Harris Lecture 9 notes (1 per page) - Signaling via protein...

Unformatted text preview: Signaling via protein modifications 1. Enzyme-coupled receptors (and protein phosphorylation) 2. Use of proteolysis in signaling 1 1 Signaling via protein modifications 1. Enzyme-coupled receptors (and protein phosphorylation) There are six main classes of enzyme-coupled receptors i) ii) iii) iv) v) vi) Receptor tyrosine kinases RTKs Tyrosine-kinase-associated receptors Receptor serine/threonine kinases Histidine-kinase-associated receptors Receptor guanylyl cyclases Receptorlike tyrosine phosphatases 2 2 Receptor tyrosine kinases RTK ~60 encoded in the human genome Some bind secreted proteins and others bind cell surface proteins tyrosine kinase domain T M proteins 3 3 Receptor tyrosine kinases mediate essential functions cell growth and proliferation (related to cancer) 4 4 one phosphorylate another Signaling molecules induce the transautophosphorylation of receptor tyrosine kinases Ligand binding dimerizes the receptor Their kinase domains are brought together and phosphorylate each other dimerize bring 2 RTK together kinase domains are together== activated Phosphorylation of the kinase domains enhances their activity Phosphorylation of other regions creates docking sites to assemble a signaling complex 5 5 If a receptor tyrosine kinases with a defective kinase domain was expressed together with the normal receptor what would happen? A) The cell would respond normally to the signal B) The cell would have no response to the signal C) The cell would have a reduced response to the signal depends on how much the mutant form is expressed. If there are a lot of mutant receptors, the cell will have no response 6 6 Phosphorylated receptor tyrosine kinases recruit proteins transduce the signal into the cytoplasm which mediate downstream signaling 7 7 The recruited proteins contain domains (SH2 or PTB domains) that bind to phosphotyrosine and neighbouring sequences Many molecules in the cell have phosphotyrosine. This binding site adds specificity. 3D structure of an SH2 domain The basic functionality of an SH2 domain 8 8 Examples of proteins recruited to phosphorylated PDGF receptor The proteins often contain additional domains (e.g. SH3 domains which bind proline-rich regions) They can function as adaptors to recruit proteins without SH2 domains9 9 Genetic studies in the Drosophila eye identified core components of the receptor tyrosine kinase signaling pathway The Drosophila compound eye consists of ~800 ommatidia little circles that collect light The ommatidia are composed of 8 photoreceptor cells and 12 support cells They arise from a simple epithelial sheet through the sequential differentiation of the photoreceptor cells The R7 photoreceptor cell is needed to detect UV light A screen was performed to identify mutants that failed to specify R7 > based on their 10 insensitivity to UV light 10 Genetic studies in the Drosophila eye identified core components of the receptor tyrosine kinase signaling pathway The first mutant identified was called Sevenless (Sev) the normal Sevenless protein was shown to be a receptor tyrosine kinase expressed in R7 cells Sevenless protein = RTK in R7 cell 11 11 Genetic studies in the Drosophila eye identified core components of the receptor tyrosine kinase signaling pathway The second mutant identified was called Bride-of-sevenless (Boss) the normal Bride-of-sevenless protein was shown to be the ligand for Sevenless expressed on R8 cells 12 12 Genetic studies in the Drosophila eye identified core components of the receptor tyrosine kinase signaling pathway Drk and Son-of-sevenless (Sos) were identified in subsequent screens Drk links Sevenless to Son-of-sevenless Son-of-sevenless is a GEF for Ras 13 Drk SH3 domain that binds to proline rich region of Sos (Ras GEF) 13 Genetic studies in the Drosophila eye identified core components of the receptor tyrosine kinase signaling pathway This basic mechanism is used in different contexts in all animals What is Ras? 14 14 molecular switch 15 15 Ras is a molecular switch downstream of receptor tyrosine kinases •monomeric GTPase •attached to the cytoplasmic face of the plasma membrane by a lipid anchor •activated by Ras-GEFs and inactivated by Ras-GAPs •its activity leads to cell proliferation or differentiation •30% of human tumours have hyperactive mutant forms of Ras 16 16 Ras activates a mitogen-activated protein kinase module (MAP kinase module) to change protein activity and gene expression Downstream of Ras is a cascade of kinases (MAP kinase module). Mitogen is a secreted protein that binds to a receptor and induces cell proliferation. Mitogen binds to Tyrosine Kinase and start signalling. Ras activates MAP kinase module. 17 17 At least 5 parallel MAP kinase modules can operate in a mammalian cells (there are at least 12 MAPKs, 7 MAPKKs, and 7 MAPKKKs) How is non-specific cross-talk controlled? Why wouldn't MAPKKK phosphorylate all MAPKK in the cell? In yeast, scaffolds bind specific MAP kinase modules, insulating them from other modules and increasing response specificity 18 18 Other pathways downstream of RTKs and overlap with GPCR pathways DAG 19 19 Other pathways downstream of RTKs and overlap with GPCR pathways 20 20 Signaling via protein modifications 1. Enzyme-coupled receptors (and protein phosphorylation) There are six main classes of enzyme-coupled receptors i) ii) iii) iv) v) vi) Receptor tyrosine kinases Tyrosine-kinase-associated receptors Receptor serine/threonine kinases Histidine-kinase-associated receptors Receptor guanylyl cyclases Receptorlike tyrosine phosphatases Tyrosine-kinase-associated receptors interact non-covalently with a cytoplasmic tyrosine kinase… The receptor itself does not have a kinase domain. It pulls a TK and interact with it non-convalently. …but their mechanisms of action are similar to receptor tyrosine kinases 21 21 Signaling via protein modifications 1. Enzyme-coupled receptors (and protein phosphorylation) There are six main classes of enzyme-coupled receptors i) ii) iii) iv) v) vi) Receptor tyrosine kinases Tyrosine-kinase-associated receptors Receptor serine/threonine kinases Histidine-kinase-associated receptors Receptor guanylyl cyclases Receptorlike tyrosine phosphatases 22 22 Receptor serine/threonine kinases -the largest class of cell surface receptors in plants (also function in animals) ~6 major families in plants the largest is the leucine-rich repeat receptor kinase family (175 members in Arabidopsis [e.g. the Clavatal1/Clavatal2 complex]) Clv1/Clv2 signaling suppresses the development of stems, leaves and flowers It's suppressing the differentiation of stem cells into flowers, leaves… Gene regulation can remove the P when these cells left the meristem and need to differentiate. Clv3 travels to cells with Clv2 and Clv1. Clv3 binds to Clv2 and Clv1. Clv2 and Clv1 phosphorylate themselves and sends signal onwards (Rho-family GTPase) 23 Rho family GTPase is a switch. It will block genes that will form stem, leaves and flowers. 23 Histidine-kinase-associated receptors -activate a “two-component” signaling pathway -used by bacteria, yeast and plants, but not animals e.g. regulation of bacterial chemotaxis flagella rotate counterclockwise, they will propel the bacteria forward. attractant flagella rotate clockwise. repellent CheA is the histidine kinase CheZ can remove the Pi and cell can move forward (counterclockwise) It phosphorylates itself and then transfers the phosphate to an aspartic acid on CheY 24 24 Signaling via protein modifications 1. Enzyme coupled receptors and protein phosphorylation 2. Use of proteolysis in signaling Notch signaling Hedgehog signaling Wingless signaling TNF / NF B signaling 25 25 Lateral inhibition by Notch signaling 26 26 The force of endocytosis pulls away Notch segment, When Notch binds to Delta, endocytosis happens. Delta binding leads to Notch 1st cleavage is on Golgi cleavage… noncovalent interaction a fragment of Notch enters the nucleus. …and movement of Notch fragment to the nucleus 27 27 Organizer function in vertebrate limb development Source of the morphogen sonic hedgehog (Shh) Shh spreads from this source The Shh gradient controls the formation of distinct digits POSTERIOR 28 28 Without hedgehog signaling the transcriptional activator Ci is sequestered in the cytoplasm by a microtubule-associated complex Plus, this complex promotes the proteolysis of Ci to create a transcriptional repressor Hedgehog is always off when 29 there is no Hedgehog signalling. 29 Patched sequesters Smoothened. At the same time, the Smoothened protein is sequestered in intracellular vesicles by Patched 30 30 Hedgehog binds to patch Smoothened translocates onto the plasma membrane. The binding of Hedgehog to Patched allows Smoothened to transfer to the plasma membrane where it releases Ci from its inhibitory complex Smoothened+Pi inhibits Ci proteolysis and Ci can be released and activate transcription 31 31 Signaling via protein modifications 1. Enzyme coupled receptors and protein phosphorylation 2. Use of proteolysis in signaling Notch signaling Hedgehog signaling Wingless signaling TNF / NF B signaling The release of a transcriptional activator from an inhibitory complex is also at the core of Wingless and TNF / NF B signaling 32 32 How can we understand all of the signaling occurring in a cell? PNAS 104:12890 e.g a network controlling human pancreatic cancer 33 33 How can we understand all of the signaling occurring in a cell? 34 34 How can we understand all of the signaling occurring in a cell? 35 35 How can we understand all of the signaling occurring in a cell? Mapping Brain Circuits: The Connectome http://www.sfn.org/index.aspx?pagename=brainBriefings_09_mapping 36 36 Lecture 9 reading 921-931 956-957 941-943 (not figure 15-70) 946-947 950-951 37 37 ...
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