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Activating and de-activating Ras

Activating and de-activating Ras. Key human members of Ras family. KRAS, HRAS, NRAS Kick-start cell growth when activated Other signaling?. Some terms. Conserved proteins, genes, etc.: Appear in many organisms, presumably “conserved” by evolution

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Activating and de-activating Ras

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  1. Activating and de-activating Ras

  2. Key human members of Ras family • KRAS, HRAS, NRAS • Kick-start cell growth when activated • Other signaling?

  3. Some terms • Conserved proteins, genes, etc.: Appear in many organisms, presumably “conserved” by evolution • Allostericsite: a site (other than the catalytic site) that affects the rate of a catalytic reaction • Hydrolysis: cleavage of chemical bonds by the addition of water • GTPase: a protein that catalyzes hydrolysis of GTP to GDP

  4. A Video: GTPases as Molecular Switches

  5. Turning Ras On and Off • Ras is normally tightly bound to GDP • Ras is activated by binding to GTP instead of GDP • Ras is a weak GTPase (i.e., it encourges hydrolysis of GTP into GDP) • A “guanine nucleotide exchange factor” helps Ras to exchange GTP for GDP • GEF is the generic term • Sos1 is the specific GEF we will look at • A GTPase Activating Protein speeds up RasGTPase activity • GAP is the generic term • RASA1 is the specific GAP we will look at

  6. Turning Ras On and Off Ras-GDP Ras-GDP is the “off” state of Ras

  7. Turning Ras On and Off Ras-GDP Ras-GTP Ras-GTP is the “on” state of Ras

  8. Turning Ras On and Off Ras-GDP Ras-GTP The entire reversible reaction (????)

  9. Turning Ras On and Off GDP Ras-GDP Ras-O Ras-GTP GTP Ras can bind to either GDP or GTP (or not at all)

  10. Turning RasOn GDP Ras-GDP Ras-O GEF Ras-GTP GTP A guanine nucleotide exchange factor (GEF) helps Ras exchange a GDP for a GTP

  11. Turning Ras On and Off GDP Ras-GDP Ras-O GEF GAP Ras-GTP GTP A GTPase Activating Protein (GAP) helps Ras turn the GTP back into GDP

  12. But wait: There’s More!! Ras-GDP GAP GEF Ras-GTP

  13. How Fast Does Ras Turn On? Ras-GDP Sos1 Ras-GTP

  14. Guanine Nucleotide Exchange Rate Ras-GDP Ras-GDP Cdc25 REM Rate: kcat, Km Sos1 Part of Sos1

  15. Guanine Nucleotide Exchange Rate GDP Ras-GDP Ras-O Cdc25 REM Rate: kcat, Km Sos1 Part of Sos1

  16. Guanine Nucleotide Exchange Rate GTP Ras-GTP Ras-O Cdc25 REM Rate: kcat, Km Sos1 Part of Sos1

  17. Guanine Nucleotide Exchange Rate GDP GTP Ras-GDP Ras-GTP Ras-O Cdc25 REM Sos1 Part of Sos1

  18. Guanine Nucleotide Exchange Rate GDP GTP Ras-GDP Ras-GTP Ras-O Ras-GDP Cdc25 REM Rate: 5 times as fast Sos1 Part of Sos1

  19. Guanine Nucleotide Exchange Rate GDP GTP Ras-GDP Ras-GTP Ras-O Ras-GTP Cdc25 REM Rate: 75 times as fast Sos1 Part of Sos1

  20. Ras Deactivation (Hydrolysis) Ras-GDP RASA1 Ras-GTP

  21. How Fast Does Ras Turn Off Ras-GDP Ras-GTP Ras-GTP is a GTPase: It hydrolyzes GTP to GDP (slowly)

  22. Ras Deactivation (Hydrolysis) Ras-GDP RASA1 Ras-GTP RASA1 speeds up the hydrolysis by ???

  23. Turning Ras On and Off: The Whole Story (… or is it?) GDP Ras-GDP Sos1 slow Ras-O fast RASA1 Super fast Ras-GTP GTP

  24. Summary • Ras is normally tightly bound to GDP • Ras is activated by binding to GTP instead of GDP • A “guanine nucleotide exchange factor” helps Ras to exchange GTP for GDP • GEF is the generic term • Sos1 is the specific GEF we will look at • Sos1 has an allosteric site that affects the reaction rate • A GTPase activating protein speeds up RasGTPase activity (ie, hydrolyzing the GTP so it becomes GDP) • GAP is the generic term • RASA1 is the specific GAP we will look at

  25. YouTube Lectures on Ras • Wittinghofer Part 1 • Wittinghofer Part 2

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