC on the plasma membrane•IP3: see Ca++ signaling!!–Glucocorticoids: inhibit PLA2 by transcriptionally inducing Lipocortin, a protein which binds to PLA2 and blocks its activity.–Phorbol esters: strongest known tumor promotors; mimic DAG => bind PKC and activate it. Also potent activator of Ca2+ influx, MAPK pathway etc.BIMM118Phospholipids, Phosphoinositols & EicosanoidsLipid kinases:•PI3-kinase:–binds to and becomes tyrosine phosphorylated in response to activation of growth factor receptors or immune receptors–85 kDa regulatory subunit (pY) and a 110 kDa catalytic subunit–regulatory subunit contains SH2 and SH3 domains–PIP3-phosphates can bind to the pleckstrin homology (PH) domain of Akt=> Akt activation => phosphorylation of BAD, which dissociates from the antiapoptotic protein bcl-2 => inhibition of apoptosis•Wortmannin: fungal metabolite, potent, irreversible inhibitor of PI3Kinase•Ly290004: synthetic compound, blocks ATP binding site of PI3KinaseBIMM118Arachidonic Acid Metabolism•Eicosanoids: collective name for derivatives of arachidonic acid (=5,8,11,14 - eicosatetraenic acid)–AA is mainly generated through the action of PLA2 and DAG-lipase.–Rapidly metabolized by cyclooxygenase and lipoxygenase into prostaglandins and leukotrienes:BIMM118Arachidonic Acid Metabolism•Prostaglandins:–First observed in seminal fluid => name–Structure of cyclopentane ring defines letter–Double bonds in side chains account for number–Greek letter refers to the spatial position of the OH-group at C-9Initial step in PG synthesis catalyzed by PGH-synthase which has dual enzymatic activity:cyclooxygenase (closes ring =>PGG2) and peroxidase (=> 15-OH)BIMM118Arachidonic Acid MetabolismBiological functions of PGs:•Vascular tone Relaxation: PGs E1, E2, F2 and I2Constriction: PGs F2, TxA2•Platelet aggregation Increase: PGs E1, TxA2Decrease: PGs E2, I2•Uterus tone Increase: PGs E1, E2, F1•Bronchial muscle Constriction: PGFsRelaxation: PGEs•Gastric secretion Inhibition: PGs E1, E2, I2•Temperature and pain Increase: PGEsBIMM118Arachidonic Acid Metabolism•Leukotrienes:–First found in leucocytes; contain 3 conjugated double bonds–Lipoxygenase generates Hydroperoxyeicosatetraenoic acid (HPETE)–LTC4, D4 and E4 mediate allergic reaction: Slow Reacting Substance of Anaphylaxis (SRS-A) => mediates anaphylactic shock 10,000 fold more potent than histamine!!! => constricts bronchi, dilate blood vessels–LTB4 is a very strong chemoattractant for macrophagesBIMM118Growth Factor Receptors•Many growth factors (EGF, PDGF, IGF-1, CSF-1, ...) signal through receptors with intrinsic tyrosine kinase activity•Common features:–Large, glycosylated ligand binding domain–Single hydrophobic transmembrane domain–Activation occurs through ligand mediated oligomerization–Undergo ligand induced downregulation by internalization–Cytoplasmic tyrosine kinase domain: •most highly conserved region•GlyXGlyXXGlyX(15-20)LysLys is critical for ATP binding - mutation renders receptor kinase inactive, which abrogates all cellular responses => signaling depends on tyrosine phosphorylation of receptor and cytoplasmic substrates•Tyrosine kinase receptors also bind and activate cytoplasmic tyrosine kinases–Autophosphorylation sites:•conserved in the C-term of each receptor class•autophosphorylation does not effect Km of receptor kinase activity•provide docking sites for SH2 domain containg signaling proteinsBIMM118Growth Factor Receptors•Three subclasses:–Class I: two Cys-rich region in the EC, monomeric ligand EGF-R, erbB2, erbB3, erbB4 (heregulin receptors)–Class II: heterotetrameric: 2 and 2 chains stabilized through S-S bonds: monomeric ligand Insulin-R, IGF-1-R–Class III: Repeats of mmunoglobulin-like structure, dimeric ligand FGF-R, NGF-R, PDGF-R, CSF-1-R, c-kitBIMM118Growth Factor ReceptorsSignaling through Adapter proteins:•grb2: adapter with one SH2 domain which binds PY residue on RTK, and two SH3 domains which bind to•Sos: “Son of Sevenless” (mutation in drosophila prevents development of the R7 photoreceptor cell). Functions as a GEF to facilitate GTP loading of •ras: GTP binding protein, farnesylated; protooncogene, provides a docking site on the plasma membrane for raf.•ras-GAP: negative regulator of groth factor signaling: promotes GTP hydrolysis through the GTPase activity of ras.BIMM118Growth Factor ReceptorsSignaling through Adapter proteins:•raf: ser/thr kinase of the MAPKKK/MEKK family (MEKK does normally NOT phosphorylate MEK, but rather MKK4/7 => Stress pathway); requires context of plasma membrane for activation (mixing GTP-ras and raf in a test tube fails to activate raf) => raf likely to be phosphorylated at the plasma membrane. Activated raf
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