John Backer

1. mTORC1 and the Endosomal System JON BACKER DEPT. OF MOLECULAR PHARMACOLOGY ALBERT EINSTEIN COLLEGE OF MEDICINE BRONX, NEW YORK

2. Cell Growth The mTOR Protein Kinase Ma and Blenis, Nat. Rev. Mol. Biol. 2009 Nutrients

3. LKB1 PTEN TSC1 TSC2

4. Guertin and Sabatini, Cancer Cell 2007 mTOR and Angiogenesis

5. Dancey et al., Sem. Oncology 2009

6. Dancey et al., Sem. Oncology 2009

7. Dancey et al., Sem. Oncology 2009

8. Liu et al, Nat. Rev. Drug Discovery 2009 Anti-Signaling Drugs in Clinical Trials

9. S6K1 TORC1 Rheb(GTP) TSC1/2 Akt Insulin 4EBP1 mTOR and Translational Control Ribosomal S6 Translation initiation Amino Acids hVps15/ hVps34 Glucose AMPK

10. Vps34 Vps15 Rab5 GTP PI[3]P Christoforidis et al., Nature Cell Biol. 1999 Murray et al., Traffic (2002) hVps34 Regulates Early Endosomal Fusion P P hVps34 Class III PI[3]P PX FYVE hVps15

11. Former Labmates Maya Byfield James Murray Gary Gerfen Mark Girvin Ana Maria Cuervo Invasion and Motility PO1 Anne Bresnick Diane Cox John Condeelis Jeff Pollard Jeff Segall Richard Stanley Roger Williams (Cambridge) Somasekar Seshagiri (Genentech) Tom Neufeld (Univ. Minnesota) Kevan Shokat (USCF) Bart Vanhaesebroeck (London Sch. Med.) Funding: NIH, NCI, ADA

12. CCV EE RC LE Lysosome TGN Plasma Membrane 2 1 3 4 5 5 6 Is mTORC1 Inhibited by Disruption of the Endocytic System? Mutant Effect 1) Rab5 CA Inhibit 2) Eps15 No Effect 3) Rab11 Mutants No Effect 4) Rab7 Mutants No Effect 5) PIKFyve DN No Effect 6) SKD1 DN No Effect

13. Constitutive activation of Rab5 blocks mTORC1/S6K1 activation AA Stimulated S6K1 in HEK293T Flinn et al. Mol. Biol. Cell. (2010)

14. CCV EE RC LE Lysosome TGN Plasma Membrane 2 1 3 4 5 5 6 Is mTORC1 Inhibited by Disruption of the Endocytic System? Mutant Effect 1) Rab5 CA Inhibit 2) Eps15 No Effect 3) Rab11 Mutants No Effect 4) Rab7 Mutants No Effect 5) PIKFyve DN No Effect 6) SKD1 DN No Effect

15. Rab5CA blocks early/late endosomal conversion producing hybrid early/late endosomes Flinn et al. Mol. Biol. Cell. (2010)

16. Blocking early/Late endosomal conversion by hVps39 KnDn inhibits mTORC1/S6K1 EEA1 LAMP2 5 micron 5 micron 20 micron 20 micron Hela Flinn et al. Mol. Biol. Cell. (2010) S6K1 Phosphorylation

17. Sancak et al, Science. 2008 mTOR Activation by Amino Acids Requires Rag-mediated Endosomal Translocation

18. Amino Acids Induce mTOR translocation Flinn et al. Mol. Biol. Cell. (2010)

19. Rab5CA leads to mTOR localization to hybrid early/late endosomes Flinn et al. Mol. Biol. Cell. (2010)

20. mTOR, hVps34 and the Endosomal System EE LE MVB Lys Rab5 V34 V15 Rab7 V34 V15 HOPS Lamp2 Rheb Rab5

21. mTOR, hVps34 and the Endosomal System EE LE MVB Lys Rab5 V34 V15 Rab7 V34 V15 Rheb mTOR (off) mTOR (on) Amino Acid Starved Amino Acid Replete Lamp2 HOPS Rab5

22. MVB Lys mTOR (off) Amino Acid Replete EE/LE Rab5CA V34 V15 Rab7 Rab5CA mTOR, hVps34 and the Endosomal System LAMP2

23. MVB Lys Rheb mTOR (off) EE/LE Rab5CA V34 V15 Rab7 Rab5CA mTOR, hVps34 and the Endosomal System X LAMP2

24. Activation of Endogenous Rheb Does not Rescue Rab5CA Inhibition of mTOR TSC2-/- MEFs Flinn et al. Mol. Biol. Cell. (2010)

25. MVB Lys mTOR (off) EE/LE Rab5CA V34 V15 Rab7 Rab5CA mTOR, hVps34 and the Endosomal System X TSC1/2 X Rheb LAMP2

26. MVB Lys Rheb mTOR (off) EE/LE Rab5CA V34 V15 Rab7 Rab5CA mTOR, hVps34 and the Endosomal System Rheb Rheb Rheb Rheb Rheb Rheb Rheb Rheb Rheb X LAMP2

27. Endosomal Alkalinization blocks mTOR Activation

28. HEK293T AA Stimulated S6K1 Exogenous Rheb renders mTORC1 insensitive to ConA/NH4Cl AA:

29. Increasing intraluminal endosomal pH inhibits mTORC1 signaling

30. Endosomal Alkalinization inhibits amino acid-stimulated mTORC1/S6K1 signaling Hela AA Stimulated S6K1