1. Molecular and Cellular Mechanisms of Biosignaling Prof. Yong Tae Kwon Research interest: ubiquitin-proteasome system and autophagy in mammals Part I. Basics of Biosignaling (~2 lectures) Ligand, receptor (GPCR, RTK), intracellular signaling pathways, second messenger Part II. Signaling, Cell Cycle, and Cancer (~3 lectures) Growth factor-induced signaling, cell cycle, cell cycle checkpoint, apoptosis, DNA repair, oncogenesis, cancer treatment Part III. Molecular Cellular Physiology of Biosignaling (~3 lectures) Intercellular and intracellular signaling in the context of cell type- specific processes: nervous signaling, photosignaling, muscle contraction, and coagulation Part IV. Biosignaling in neuroendocrine system and metabolism (~2 lectures) Topics will include metabolism, obesity, diabetes, etc.

2. Each Student will present 1-2 slides at the end of my lecture. Lehninger Principles of Biochemistry, 5 th Ed Reading materials for additional information and examination

3. N-terminal amino acid as degradation signal The N-end rule pathway E3 X substrate X Ubiquitin-dependent proteolysis Ub

4. Components involved in this pathway in mammals? Physiological functions of identified components? Substrates underlying identified functions? Human diseases caused by mutations in the pathway? Biochemical principle in UPS? Inhibitors to control pathophysiological conditions? Is this physiologically meaningful?

5. Components and structure of N-end rule pathway Kwon, Nat Rev Mol Cell Biol 2011 Kwon, Ann Rev Biochem 2012 UBR1UBR3 UBR2UBR6 UBR4UBR7 UBR5

6. The Cell as a starting point to study All Biomedical Sciences Nucleus Nucleolus DNA Gene Precursor RNA Mature RNA Endoplasmic reticulum (ER) Protein synthesis Ribosome mRNA tRNA

7. Splicing DNA (exon and intron) Precursor RNA vs. mRNA: splicing mRNA vs. cDNA protein Endoplasmic reticulum (ER) Complex: ribosome, mRNA, tRNA ~35,000 genes 1 million proteins ???

8. Protein expression: mRNA, ER, ribosome, tRNA, polysome

9. Signaling between cells is usually mediated by interaction of a ligand with a receptor. The ligand-bound receptor activates intracellular signaling pathway to control various cellular functions. Basic elements of signal transduction Signal Transduction

10. Hydrophobic molecules (steroid hormones, retinoic acids, etc) enter cells and bind to intracellular receptors Hydrophilic or big molecules (proteins or polypeptide hormones) bind to cell surface receptors Majority Intracellular vs. cell surface receptors

11. Major types of intercellular signaling Contact dependent signaling: gap junction or ligand-receptor interaction on cell surface. Endocrine signaling: A sender cell secretes ligands (hormones) into the blood for long distance transport to target receptors. Paracrine signaling: A sender cell secretes ligands that are diffused (up to ~0.1 mm) to bind to target receptors. Synaptic signaling: An axon terminus secretes ligands (neurotransmitters) Into restricted environment to target nerves or muscles.

13. Cell surface receptors categorized by action modes Ligand binding to ligand-gated ion channel receptor permits ions (e.g., Na+) to permeates through the receptor in nervous cells. Enzyme-linked receptors themselves are kinases that phosphorylate effectors. Cytokine binding to cytokine receptors (without kinase activity) activates a kinase effect. G-protein-coupled receptors (GPCR) ~50% of all known drugs

14. Major Features of Signal Transduction: Specificity Numerous types of ligands Ligand specificity of receptors: signal to noise

15. Major Features of Signal Transduction: Amplification The initial ligand signal can be amplified ~10,000 times. Through a cascade of signaling molecules. Usually, from the plasma membrane toward the nucleus.

16. Major Features of Signal Transduction: Desensitization Homeostasis in signaling Negative feedback

17. Major Features of Signal Transduction: Integration Various signals are integrated and merged into common regulatory machinery in the cytoplasm and nucleus. Downstream signaling pathways are usually simpler (e.g., on vs. off).

18. Cells can sense millions of ligands.

19. G-Protein-Coupled Receptors (GPCRs) Represent ~5% of all proteins Targets of ~50% of all known drugs Hormones, neurotransmitters, Local mediators Heterotrimeric GTP-binding proteins (G proteins)

20. Heterotrimeric G proteins coupled with GPCR  s : stimulates adenylate cyclase, cAMP up.  i : inhibits adenylate cyclase, cAMP down  q : phospholipase C IP3, DAG, Ca++  12 : guanine-nucleotide exchange factors (GEFs)

21. Extracellular ligands Epinephrine (adrenaline) and its synthetic analogs

22. Transduction of the epinephrine signal: the  -adrenergic pathway GTP and cAMP

23. Phosphorylation and GTP-binding as major molecular switches Phosphorylation: the most widely used protein modification by kinases GTP-binding: switch on and off by the status of GTP-binding of G-proteins Molecular switches that turn on and off proteins

25. Inactivation of G-proteins by GAP GTP-bound: active Bound GTP hydrolyzed by GTPase activities (GAP) of Ras and its GAP Switch I and switch II relaxed into an inactive conformation Not interact with downstream targets such as Raf.

26. Activation and inactivation of G proteins  -GTP (active) +  (active)  -GDT (inactive) 1 GPCR vs. 10-100 G-proteins G proteins activate downstream effectors GTPase activator proteins (GAPs) and regulators of G protein signaling (RGSs) inactivate G proteins By modulating the GTPase activity

28. Constitutive activation of G  s by cholera toxin Cholera toxin The bacterium Vibrio cholerae Responsible for the harmful effects of cholera infection  s: constitutive activation Adenylate cyclase/cAMP ADP-ribosylation of G  s Chloride channel in intestine Diarrhea

30. Adenylate cyclases regulated by Gs and Gi

31. Ligand-bound receptors may mediate signaling via second messengers, small intracellular molecules. Fast signaling for the entire population of proteins that are present in the cell. Second Messengers

32. Synthesis and degradation of cAMP cAMP as a second messenger for GPCR The GPCR effector adenylate cyclase Breakdown by cyclic nucleotide phosphodiesterases (PDE) Activated by Gs and inhibited by Gi Basal conc.: 10 -7 Activated: 2-100 fold

33. Activation of cAMP-dependent protein kinase (PKA) When [cAMP] is low, two regulatory (R) subunits associate with catalytic subunits. The complex is catalytically inactive. When [cAMP] rises in response to a hormonal signal, each R subunit binds two cAMPs. Dramatic reorganization that pulls its inhibitory sequence away from the C subunit. Open up the substrate-binding cleft and releasing each C subunit in its catalytically active form.

34. Activation of cAMP-dependent protein kinase (PKA)

35. PKA controlled by cAMP cAMP function is mediated by cAMP-dependent protein kinase (PKA) PKA phosphorylates many other proteins cAMP-regulated gene regulatory proteins (CREBs) cAMP-sensitive regulatory elements (CRE)

38. Amplification in epinephrine cascade Epinephrine triggers a series of reactions in hepatocytes. Catalysts activate catalysts, resulting in great amplification of the signal.

39. Fig. 13.23. Phosphorylation to desensitize GPCR Protein kinase A (PKA) GPCR-specific protein kinases (GRKs) Unbound / Inactive Bound / ActiveBound / Inactive

40. Fig. 13.10. Termination of receptor-dependent signal transduction is essential to return to basal (pre-activation) state or to prevent overactivation fast Phosphorylation slow

41. Desensitization of the  -adrenergic receptor in the continued presence of epinephrine1  -adrenergic protein kinase (  ARK)  -arrestin (  arr).

42. Gq phospholipase C and IP 3 Two intracellular second messengers are produced in the hormone-sensitive phosphatidylinositol system: inositol 1,4,5-trisphosphate (IP 3 ) and diacylglycerol. Both contribute to the activation of protein kinase C. By raising cytosolic [Ca 2+ ], IP 3 also activates other Ca 2+ -dependent enzymes.

44. Ca++ signaling In response to many hormones and neurotransmitters By binding to Ca++-dependent regulators 10 -3 M (out, ER, SR) vs. 10 -7 M (in) Na+/Ca++ exchanger Ca++ pump: ATP Ca++ as a 2 nd messenger

45. Fig. 13.29. Ca++ signaling and its action

46. Triggering of oscillations in intracellular [Ca 2+ ] by extracellular signals A dye (fura) that undergoes fluorescence changes when it binds Ca 2+. Fluorescence intensity represented by color. The cells are heterogeneous in their responses. Some have high intracellular [Ca 2+ ] (red), others much lower (blue). Feedback mechanism

47. Triggering of oscillations in intracellular [Ca 2+ ] by extracellular signals Fura is used in a single hepatocyte. Norepinephrine (added at the arrow) causes oscillations of [Ca 2+ ] from 200 to 500 nM. Similar oscillations are induced in other cell types by other extracellular signals.

48. Transient and highly localized increases in [Ca 2+ ] A weak [IP 3 ]-producing stimulus may cause a single [Ca 2+ ] channel to open briefly. A somewhat stronger stimulus may cause all the Ca 2+ channels in a cluster to open. A sufficiently large puff produces elevated [Ca 2+ ] over an area great enough to include neighboring clusters of Ca 2+ channels. Opening of the channels in neighboring clusters propagates this effect, resulting in a wave of elevated [Ca 2+ ] moving along the ER.

50. Ca++ and calmodulin (CaM) Calmodulin-dependent kinases Auto-phosphorylation: sustained activity

51. Receptor tyrosine kinase Growth factors: EGF, PDGF etc

53. Receptor tyrosine kinases Tyr kinase domain vs. Ligand-binding domain Growth factor receptors for insulin (INS-R), vascular epidermal growth factor (VEGFR), platelet-derived growth factor (PDGF-R), epidermal growth factor (EGF-R), nerve growth factor (NGF-R), and fibroblast growth factor (FGF-R).

54. Many signaling processes are mediated by an assembly of multiple signaling molecules. Assembly of signaling molecules

55. SH2 domain interacting with P-Tyr + 3 residues of substrate

56. Some binding modules of signaling proteins

57. ~30% of all cancers involve Ras mutations. RTS signaling and Ras GTPase

58. RTK-RAS-MAPK cascade RTK: kinase receptor Ras: GTPase MAPKKK: kinase MAPKK: kinase MAPK: kinase Transcription factors (e.g., c-Fos) Why multi-step in signaling? Signal amplified in number and duration Signal fine regulated Signal cross talks

59. Regulation of gene expression by insulin through a MAP kinase cascade Insulin receptor: INS-R INS-R autophosphorylation Insulin receptor substrate -1 (IRS-1) Ras Raf-1 MEK: MAPKK ERK: MAPK Elk1 Serum response factor (SRF)

60. Receptor Serine/Threonine Kinases Fig. 13.18. TGF  -activated receptor serine/threonine kinase cascade TGF  and BMPs Tissue development and differentiation Mutations in these pathways found in cancers SMAD-dependent transcription

61. The JAK-STAT transduction mechanism for the erythropoietin receptor Erythropoietin (EPO) Dimerization of EPO receptor. JAK phosphorylates EPO-R. (a)STAT5 binds to P–EPO-R JAK - p-STAT STAT5 dimer Exposing a nuclear localization sequence (NLS). (b) Grb2 binds P–EPO-R and triggers the MAPK cascade. Cytokines: paracrine/autocrine polypeptides Interleukins: immune response in leukocyte Interferons: immune response against viruses or bacteria

62. Fig. 13.21. Heterotrimeric G proteins coupled with GPCR G proteins  s : stimulates adenylate cyclase, cAMP up.  i : inhibits adenylate cyclase  q : phospholipase C IP3, DAG, Ca++  12 : GEFs

63. Cross talk between GPCR and RTK Insulin - INS-R Phosphorylates  -adrenergic receptor. PKB Internalization of adrenergic receptor. Alternatively, INS-R–catalyzed phosphorylation of a GPCR INS-R uses GPCR to enhance its own signaling.

64. cGMP as a second messenger

65. Two isozymes of guanylyl cyclase that participate in signal transduction cGMP synthesized by guanylate cyclase Membrane-spanning forms that are activated by their extracellular ligands: atrial natriuretic factor (ANF) and guanylin. A soluble heme-containing enzyme. Activated by intracellular nitric oxide (NO).

66. Guanylate cyclases cGMP synthesis activated by NO Phosphodiesterase (PDE) cGMP degradation NO as a signaling molecule 1998 Nobel Prize

67. Endothelial cells: NO Vascular smooth muscle cells guanylate cyclase cGMP cGMP-sensitive kinase Viagra Erectile dysfunction PDE5 inhibitor [NO] increase 80-4000-fold less potent than PDE3 (cardiac muscle) 15-fold less potent than PDE6 (retina) NO, Vasodilation, and Drug

68. Each student is required to present a slide shown below at the end of class Please use propagated materials, websites, or slides themselves

69. Hydrophobic molecules (steroid hormones, retinoic acids, etc) enter cells and bind to intracellular receptors Hydrophilic or big molecules (proteins or polypeptide hormones) bind to cell surface receptors Majority Intracellular vs. cell surface receptors

70. Major types of intercellular signaling Contact dependent signaling: gap junction or ligand-receptor interaction on cell surface. Endocrine signaling: A sender cell secretes ligands (hormones) into the blood for long distance transport to target receptors. Paracrine signaling: A sender cell secretes ligands that are diffused (up to ~0.1 mm) to bind to target receptors. Synaptic signaling: An axon terminus secretes ligands (neurotransmitters) Into restricted environment to target nerves or muscles.

71. Cell surface receptors categorized by action modes Ligand binding to ligand-gated ion channel receptor permits ions (e.g., Na+) to permeates through the receptor in nervous cells. Enzyme-linked receptors themselves are kinases that phosphorylate effectors. Cytokine binding to cytokine receptors (without kinase activity) activates a kinase effect. G-protein-coupled receptors (GPCR) ~50% of all known drugs

72. G-Protein-Coupled Receptors (GPCRs) Represent ~5% of all proteins Targets of ~50% of all known drugs Hormones, neurotransmitters, Local mediators Heterotrimeric GTP-binding proteins (G proteins)

73. Heterotrimeric G proteins coupled with GPCR  s : stimulates adenylate cyclase, cAMP up.  i : inhibits adenylate cyclase, cAMP down  q : phospholipase C IP3, DAG, Ca++  12 : guanine-nucleotide exchange factors (GEFs)

74. Activation and inactivation of G proteins  -GTP (active) +  (active)  -GDT (inactive) 1 GPCR vs. 10-100 G-proteins G proteins activate downstream effectors GTPase activator proteins (GAPs) and regulators of G protein signaling (RGSs) inactivate G proteins By modulating the GTPase activity

75. Adenylate cyclases regulated by Gs and Gi

76. PKA controlled by cAMP cAMP function is mediated by cAMP-dependent protein kinase (PKA) PKA phosphorylates many other proteins cAMP-regulated gene regulatory proteins (CREBs) cAMP-sensitive regulatory elements (CRE)

77. Gq phospholipase C and IP 3 Two intracellular second messengers are produced in the hormone-sensitive phosphatidylinositol system: inositol 1,4,5-trisphosphate (IP 3 ) and diacylglycerol. Both contribute to the activation of protein kinase C. By raising cytosolic [Ca 2+ ], IP 3 also activates other Ca 2+ -dependent enzymes.

78. Fig. 13.29. Ca++ signaling and its action

79. Ca++ and calmodulin (CaM) Calmodulin-dependent kinases Auto-phosphorylation: sustained activity

80. Receptor tyrosine kinase Growth factors: EGF, PDGF etc

81. ~30% of all cancers involve Ras mutations. RTS signaling and Ras GTPase

82. RTK-RAS-MAPK cascade RTK: kinase receptor Ras: GTPase MAPKKK: kinase MAPKK: kinase MAPK: kinase Transcription factors (e.g., c-Fos) Why multi-step in signaling? Signal amplified in number and duration Signal fine regulated Signal cross talks

83. Regulation of gene expression by insulin through a MAP kinase cascade Insulin receptor: INS-R INS-R autophosphorylation Insulin receptor substrate -1 (IRS-1) Ras Raf-1 MEK: MAPKK ERK: MAPK Elk1 Serum response factor (SRF)

84. Receptor Serine/Threonine Kinases Fig. 13.18. TGF  -activated receptor serine/threonine kinase cascade TGF  and BMPs Tissue development and differentiation Mutations in these pathways found in cancers SMAD-dependent transcription

85. Guanylate cyclases cGMP synthesis activated by NO Phosphodiesterase (PDE) cGMP degradation NO as a signaling molecule 1998 Nobel Prize

86. Endothelial cells: NO Vascular smooth muscle cells guanylate cyclase cGMP cGMP-sensitive kinase Viagra Erectile dysfunction PDE5 inhibitor [NO] increase 80-4000-fold less potent than PDE3 (cardiac muscle) 15-fold less potent than PDE6 (retina) NO, Vasodilation, and Drug