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CB201 : Molecular Biology of the Cell

CB201 : Molecular Biology of the Cell. Lipids and Signaling March 31, 2014 Brendan D. Manning Dept of Genetics & Complex Diseases Harvard School of Public Health. The diverse role of lipids in signal transduction . Isoprenoids. Acyl- glycerols. Fatty Acids. Sterols. DAG PIPs.

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CB201 : Molecular Biology of the Cell

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

  2. The diverse role of lipids in signal transduction Isoprenoids Acyl-glycerols Fatty Acids Sterols DAG PIPs Leukotrienes Prostaglandins hGH VitD Estrogen Cholesterol Farnesyl LPA Nuclear Receptors (ER, PPAR, etc) GPCR Ligand Intracellular Signaling (2nd Messengers) Lipid Rafts Small G Proteins

  3. Stimuli Receptors Transducers Amplifiers Signaling Pathway Messengers Sensors / effectors CELLULAR RESPONSES

  4. Signals are amplified by the transducing pathway Hormone Receptor (RTKs and GPCRs) Amplifier Second Messenger Effector Ser/Thr Kinase Targets

  5. Second Messengers in Signaling • Second messengers interact with and induce alterations in the activity and/or cellular location of signaling proteins • Messengers must: • Accumulate rapidly • Accumulate locally • Be mobile • Be removed by active mechanisms examples MESSENGERS Ca2+, Zn2+, Mn2+ metal ions cyclic nucleotides cAMP, cGMP dissolved gas NO lipids PIPs, DAG

  6. Calcium is rapidly mobilized to and from intracellular stores upon stimulation Ca2+ oscillations in histamine-stimulated cells Imaged with Fura-2: Ca2+-binding fluorescent dye

  7. Ca2+stimulates diverse cellular responses in a time-dependent manner

  8. cAMP Pathways Stimulatory Signal Amplifier (AdenylateCyclase) 2nd Messenger CFTR GPCR  cAMP q + AC PKA  GTP ATP CREB: cAMP Response element binding protein PDE: Phosphodiesterases PK: Phosphorylase kinase CFTR: Cystic fibrosis transmembrane regulator PK CREB PDEs Lipase

  9. The Human Kinome Many AGC family kinases are regulated by 2nd messengers cAMP PKA PKC C2 C1 DAG Ca2+ • Akt/PKB • PH PIP3

  10. Lipids 101 Fatty Acid HO O Glycerol OH HO HO O O Monoacylglycerol (MAG) HO HO O Lysophosphatidic Acid (LPA) O O LPAis the most abundant growth factor/mitogen in serum, and signals through GPCRs HO OH O P OH

  11. Lipids 101 Fatty Acid HO O Glycerol OH HO HO O O O Diacylglycerol (DAG) O HO DAGs and TAGs are stored in lipid droplets O O O Triacylglycerol (TAG) O O O

  12. Lipids 101 Phosphatidic Acid (PA) O O O Serine Ethanolamine Choline Inositol O O OH O P PA is a precursor for membrane phospholipids OH Phosphatidylserine Phosphatidylethanolamine Phosphatidylcholine Phosphatidylinositol

  13. Lipids 101 Phosphatidic Acid (PA) O O O Serine Ethanolamine Choline Inositol O O OH O P PA is a precursor for membrane phospholipids OH OH OH 5 6 Phosphatidylserine Phosphatidylethanolamine Phosphatidylcholine Phosphatidylinositol HO OH 4 1 3 2 OH OH

  14. Phosphatidylinositol phosphates (PIPs) are of very low abundance • but act as key second messengers in signaling Phosphatidylinositol-4,5-bisphosphate (PtdIns-4,5-P2 or PIP2) O O O OH OPO32- O O 5 6 O O P OPO32- 4 1 3 2 OH OH OH Lipid % of total Phophatidylserine…………………………..8.5% Phosphatidylinositol………………………..1.0% Phosphatidylinositol-4,5-bisphosphate…..0.05%

  15. PhosphoinositideKinases and Phosphatases Control the localized production and removal of PIPs PtdIns-4-P PtdIns-3,4-P2 Kinase Phosphatase PtdIns PtdIns-3-P PtdIns-4,5-P2 PtdIns-3,4,5-P3 PtdIns-5-P PtdIns-3,5-P2

  16. PIP2 Controls Many Cellular Processes

  17. Phospholipase C hydrolyzes PIP2 to produce • two second messengers, DAG and IP3 Phosphatidylinositol-4,5-bisphosphate (PtdIns-4,5-P2 or PIP2) O Phospholipase C (PLC) O O OH OPO32- O O 5 6 O O P OPO32- 4 1 3 2 OH OH OH O O O OH OPO32- O O 5 6 O OH HO P OPO32- 4 1 Diacylglycerol (DAG) 3 2 OH OH OH Inositol-1,4,5-Trisphosphate (IP3)

  18. Phospholipase C hydrolyzes PIP2 to produce • the two second messengers DAG and IP3 Stimulatory signal PIP2 DAG GPCR  q + PLCb C2 C1 GTP  IP3

  19. Phospholipase C hydrolyzes PIP2 to produce • the two second messengers DAG and IP3 Stimulatory signal PIP2 DAG RTK Ras PLCe C2 C1 IP3

  20. Phospholipase C hydrolyzes PIP2 to produce • the two second messengers DAG and IP3 Stimulatory signal PIP2 DAG RTK PLCg C2 C1 PDGFR IP3

  21. Phospholipase C hydrolyzes PIP2 to produce • the two second messengers DAG and IP3 Stimulatory signal DAG binds to C1 domains on downstream effectors PIP2 DAG RTK PLCg PKC C2 C1 The IP3 receptor on the ER is an IP3-gated Ca2+ channel Ca2+-binding Effectors Ca2+ IP3 Ca2+ ER Time (Min) Adapted from Newton J Lipid Res 2009

  22. PKCs, a Large Family of DAG Receptors Conventional Novel Atypical Substrates: Cell proliferation Cell survival Cytoskeletal dynamics Membrane trafficking

  23. PKC activates Erk signaling and • feedback inhibits PLC activity Stimulatory signal PIP2 DAG GPCR  q + PLCb PKC C2 C1 GTP  Raf MEK Erk Cell Proliferation Ca2+ IP3

  24. Phosphatidylinositol phosphates (PIPs) are second messengers • that relay spatial cues to signaling pathways O Phosphatidylinositol O O OH OH O O 5 6 O O P OH 4 1 3 2 OH OH OH Kutateladze et al., Nat ChemBiol 2010

  25. Many proteins contain domains that bind to specific PIPs, thereby • recruiting these proteins to specific membrane compartments O Phosphatidylinositol O O OH OH O O 5 6 O O P OH 4 1 3 2 OH OH OH Signaling, Cytoskeletal, and Trafficking Proteins Kutateladze et al., Nat ChemBiol 2010

  26. These PIP-binding domains contain conserved residues that • engage different combinations of phosphates on the inositol ring O Phosphatidylinositol O O OH OH O O 5 6 O O P OH 4 1 3 2 OH OH OH PI3P FYVE Domain (Fab1,YOTB,Vac1,Eea1) PI3,4,5P3 or PIP3 PH Domain (PleckstrinHomology) PI3P PX Domain (Phox Homology) Kutateladze et al., Nat ChemBiol 2010

  27. PhosphoinositideKinases and Phosphatases Control the localized production and removal of PIPs PtdIns-4-P PtdIns-3,4-P2 Kinase Phosphatase PtdIns PtdIns-3-P PtdIns-4,5-P2 PtdIns-3,4,5-P3 PtdIns-5-P PtdIns-3,5-P2

  28. 3 classes of phosphoinositide 3-kinases have different substrate specificities PI3K (class I or II) PtdIns-4-P PtdIns-3,4-P2 Kinase Phosphatase PI3K (class III) PI3K (class I) PtdIns PtdIns-3-P PtdIns-4,5-P2 PtdIns-3,4,5-P3 PtdIns-5-P PtdIns-3,5-P2

  29. Phosphoinositide 3-kinase produces PIP3 • in response to growth factors Phosphatidylinositol-4,5-bisphosphate (PtdIns-4,5-P2 or PIP2) O O O OH OPO32- O O 5 6 O O P OPO32- 4 1 3 2 OH OH OH Phosphatidylinositol-3,4,5-triisphosphate (PtdIns-3,4,5-P2 or PIP3) PI3K O O O OH OPO32- O O 5 6 O O P OPO32- 4 1 3 2 Lipid % of total Phophatidylserine………………………………..8.5% Phosphatidylinositol……………………………..1.0% Phosphatidylinositol-4,5-bisphosphate………..0.05% Phosphatidylinositol-3,4,5-bisphosphate……..0.0001% OH OH OPO32- 10-fold Increase upon stimulation

  30. Class IB PI3K is a downstream effector of GPCRs Stimulatory signal PIP3 PIP2 GPCR  s p110g + C2 C1 p101 GTP  Class IB PI3K

  31. Class IA PI3K is a downstream effector of RTKs and Ras Stimulatory signal PIP3 PIP2 RTK p110a Ras C2 C1 p85 Class 1API3K (p110 a / b / d) p110 p85

  32. Class IA PI3K is a downstream effector of RTKs and Ras Stimulatory signal PIP3 PIP2 RTK p110a p85 Class 1API3K (p110 a / b / d) p110 p85 The SH2 domains of p85 bind to phospho-tyrosineresidues on RTKs and scaffolding adaptors to bring PI3K to its substrate PI4,5P2 at the plasma membrane

  33. Class I PI3Ks generate the lipid second messengerPIP3, • which recruits specific downstream effectors Stimulatory signal • EGF PIP3 PIP2 RTK p110a • Effector p85 PTEN • PH • PH • wortmannin Time-lapse microscopy (10s intervals): 60s stimulation with EGF Followed by wortmannin treatment Cullen et al., BJ (1998) 335; 139-146. • GFP

  34. Class I PI3Ks generate the lipid second messengerPIP3, • which recruits specific downstream effectors Stimulatory signal PIP3 PIP2 RTK p110a • Effector p85 PTEN • PH • PH • >> PH domains found in • 275 human proteins • >> 10-20% have strong preferences • for specific PIP species • (PI4,5P2 / PI3,4P2 / PI3,4,5P3) • >> PH domains engage phosphates • on the inositol head group • via three variable loops Kutateladze et al., Nat ChemBiol 2010

  35. Class I PI3Ks generate the lipid second messengerPIP3, • which recruits specific downstream effectors Stimulatory signal PIP3 PIP2 RTK p110a • Effector p85 PTEN • PH PIP3-bindingEffectors of PI3K • Grp1 – An ARF GEF – Vesicle trafficking • Vav2 – A Rac GEF – Cytoskeletal dynamics / Migration • Btk– A Tyr Kinase – PLC regulation (DAG/Ca2+) • Akt – A Ser/Thr Kinase – Many substrates

  36. Akt (aka protein kinase B or PKB) is a major • downstream effector of PI3K signaling Stimulatory signal PIP3 PIP3 PIP2 PIP3 T308 RTK p110a • Akt • PDK1 • mTORC2 p85 PTEN S473 • PH • PH Rapid and time dependent Dose dependent Stochastic Response (p-Akt) Debnath et al. Cell 2002

  37. Akt (aka protein kinase B or PKB) is a major • downstream effector of PI3K signaling Stimulatory signal PIP3 PIP3 PIP2 PIP3 T308 RTK p110a • Akt • PDK1 • mTORC2 p85 PTEN S473 • PH • PH Cell Survival Cell Proliferation Metabolism (cell-tissue-system) Cell Growth

  38. PI3K-Akt is constitutively activated in • over 50% of human cancers PIP3 PIP3 PIP2 PIP3 * T308 * Ras RTK p110a * • Akt • PDK1 • mTORC2 p85 PTEN * X S473 • PH • PH Cell Survival Cell Proliferation Metabolism (cell-tissue-system) Cell Growth

  39. PIK3CA and PTEN are frequently altered • in human cancers • (data from ~69 tumor genome sequencing studies) PIP3 PIP3 PIP2 PIP3 Uterine T308 Ras RTK p110a * • Akt • PDK1 • mTORC2 p85 PTEN X Prostate Cervical Breast Lung Brain Head & Neck S473 • PH • PH Ovarian Colon Bladder Melanoma Kidney

  40. Akt has many downstream targets to control diverse cellular processes Manning and Cantley2007 Cell

  41. Akt has many downstream targets to control diverse cellular processes R-x-R-x-x-S/T-Φ -x=any amino acid -Φ=large hydrophobic Akt-mediated phosphorylation often inhibits its substrates Manning and Cantley2007 Cell

  42. FOXO proteins regulate transcription downstream of Akt Akt Akt Vertebrate C. elegans Tran et al 2003 Science STKE Greer and Brunet 2005 Oncogene

  43. S/T phosphorylation, like Tyr phosphorylation, creates protein-protein binding motifs Yaffe and Elia 2001 CurrOpin Cell Biol

  44. Sequence overlap between Akt substrate preference and phospho-binding motif of 14-3-3 proteins Reinhardt and Yaffe 2013 Nat Rev Mol Cell Biol FOXO1-T24 R-x-R-x-x-S/T-Φ-x Akt substrate R-P-R-S-C-T-W-P 14-3-3 binding x-x-R-x-x-S/T-x-P P

  45. Akt Controls FOXO1 Localization Akt OFF - FOXO1 nuclear Akt ON - FOXO1 cytoplasmic { PI3K PI3K 14-3-3 P FOXO Akt P Akt P X FOXO FOXO Targets Targets Apoptosis (Bim1, FasL) Cell Survival Cell Cycle Arrest (p21, p27) Proliferation

  46. Insulin control of systemic glucose homeostasis Glucose Production Liver insulin eal glucose Pancreas insulin Muscle Fat Glucose Uptake

  47. GLUT4 PIP2 PIP3 PIP3 AKT PI3K IRS-1 PI3K binding Y608 Y628 Y891 Y935 PH PTB Insulin signaling in muscle Glucose Insulin IR P Glucose Uptake IRS P P P Glycogen Synthesis Glucose Storage Muscle Mass Protein Synthesis

  48. GLUT4 PIP2 PIP3 PIP3 AKT PI3K Insulin signaling in muscle Glucose Insulin IR P P P P Glucose Uptake IRS AS160 P P P P GSK3 Glycogen Synthesis P P P TSC2 Protein Synthesis

  49. GLUT4 GLUT4 AKT Glut4 is the major glucose transporter in muscle and adipose and rapidly translocates to the plasma membrane in response to insulin Glut4 AS160/TBC1D1 is a Rab GAP P P P AS160 Rab Rab GDP GTP Glut4 Vesicle Watson and Pessin 2006 TiBS

  50. GLUT4 PIP2 PIP3 PIP3 AKT PI3K Insulin signaling in muscle Glucose Insulin IR P P P P Glucose Uptake IRS AS160 P P P P GSK3 mTORC1 Glycogen Synthesis P P P TSC2 P P Glycogen Synthase P P Protein Synthesis Lipid Synthesis

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