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Chapter 15 Signal Transduction 1) Endocrine, paracrine , autocrine signaling, and cell-cell contact (Fig. 15-2). Endocrine signaling is long distance signaling. An example would be pancreatic cells secreting insulin. Paracrine signaling is for close proximity. An example would be a nerve cell releasing neurotransmitters. In autocrine signaling the cell that produces the ligand also contains the receptor for that ligand. This is how cancer cells work. In signaling by plasma membrane attached proteins, the target cell does something in response to direct contact from the signaling cell. ) List examples of 1) steroid hormones and 2) amino acid derivatives that act as ligands. What are the catecholamines, and which amino acid are they derived from? Steroid hormones bind cytosolic receptors. They include cortisol, progesterone, estradiol, testosterone, thyroxine and retinoic acid. Steroid receptor complexes increase or decrease the transcription rates of certain genes. Dopamine, norepinephrine, epinephrine, serotonin and histamine are ligands that are derived from amino acids. Catecholamines are ligands derived from the amino acid tyrosine. 3) What’s an agonist?
What’s an antagonist? A doctor prescribes isoproterenol to his patient – why? Why not epinephrine? Another patient receives alprenolol – why? (See page 629 and Fig. 15-5). Agonist= structural analog, antagonist=inhibitor. Isoproterenol has lower Kd (higher affinity) than epinephrine, and will inc. smooth heart muscle contraction. Alprenol is an antagonist (“beta blocker”) and slows heart contractions 4) What are the five kinds of second messengers we described in lecture. (Fig. 15-9 shows only four): cAMP, cGMP, DAG, IP3 and Ca2+ 5) What are GTP-binding (switch) proteins?
When are they on? When are they off? (Fig 15-8). GEFs help turn them on. GAPs help turn them off. Are “on” when bind GTP, and “off” when bind GDP. Ex: Ras, Ran, trimeric G proteins 6) What are kinases versus phosphatases? Kinases phosphorylate, Phosphatases dephosphorylate 7) What are the main features of a G-protein coupled receptor (GPCR, Figs. 15-10, 15-12, and 15-13)? What is epinephrine and what kinds of receptors does it bind on what cells to induce what responses? GPCRs= “seven-pass” receptors with amino terminus outside cell and carboxy terminus inside cell.
Epinephrine released when glucose needed quickly (inc. glycogenolysis and lipolysis); binds to GPCR receptor , which activates a G protein (switch protein), which activates an effector protein (adenylyl cyclase) producing cAMP NOTE: cAMP does not involve RTK (tyrosine), but uses Ser/Thr kinases! PDE degrades cAMP 8) Describe the three G proteins ? , ? , and ?. Which one binds GTP/GDP (hint for question 6 above). G? ???binds GDP, is tethered to inner leaflet of plasma membrane, but dissociates from ? and ? to activate effector protein (adenylyl cyclase). G? and G? never separated!
Are tethered to inner leaflet and work as a unit. 9) Describe FRET (Fig. 15-14). 10) What is adenylyl cyclase? Figs 15-21 and 15-22. What does it do? How is adenylyl cyclase positively and negatively controlled? Positive: epinephrine binds ? adrenergic receptors to activate Gs, actvating adenylyl cyclase; Negative: PGE binds to ? adrenergic receptors to activate Gi which then inhibits adenylyl cyclase. 11) The complete “Fight or Flight” road map. YIKES!! What happens when cAMP rises? What happens when cAMP drops? 12) T/F: Second messengers are long-lived in their signaling effectiveness?
How is cAMP degraded? PDE 13) What is cAMP-dependent protein kinase and how does it work? (Figs. 15-23 and 16-31) Do not involve tyrosine kinases (RTK), but use Ser/Thr kinases (binding of cAMP releases catalytic sites…) 14) What do we mean by amplification in signal transduction? Fig. 15-26. So many steps involved in signal transduction b/c you’re amplifying signal at every step fast response 15) PIP2, DAG, IP3, and the release of Calcium from the endoplasmic reticulum. (Fig. 15-30). Each PI kinase phosphorylates inositol ring: PI PIP PIP2, and cleavage of PIP2 by Phospholipase C yields DAG and IP3.
Phospholipase C is activated by a hormone binding to GPCRs and activation of G proteins. IP3 releases Ca2+ back into cytosol to transduce a signal (Ca2+ binds to PKC which binds to DAG phosphorylates substrates). Ca2+ pumps normally pump Ca2+ (from cytosol) into ER or out to exterior; yet IP3 causes ion channels to open and release Ca2+ into cytosol. Once Ca2+ released, it positively feeds back on channels to allow more Ca2+ to flow out. But once Ca2+ becomes depleted from ER and at high conc. in cytosol, it inhibits channels. ALSO: once Ca2+ rises in cytosol, acts as a 2nd messenger to trigger insulin release 6) Calmodulin. Activated by binding of 4 Ca2+ molecules, it then activates: PDE (to degrade cAMP), glycogen phosphorylase kinase GPK (to break down glucose, activates this path without cAMP! ), other protein kinases, and Nitric Oxide (NO) synthase (involved in acetylcholine relaxation of smooth muscle in conjunction with cGMP) 17) How are blood vessels dilated by acetylcholine (Fig. 15-31)? BTW, what does Viagra do? Acetylcholine binds acetylcholine GPCR, which activates phospholipase C, which makes IP3, which binds to Ca2+ (leading it to inc. n cytosol), and Ca2+ binds calmodulin, which activates NO synthase that produces NO. The NO is then released by paracrine signaling into muscle cells and binds NO receptor that converts GTP to cGMP, which activates protein kinase G relaxation of muscle cell and through endocrine signaling causes blood vessel dilation. VIAGRA blocks degradation of cGMP by PDE (may cause blindness b/c rod cells kept open by cGMP) 18) Beta arrestin in receptor desensitization. If receptor constantly exposed to epinephrine, may itself become phosphorylated by PKAblocking transducing signal, downregulating ALL GPCRs.
Once ? -adrenergic receptor is phosphorylated by BARK (? -adrenergic receptor kinase) ? -arrestin binds the receptor to block its activation of Gs, as well as promotes formation of *clathrin-coated vesicles for endocytosis of the bound receptor (to deplete surface receptors) CHAPTER 16: Signal Transduction and Gene Expression 1) List several ligands that bind to Receptor Tyrosine Kinases (RTKs). * Nerve growth factor (NGF), platelet derived growth factor (PDGF), fibroblast growth factor (FGF), epidermal growth factor (EGF), insulin, and more
List several kinds of general responses that could occur. What characteristics are different between RTKs and GPCRs (i. e. their protein structure and function)? * RTK= receptor tyrosine kinases; havee components: extracellular ligand-binding site, a single hydrophobic trans-membrane ? helix, and a cytosolic catalytic domain. Ligand binding causes a conformational change that promotes formation of a functional dimeric receptor, bringing together two poorly active kinases that then phosphorylate each other on a tyrosine residue in the activation lip.
Phosphorylation causes the lip to move out of the catalytic site, thus allowing ATP or a protein substrate to bind. The activated kinase then phosphorylates other tyrosine residues in the receptor’s cytosolic domain. The resulting phophotyrosines function as docking sites for various signal-transduction proteins. * GPCR= G-protein coupled receptors. Binding of ligand triggers the exchange of GTP for GDP on the G? subunit and dissociation of G? ???GTP from the G?? complex, and G? subunit transduces the signal, but in yeast pheromone receptors it’s the G?? complex. G?? unctions by triggering a kinase cascade (similar to the one for Ras). Its proteins are involved in mating-specific cellular responses. 2) What are adaptor proteins? Specifically what are SH2, SH3, and PTB domains and how do they work. (Figs. 16-19, 16-20) No intrinsic enzyme activity; have docking sites for other effector proteins, such as SH2, SH3, or PTB domain (Phospho-Tyrosine Binding). These docking proteins pass the signal onto Ras. 3) Growth FactorRTK>GRB2>SOS>Ras>Raf>MEK>MAP>differential gene expression for cell division or specific cell type differentiation.
What happens at each step? (Figs. 16-21, 16-22, 16-25, 16-27). Why so many steps (see question 16 in the previous section)? Sev gene regulates R7 development and in mutants R7 is missing cell differentiates into a cone instead and flies now sensitive to UV light. The Sev gene product is RTK and Boss (in R8 cells) is the ligand for this RTK. Once Boss binds/activates Sev RTK it causes GRB2 (with SH subunits) to bind receptor, leading to relocation of SOS (the GEF for Ras) from the cytosol to the membrane where Ras-GDP resides and it activates it… ) Phosphatidylinositol 3,4,5 tri-phosphate and Protein Kinase B (Fig. 16-29 and 16-30). PI-3,4,5-triphosphate ( made by phosphorylation of PIP2 at #3 OH by PI-3 Kinase) is docking site for Protein Kinase B. PKB bound to PI 3,4,5-triphosphate and PDK1 (also bound to PI 3,4,5triphosphate) diffuse into membrane and PDK1 phosphorylates/activates PKB Ras-independent insulin signaling 5) Insulin versus glucagon. (Slide from lecture shows a Table comparing and contrasting the two ligands and their effects on serum glucose. ) Insulin: synthesized in ? ells and when there’s high blood glucose activate GLUT4 (glucose transporter) and inc activity of glycogen synthase removal of glucose from blood and its storage as glycogen. Glucagon: reacts to a decrease in blood glucose, stimulating release of glucagon, activating adenylyl cyclase, activating glycogen phosphorylase and inhibiting glycogen synthase degradation of glycogen and release of glucose into blood. Chapter 20: The Cell Cycle 1) Who were the three Nobel Prize winners in Physiology and Medicine for 2001? * Leland Hartwell * Tim Hunt * Paul Nurse ) Review the gross morphological events of prophase, metaphase, anaphase, and telophase. * Prophase * Chromosomes condense to the 30nm solenoid fiber * Chromatids remain attached at the centromeres and the spindle forms * The nuclear envelope disassembles in most eukaryotic cells (called “open” mitosis). Yeasts and other fungi have a “closed” mitosis * ER and Golgi turn into vesicles * Metaphase – Condensed chromosomes align in a straight line that is referred to as the metaphase “plate” * Anaphase * Sister chromatids separate from each other The spindle is critical for chromatid movement to opposite poles * Molecular motors generate force and movement * Telophase * Beginning of the next interphase * Chromosomes begin to decondense * The nuclear envelop and the nucleolus begin to reassemble * Cytokinesis * Cytoplasm divides * Golgi and ER reform from vesicle fusion 3) Figure 20-2 is a good summary. 4) Three major classes of Cdk/cyclin complexes: Where they work in the cell cycle, and what do they do. What are the three critical steps in the cell cycle? * G1 cyclin-CDKs Expressed when growth factors (EGF, PDGF, NGF, etc) signal the cells to divide * Phosphorylates the retinoblastoma protein in mammalian cells * S-phase cyclin-CDKs * Form during G1, but are held silent by an inhibitor * The inhibitor is destroyed by ubiquitin-mediated proteolysis. Then, the cell progresses into S-phase * Mitotic cyclin-CDKs * Also called MPF (maturation/mitosis promoting factor) * Form in S-phase and G2, but are held silent until late G2 * Once activated, mitotic Cdk-complexes initiate mitosis * Chromosome condensation Nuclear envelope disassembles and the spindle forms * Chromosomes align on the metaphase plate * ER and Golgi turn into vesicles * Partially activates anaphase promoting complex (APC) 5) Classic experiments: * What happens when you fuse a G1 cell to a M-phase cell? Fig. 20-3 * Interphase cells advance prematuring into M-phase * Now we know that the diffusible regulators are the mitotic Cdk-complexes (MPF) * What happens when you fuse a G1 cell to a S-phase cell? * G1 nuclei begin to replicate their DNA prematurely Used [3H]-thymidine incorporation and autoradiography to visualize DNA synthesis * Now know that diffusible S-phase Cdk-complexes activated the pre-replication complexes on DNA origins of replication in the G1 nuclei * What happens when you fuse a G2 cell to a S-phase cell? * Re-replication of G2 DNA does not occur * Once DNA is replicated, it cannot be re-replicated in that same cycle * What’s the diffusible regulator in the first experiment? MPF 6) What two species of yeast were used to decipher the genetics of the cell ycle? What’s a closed mitosis versus and open mitosis? * Budding and fission yeast * In open mitosis, the nuclear envelope disassembles during mitosis. In closed mitosis, the nuclear envelop does not disassemble. 7) What is “cloning by complementation”? (Fig. 20-4). This is the same as functional complementation. * Many cdc mutations identified are temperature sensitive * Grow and divide at permissive temperatures * Fail to divide at non-permissive temperatures * We can select cDNAs by functional complementation ) What is MPF (what two proteins make up MPF) and where did its name come from (i. e. what organism and cell type)? (Figs. 20-5 and 20-6) * MPF is the maturation promoting factor. It is comprised of Cdk1-Cyclin B * The name came from studying frog oocyte maturation in vitro 9) The pathway to MPF destruction: What is Anaphase Promoting Complex (APC)? What activates APC? What does APC then do and how does it do it? Fig. 20-10 * MPF is a kinase that phosphorylates many different substrates to initiate mitotic events * To exit mitosis, MPF must be destroyed Destruction of MPF depends on the destruction of Cyclin B * Destruction of Cyclin B is via the ubiquitin pathway * Ubiquitin is covalently linked to lysines behind the destruction box * Cyclin B without the destruction box will not be destroyed * APC destroys MPF, but APC was actually activated earlier at anaphase by MPF 10) You have to know Fig. 20-13 and 20-14!! 11) Molecular events at the onset of mitosis: a) Nuclear envelope disassembly: what are the lamin proteins, how do they disassemble, and where do they go when they disassemble? (Figs. 0-16, 20-17) * The nuclear lamina supports the nuclear envelope. It is found on the underside of the inner envelope membrane. * The nuclear lamina is made of three lamin proteins: A, B and C * All three lamin protein form coiled-coil dimers * Two dimers form a tetramer with head-to-head or tail-to-tail orientations * MPF phosphorylation of Ser residues causes disassembly * A and C diffuse into the cytoplasm. B remains bound to the membranes that form vesicles during mitosis b) Condensation of chromatin: what are the SMC protein (condensins)? * SMC proteins in yeast Structural maintenance of chromosomes (SMC) * Large proteins that form coiled-coils * ATPase activity in their C-terminus * Function in the normal segregation of chromatids * SMC proteins in frogs * Part of a complex called condensin that becomes phosphorylated at the onset of mitosis * Condensins bind DNA and wind it into “supercoils” with ATP hydrolysis * Several condensins bind along the lenth of the chromosomes to form coiled-coils to compact the DNA c) Spindle assembly due to MPF phosphorylation of microtubule-associated proteins d) ER and Golgi vesiculation ??? due to direct MPF phosphorylation
What are the cohesins – what do they do and what regulates their activity? How does APC play a role in this regulation? (Fig. 20-21, 20-22) * Cohesins hold sister chromatids together * Separation of chromatids is not dependent on MPF destruction * Cohesin function is regulated by an anaphase inhibitor called securing. This inhibitor is a target for APC ubiquitination 12) Yeast cell cycle (Figs. 20-29 and 20-28) * Sic1 is the S-phase inhibitor destroyed by ubiquitination * E3 for the ubiquitination is cdc34 * The ubiquitination complex is called SCF Once Sic1 is destroyed, Cdc28/Clb5 + 6 phosphorylate substrates to initiate DNA replication * G1 cyclin-Cdc28 phosphorylated Sic1, enabling its recognition and ubiquitination by Cdc34 and SCF * Cln1 and Cln2 arise early on in the cell * Cdc28 is only in yeast * Clb5 and Clb6 arise late in G1. They are called S-phae cyclins. They are rapidly turned on by the destruction of Sic1 13) Why chromosomes replicate only once per cell cycle (Fig. 20-30) * Protein degradation makes cell cycle progression move forward, not reverse * Sic1 is destroyed at G1 to S-phase transition Anaphase inhibitory (securing) is destroyed at the metaphase to anaphase transition * Cyclin B is destroyed at the mitosis to G1 transition 14) Early and late mammalian response genes. 15) Mammalian cell cycle (Fig. 20-32) * Growth factor hormones are called mitogens * The absence of mitogens cases cells to arrest in G1 or G0 * If mitogens are added, cells advance past the restriction point and are committed to S-phase and mitosis * Mammalian cells have several Cdks * Cdk 1, 2, 4, and 6 are used for the cell cycle * Cdk 1 complements Cdc2 * Mammalian cells also have multiple cyclins: D, E, A and B 6) D and E cyclins and their function, the Rb and E2F proteins (Figs. 20-33) * D type cyclins come from proto-oncogenes * Cyclin E is the principle player getting the cell past the restriction point * Cyclin D-Cdk4 or 6 is activated first and then Cyclin E-Cdk2 * Once CyclinD-Cdk4/6 is activated in phosphorylates retinoblastoma protein (Rb) which releases E2F * E2F now acts as a transcription activator. Cyclin E-Cdk2 then phosphorylated even more Rb/E2F via positive feedback loop 17) Overview of mammalian check points, p53 (Figs. 20-34 and 20-35)