CB201 : Molecular Biology of the Cell

1. CB201: Molecular Biology of the Cell Cell Growth and Nutrient Sensing March 31, 2014 Brendan D. Manning Dept of Genetics & Complex Diseases Harvard School of Public Health

2. Secreted Factors (growth factors, mitogens, hormones, cytokines) Growth Differentiation Proliferation Death Nutrients Stress

3. Cardiac Myocytes NTg Pancreatic b-cells caAkt The PI3K-Akt pathway plays an evolutionarily conserved role in the control of cell growth Drosophila Cell Growth PI3K Akt PTEN Control DN-PI3K PTEN

4. Overexpression Gene Disruption WT tsc1-/- The PI3K-Akt pathway plays an evolutionarily conserved role in the control of cell growth Drosophila Cell Growth PI3K Akt PTEN TSC1/2 Tapon et al. 2001 Cell Potter et al. 2001 Cell Gao and Pan 2001 Genes Dev Control DN-PI3K PTEN C Tsc1 Tsc2 Tsc1/2

5. Gene Disruption WT tsc1-/- The PI3K-Akt pathway plays an evolutionarily conserved role in the control of cell growth Drosophila Cell Growth PI3K Akt PTEN TSC1/2 Tuberous Sclerosis Complex Control DN-PI3K PTEN Tuber Normal

6. Tuberous Sclerosis Complex TSC affects ~1 in 6,000 individuals (children). Characterized by widespread benign tumors (hamartomas). Abundant facial fibromas and kidney and lung malfunction due to tumor burden. CNS manifestations: epilepsy, cognitive deficits, and autism spectrum disorders. Mapped to germline mutations in TSC1 (~15%) or TSC2 (~70%). Brain Skin Kidney Lung (LAM)

7. PIP2 PIP3 PIP3 PIP3 PIP2 PIP3 AKT PDK1 PI3K The PI3K-TSC-mTOR Pathway RTK P IRS1 PTEN P Manning et al. Mol Cell 2002 Inoki et al. Nat Cell Biol2002 P P P TSC2 TSC1 TSC3? TBC1D7 Dibble et al. Mol Cell 2012 J Med Genet 2013 Hum Mut2013 Cell Growth

8. PIP2 PIP3 PIP3 PIP3 PIP2 PIP3 AKT PDK1 PI3K The PI3K-TSC-mTOR Pathway RTK P IRS1 PTEN P Rheb P P P GDP TSC2 TSC1 Rheb TBC1D7 GTP Rapamycin mechanistic (or mammalian) Target Of Rapamycin mTOR Cell Growth

9. Rapamycin is a very specific natural inhibitor of mTOR Easter Island (Rapa Nui) Rapamycin is an allosteric and partial inhibitor of mTOR

10. Rapamycin is a very specific natural inhibitor of mTOR Rapalogs: Rapamycin= Sirolimus/Rapamune CCI-779 = Temsirolimus RAD001 = Everolimus MK8669 = Ridaforolimus Rapamycin is an allosteric and partial inhibitor of mTOR mTOR kinase inhibitors (Torin, KU0063794, AZD8055, GDC0349, XL388)

11. mTOR complex 1 senses physiological and pathological growth signals and is widely dysregulated in human diseases Growth Factors Insulin Glucose Cytokines ATP Obesity Oncogenes Oxygen mTORC1 Tumor Suppressors Amino Acids Infectious Agents mTOR Stress mLST8 Raptor Cancer Type-2 Diabetes Asthma Tuberous Sclerosis Complex Autism Spectrum Disorders Obesity Systemic Lupus Erythematosus Fragile X Syndrome Lymphangioleiomyomatosis Epilepsy Non-alcoholic Fatty Liver Disease Arthritis Neurofibromatosis Huntington’s Cowden Syndrome Autoimmune Lymphoproliferative Syndrome Cardiovascular Disease Alzheimer’s Proteus Syndrome Peutz-Jeghers Syndrome Polycystic Kidney Disease Parkinson’s Progeria Aging

12. The PI3K-mTOR Pathway PIP3 PIP2 PIP3 PIP2 PIP3 PIP3 AKT PDK1 PDK1 PI3K RTK T308 P IRS1 S473 PTEN P Rheb P P P GDP TSC2 mTORC2 TSC1 Rheb mTOR mLST8 TBC1D7 GTP SIN1 Rictor mTORC1 Rapamycin mTOR mLST8 Raptor P P 4E-BP1 T229 P P P P S6K1/2 T389 Cell Growth

13. Activation Loop Hydrophic Motif mTORC1 mTOR mLST8 Raptor PDK1 PDK1 T229 T389 P P mTORC2 mTOR mLST8 SIN1 Rictor S473 T308 P P The Human Kinome S6K Kinase PIP3 AKT PH Kinase P P SGK1 Kinase P P PKCa Kinase

14. The PI3K-mTOR Pathway PIP3 PIP2 PIP2 PIP3 PIP3 PIP3 AKT PDK1 PI3K RTK P IRS1 PTEN P Rheb P P P GDP TSC2 TSC1 Growth Rheb TBC1D7 GTP mTORC1 Rapamycin mTOR mLST8 Raptor Proliferation P P 4E-BP1 Rapamycin reduces cell size and stalls cells in G1 P P P P S6K1/2 Cell Growth

15. The PI3K-mTOR Pathway PIP2 PIP3 PIP3 PIP3 PIP2 PIP3 AKT PDK1 PI3K RTK P IRS1 PTEN P Rheb P P P GDP TSC2 TSC1 Rheb TBC1D7 GTP mTORC1 mTOR mLST8 dS6K KO Raptor P Montagne et al. 1999 Science P 4E-BP1 P P ? P P S6K1/2 Cell Growth

16. Anabolic Growth & Proliferation DNA RNA Proteins Organelles Plasma membrane Nucleotides Nucleotides Amino Acids Lipids Lipids The metabolic challenges of growth and proliferation ATP + NADPH Increased production of biomass

17. mTORC1 Promotes Anabolic Metabolism Rheb GDP TSC2 TSC1 Rheb TBC1D7 GTP mTORC1 mLST8 mTOR Raptor Macromolecules CAD 4E-BP S6K ULK1 HIF1 SREBP Autophagy mRNA Translation Aerobic Glycolysis De novo Lipid Synthesis Pentose Phosphate Pathway De novo Nucleotide Synthesis Ribosome Biogenesis ATP Nutrients Biosynthetic Precursors NADPH Protein Ribose AAs Glycerol Membranes Nucleic Acids Anabolic Cell Growth and Proliferation

18. mTORC1 promotes cap-dependent translation P P P P mTORC1 4E-BP 4E-BP eIF4E 7-mGTP AUG AAAAAA 5’-UTR

19. mTORC1 promotes cap-dependent translation P P P P 4E-BP eIF4G Unwinding 2° structure eIF4E 4A 4B 7-mGTP AUG AAAAAA P S6K1 mTORC1

20. mTORC1 promotes cap-dependent translation P P P P 4E-BP eIF4G Unwinding 2° structure eIF4E 4A 4B 7-mGTP AUG AAAAAA P 40S S6 P P Increased translation of mRNAs with complex 5’-UTRs (Cyclin D, C-Myc) P P S6K1 mTORC1

21. mTORC1 promotes cap-dependent translation P P P P 4E-BP eIF4G eIF4E CCUUUCU 7-mGTP AUG AAAAAA mTORC1 Translation of 5’-terminal oligopyrimidine (TOP) mRNAs: >>All ribosomal proteins >>Translation factors Global increase in protein synthesis (proteins make up ~50% of cell mass)

22. mTORC1 Promotes Anabolic Metabolism Rheb GDP TSC2 TSC1 Rheb TBC1D7 GTP mTORC1 mLST8 mTOR Raptor Porstmann et al. 2008 Cell Metab Macromolecules CAD 4E-BP S6K ULK1 HIF1 SREBP Autophagy mRNA Translation Aerobic Glycolysis De novo Lipid Synthesis Pentose Phosphate Pathway De novo Nucleotide Synthesis Ribosome Biogenesis ATP Nutrients Biosynthetic Precursors NADPH Protein Ribose AAs Glycerol Membranes Nucleic Acids Anabolic Cell Growth and Proliferation

23. The sterol-regulatory element binding proteins (SREBPs) are global transcriptional regulators of de novo lipid synthesis Horton et al., 2002 J. Clin Invest

24. mTORC1 signaling stimulates SREBP processing and nuclear accumulation S2P - INSIG SREBP SCAP Lumen - ER Cytosol S1P mTORC1 Lumen Golgi Cytosol De novo Lipid Synthesis Gene Targets Nucleus Düvelet al. 2010 Mol Cell

25. mTORC1 signaling stimulates SREBP processing and nuclear accumulation S2P - INSIG SREBP SCAP Lumen - ER Cytosol S1P mTORC1 Lumen Golgi Cytosol siCtl +Vehicle siCtl +Rap siSREBP1/2 +Vehicle Tsc2-/- MEFs Gene Targets Nucleus Rapamycin treatment or SREBP1/2 knockdown both deplete cholesterol esters in the cell StephaneRicoult, unpublished

26. AKT PI3K mTORC1 is required for insulin to activate hepatic SREBP1c and lipid synthesis Insulin PIP2 PIP3 PIP3 IRS Rheb P P P GDP TSC2 TSC1 FOXO Gluconeogenesis Rheb TBC1D7 GTP mTORC1 SREBP1c Lipogenesis mTOR mLST8 Duvel et al., 2010 Mol Cell Li et al., 2010 PNAS Yecies et al. 2011 Cell Metab Wan et al., 2011 Cell Metab Peterson et al., 2011 Cell Owen et al., 2012 PNAS Raptor P S6K1

27. Secreted Factors (growth factors, mitogens, hormones, cytokines) Growth Differentiation Proliferation Death Nutrients Stress

28. Coordination and integration of growth factor and nutrient sensing pathways EGF PDGF Insulin/IGF1 Nutrients and Energy Cytokines ATP Growth Factors Oxygen Glucose Glutamine [Systemic nutrient status] Amino Acids Signaling Pathways nutrient sensing Anabolic Metabolism Cell Growth and Proliferation

29. Coordination and integration of growth factor and nutrient sensing pathways EGF PDGF Insulin/IGF1 Nutrients and Energy Cytokines ATP Growth Factors Oxygen Glucose Glutamine Amino Acids mTORC1 mTOR mLST8 Raptor S6K1 Protein Synthesis Cell Growth and Proliferation

31. Amino acid and growth factor signals are integrated by mTORC1 at the lysosome Amino acid deplete Amino acid replete Kinase OFF “on” “off” V- ATPase V- ATPase GDP GTP RagA RagA Ragulator Ragulator +amino acids GTP GDP RagC RagC Cytosol Lysosome Lumen AA Sensor? Dubouloz et al. 2005 Mol Cell Sancak et al. 2008 Science Kim et al. 2008 Nat Cell Biol Sancaket al. 2010 Cell Zoncu et al. 2011 Science Bar-Peled et al. 2012 Cell Bar-Peled et al. 2013 Science Panchaud et al. 2013Sci Signal mTORC1 LST8 mTOR Raptor

32. Amino acid and growth factor signals are integrated by mTORC1 at the lysosome Amino acid deplete Amino acid replete Kinase OFF “on” “off” V- ATPase V- ATPase GTP GDP RagA RagA Ragulator Ragulator +amino acids GTP GDP RagC RagC Cytosol Lysosome Lumen AA Sensor? Dubouloz et al. 2005 Mol Cell Sancak et al. 2008 Science Kim et al. 2008 Nat Cell Biol Sancaket al. 2010 Cell Zoncu et al. 2011 Science Bar-Peled et al. 2012 Cell Bar-Peled et al. 2013 Science Panchaud et al. 2013Sci Signal mTORC1 mTORC1 LST8 LST8 mTOR mTOR Raptor Raptor

33. Amino acid and growth factor signals are integrated by mTORC1 at the lysosome Amino acid replete No growth factors Kinase OFF “on” P V- ATPase GTP RagA Ragulator GDP GDP TSC2 RagC RHEB Cytosol Lysosome Lumen AA Sensor? TBC1D7 TSC1 mTORC1 LST8 mTOR Raptor Menon, Dibble et al 2014 Cell

34. Amino acid and growth factor signals are integrated by mTORC1 at the lysosome PI3K Amino acid replete Growth factor stimulated Kinase ON Akt TSC2 “on” P P P P P V- ATPase GTP RagA Ragulator GDP GTP GDP TSC2 RagC RHEB RHEB Cytosol Lysosome Lumen A spatial “AND gate” or “coincidence detector” AA Sensor? TBC1D7 TBC1D7 TSC1 TSC1 mTORC1 LST8 mTOR Raptor Menon, Dibble et al 2014 Cell

35. Oncogenic PI3K signaling results in constitutive dissociation of the TSC complex from the lysosome PTEN PI3K Amino acid replete Oncogenic signaling Kinase ON Akt TSC2 “on” P P P P P V- ATPase GTP RagA Ragulator GDP GTP GDP TSC2 RagC RHEB RHEB Cytosol Lysosome Lumen PC3 – PTEN null prostate cancer cells Vehicle MK2206 TBC1D7 TBC1D7 TSC1 TSC1 TSC2-LAMP2 mTORC1 LST8 mTOR Raptor Menon, Dibble et al 2014 Cell

36. Amino acid and growth factor signals are integrated by mTORC1 at the lysosome PI3K Amino acid replete Growth factor stimulated Kinase ON Akt TSC2 “on” P P P P P V- ATPase GTP RagA Ragulator GDP GTP GDP TSC2 RagC RHEB RHEB Cytosol Lysosome Lumen AA Sensor? TBC1D7 TBC1D7 Why the lysosome? >Amino acid production and storage >Regulation of lysosomal function and autophagy TSC1 TSC1 mTORC1 LST8 mTOR Raptor

37. mTORC1 monitors the health and function of the lysosome Kinase ON “on” V- ATPase GTP RagA Ragulator P GTP GDP P TFEB RagC RHEB Cytosol Lysosome Lumen [H+] (low pH) AA Sensor? mTORC1 LST8 mTOR Raptor Settembre et al 2012 EMBO J Roczniak-Ferguson et al 2012 Science Signal

38. mTORC1 monitors the health and function of the lysosome Nucleus Kinase OFF Gene Targets TFEB V-ATPase subunits Lysosomalenyzmes Autophagy genes P P “off” V- ATPase GDP RagA Ragulator P GTP GTP P TFEB RagC RHEB Cytosol Lysosome Lumen [H+] (High pH) Amino acid depletion mTORC1 LST8 mTOR Raptor Settembre et al 2012 EMBO J Roczniak-Ferguson et al 2012 Science Signal

39. Autophagy (self eating) Nutrient depletion Adapted from Boya et al. Nat Cell Biol 2013

40. Autophagy recycles macromolecules into biosynthetic precursors mTORC1 OFF Rabinowitz and White Science 2010

41. Autophagy (self eating) Nutrient depletion ATG101 FIP200 ATG13 ULK1/2 mTORC1 LST8 mTOR Raptor Adapted from Boya et al. Nat Cell Biol 2013

42. Glucose and Energy Sensing

43. Glucose and oxygen levels are sensed, in part, through resulting effects on intracellular energy levels ATP ADP Glucose 6-P Glucose Fructose 6-P ATP ADP Fructose 1,6-bis-P Glyceraldehyde 3-P Dihydroxyacetone-P NAD NADH 1,3-Bisphosphoglycerate ADP ATP 3-Phosphoglycerate 2-Phosphoglycerate O2 ATP Phosphoenolpyruvate ADP ATP TCA Pyruvate Ox-Phos Acetyl-CoA mitochondria

44. The AMP-dependent protein kinase (AMPK) is a critical sensor of cellular energy status LKB1 P a g b AMP AMP Adapted from Hardie, D. G. J Cell Sci 2004;117:5479-5487

45. AMPK is a critical sensor of cellular energy status Energy Consumption Adapted from Hardie 2007 Nat Rev Mol Cell Biol

46. AMPK turns on ATP-producing catabolic processes and turns off ATP-consuming biosynthetic processes Metformin, Rosiglitazone, Resveratrol exercise, nutrient deprivation, hypoxia, mitochondrial poisons ( AMP/ATP) Anabolic Processes Catabolic Processes Adapted from Hardie 2007 Nat Rev Mol Cell Biol

47. Growth-promoting signals Poor growth conditions Growth factor withdrawal Starvation Energy depletion Cellular stress Tumor Suppressors Mitogens Cytokines Hormones Nutrients Energy Oncogenes Rheb GDP TSC2 TSC1 Rheb TBC1D7 GTP mTORC1 mTORC1 and AMPK reciprocally regulate anabolic & catabolic processes mLST8 mTOR Raptor Protein Synthesis Lipid Synthesis Autophagy AMPK Energy Depletion

48. PIP3 PIP2 AKT PI3K Integration of systemic and local signals reflecting nutrient and energy status Systemic Nutrients (insulin/IGF1) Ras PIP2 PIP3 PIP3 P IRS1 PTEN P Rheb P P P GDP TSC2 P Exercise TSC1 AMPK AMP Rheb Metformin TBC1D7 GTP Glucose Local Nutrients mTORC1 Rag A/B GTP Oxygen mTOR mLST8 Rag C/D GDP Raptor Amino Acids P Anabolic Cell Growth and Proliferation

49. Oncogenes EGFR HER2 MET FGFR BCR-ABL RAS RAF PI3K AKT Wnt Tumor Suppressors PTEN INPP4B NF1 LKB1 ATM APC Rheb GDP TSC2 TSC1 Rheb TBC1D7 GTP mTORC1 mTORC1 is aberrantly activated in up to 80% of human cancers mLST8 mTOR Raptor Autophagy mRNA Translation Aerobic Glycolysis De novo Lipid Synthesis Pentose Phosphate Pathway De novo Nucleotide Synthesis ATP Ribosome Biogenesis Biosynthetic Precursors NADPH Ribose AAs Glycerol Nucleic Acids Protein Membranes Cell Growth and Proliferation