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 Allosteric site: 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 Ras GTPase 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 GTP Ras- GDP Ras- GTP Ras-O Ras can bind to either GDP or GTP (or not at all)

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

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

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

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

14. Cdc25 REM Guanine Nucleotide Exchange Rate Ras- GDP Sos1 Rate: k cat, K m Part of Sos1 Ras- GDP

15. Cdc25 REM Guanine Nucleotide Exchange Rate Sos1 Rate: k cat, K m Part of Sos1 Ras- GDP GDP Ras-O

16. Cdc25 REM Guanine Nucleotide Exchange Rate Ras-O Ras- GTP Sos1 GTP Rate: k cat, K m Part of Sos1

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

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

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

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

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 Ras- GTP RASA1 RASA1 speeds up the hydrolysis by ???

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

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 Ras GTPase 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