Convergence, divergence and crosstalk


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Convergence, divergence and crosstalk

  1. 1. Convergence, Divergence and Crosstalk Among Different Signaling Pathways Herbert, Barnabas E. MB.Sc, Hse
  2. 2. Key Concept • Convergence • Divergence • Crosstalk • Signaling Pathways
  3. 3. Key Concept • Convergence • Divergence • Crosstalk • Signaling Pathways
  4. 4. CONVERGENCE Signals from a VARIETY OF UNRELATED receptors converge to activate a COMMON EFFECTOR after binding to their individual ligand.E.g Ras Raf.
  5. 5. MAP-kinase serine/threonine phosphorylation Pathway activated by Ras • Ras- activated phosphorylat ion cascade
  6. 6. CONVERGENCE • Signals usually from RECEPTORS • Examples: -G-protein coupled receptors -Receptor tyrosine kinases -Integrins
  7. 7. CONVERGENCE Signals transmitted form a G protein-coupled receptor, an integrin and a receptor tyrosine kinase all converge on Ras and are then transmitted along the MAP kinase cascade.
  8. 8. CONVERGENCE • Lead to formation of PHOSPHOTYROSINE DOCKING sites for SH2 domain • Lead to TRANSCRIPTION and PROMOTION of a SIMILAR set of growth promoting genes in target cells. • Signals transmitted from G-protein-coupled receptors on integrins, and a receptor tyrosine kinase all CONVERGE on Ras/Raf and are then transmitted along the MAP kinase cascade. • Integrins are receptors at sites of cell-substrate and cell-cell contact.
  9. 9. Key Concept • Convergence • Divergence • Crosstalk • Signaling Pathways
  10. 10. Key Concept • Convergence • Divergence • Crosstalk • Signaling Pathways
  11. 11. DIVERGENCE Signals from the same ligand diverge to activate A VARIETY OF DIFFERENT EFFECTORS leading to diverse cellular responses.
  12. 12. DIVERGENCE
  13. 13. DIVERGENCE • Effects are usually LIGAND based • Examples -EGF ligand -Insulin ligand
  14. 14. KEY CONCEPTS • Convergence • Divergence • Crosstalk • Signaling Pathways
  15. 15. KEY CONCEPTS • Convergence • Divergence • Crosstalk • Signaling Pathways
  16. 16. CROSS TALK Signals are passed BACK AND fORTH between DIFFERENT PATHWAYS Example: Cyclic Adenosine Monophosphate (cAMP)
  17. 17. How does cAMP block signals transmitted through the MAP kinase cascade? • Achieves this by: -activating PKA (a cAMP dependent kinase) -PKA phosphorylates/inhibits Raf (a protein that leads the MAP kinase cascade)
  18. 18. Crosstalk between 2 major signaling pathways. cAMP acts in some cells via cAMP-dep.kinase, PKA, to block transmission of signals from Ras to Raf which inhibits activation of MAP kinase cascade. Also both PKA and kinases of MAP kinase cascade phosphorylate transcription factor CREB on same serine residue, activating transcription factor and allowing it to bind to specifrc sites on the DNA.
  19. 19. CROSSTALKS • cAMP -Initiator of rxn cascade for CHO mobilization -Can also inhibit growth of variety of cells by blocking signals transmitted through the MAP kinase cascade.
  20. 20. • Convergence • Divergence • Crosstalk • Signaling Pathways
  21. 21. • Convergence • Divergence • Crosstalk • Signaling Pathways
  22. 22. SIGNALING PATHWAYS • Provide a mechanism for routing information through a cell • Comparable to the nervous system: -the cell receives information about its environment through the activation of various surface receptors.
  23. 23. CELL SURFACE RECEPTORS • Acts like sensors to detect extracellular stimuli • Can bind only to specific ligands • Unaffected by the presence of a large variety of UNRELATED molecules
  24. 24. Do not forget! “A single cell may have dozens of different receptors sending signals to the cell interior simultaneously!”
  25. 25. What happens when signals are transmitted into cells???
  26. 26. The signals are selectively routed along a number of different signaling pathways that may cause the cell to: -Divide (Mitosis) -Change shape -Activate a specific metabolic pathway -Apoptosis (Commit suicide)
  27. 27. CENTRAL IDEA In this way, the cell integrates information arriving from different sources and mounts an appropriate and comprehensive response
  28. 28. How are different stimuli able to evoke distinct responses, even though they utilize similar pathways?
  29. 29. Contrasting cellular responses are due to differences in the protein composition of different cell types (Different cells have different isoforms of these various proteins)
  30. 30. A working theory, not a satisfactory answer!
  31. 31. In actual fact, signaling pathways in the cell are much more complex.
  32. 32. SURMARY Signals from a variety of UNRELATED RECEPTORS can CONVERGE to activate a common effector, such as Ras/Raf; signals fro the SAME LIGAND can DIVERGE to activate a variety of DIFFERENT EFFECTORS; and signals can be passed BACK AND FORTH between pathways (Cross talk).
  33. 33. • Paroxysmal vertigo • Define the following and give examples. -Convergence -Divergence -Crosstalk QUESTION ONE
  34. 34. What happens when signals are transmitted into cells??? QUESTION TWO
  36. 36. Concentration is the Key! Keep calm!! 
  38. 38. History of Second Messengers • Before 1980 -Organic compounds e.g cAMP -Ions e.g Ca2+ • After 1980 -Inorganic gas -> Nitric Oxide (NO)
  39. 39. Nitric Oxide - Formed from L-arginine (amino acid) in a rxn catalyzed by the enz Nitric Oxide Synthase (NOS) - Discovered as second messenger by accidental observation
  40. 40. Flash Back: Acetylcholine - Known to act in the body to relax smooth muscle cells of blood vessels. - Response could not be duplicated in vitro - Binds to receptors on the surface of endothelial cells - Leads to the production and release of an AGENT that diffuse through the cell’s plasma membrane
  41. 41. Acetylcholine - Causes the muscle cells to relax - The AGENT was later discovered to be Nitric Oxide (NO)
  42. 42. NITRIC OXIDE: MOA Step one: Acetylcholine binds to the outer surface of endothelial cell Step two: Causes a rise in cytosolic Ca2+ concentration Step three: Ca2+ activates NOS to synthesize NO
  43. 43. NITRIC OXIDE: MOA Step four: NO formed in endothelial cell diffuses across the plasma membrane to adjacent muscle cells Step five: Stimulates guanyl cyclase in smooth muscle which synthesizes cGMP, a 2nd messenger similar in structure to cAMP
  44. 44. NITRIC OXIDE: MOA Step six: cGMP leads to a decrease in cytosolic Ca2+ concentration which leads to smooth muscle cell relaxation Conclusion/Discovery: “NO acts as an activator of guanyl cyclase”
  45. 45. Medical Relevance: Nitroglycerine -used to treat the pain of angina that results from an inadequate flow of blood to the heart. -metabolized to NO which stimulates the relaxation of the smooth muscles lining the blood vessels of the heart -leads to increase blood flow to the organ
  46. 46. Pharmacological Relevance: Sildenafil - Aka Viagra - Developed following the discovery of NO
  47. 47. Sildenafil: MOA • During sexual act -nerve endings in the penis release NO -causes: (a) relaxation of smooth muscle cells in the lining of penile blood vessels (b) Engorgement of the organ with blood
  48. 48. Sildenafil: MOA • Viagra -has no effect on the release of NO or the activation of guanylyl cyclase -(instead) inhibits cGMP phosphodiesterase
  49. 49. Phosphodiesterase: MOA • An enzyme • Destroys cGMP • Inhibition of this enzyme leads to: (A) maintained, elevated levels of cGMP -> (B) promotes the development and maintenance of an erection
  50. 50. Since Viagra acts to maintain elevated levels of cGMP, does it affect the heart as well???
  51. 51. Viagra -specific for one particular isoform of cGMP phosphodiesterase (PDE5) -version that acts in the penis PDE3 -plays key role in the regulation of heart muscle contraction (not inhibited by Viagra)
  52. 52. NO or N2O???
  53. 53. Do not forget! Nitric Oxide (NO) should not be confused with Nitrous Oxide (Laughing Gas)
  54. 54. Describe the steps in the signaling pathway by which nitric oxide mediates dilation of blood vessels.
  55. 55. Since Viagra acts to maintain elevated levels of cGMP, does it affect the heart as well??? Explain!
  56. 56. Information overload!!!
  57. 57. Five Minutes Break!!!
  58. 58. For everything there is a season, and a time for every matter under heaven: A time to be born, and a time to die… Ecclesiastes 3: 1f
  59. 59. For every cell, there is a time to live and a time to die…
  60. 60. Apoptosis
  61. 61. Apoptosis …Programmed cell death Discussed by: HERBERT, B.
  62. 62. Objectives • At the end of the discuss, students should be able to: – Describe the steps that occur between the time that a TNF molecule binds to its receptor and the eventual death of the cell. – Describe the steps that occur between the time a proapoptotic Bcl-2 membrane binds to the outer mitochondrial membrane and the death of the cell.
  63. 63. Cells die for two quite different reasons • Accidental death • result of mechanical trauma or exposure to some kind of toxic agent (necrosis) • only type of death seen in unicellular organisms • Deliberate death • result of an built-in suicide mechanism known as apoptosis or programmed cell death.
  64. 64. Necrosis • When cells are injured – ATP concentrations fall so low that the Na+ /K+ ATPase can no longer operate, – ion concentrations are no longer controlled – causes the cells to swell and then burst – cell contents leak out – causes the surrounding tissues to become inflamed
  65. 65. Apoptosis • A normal occurrence • An orchestrated sequence of events • Leads to death of a cell • Eliminates cells with sustained irreparable genomic damage – Important because damage to genetic blue print can result in unregulated cell division -> Cancer • Etymology: John Kerr et al., (1972)
  66. 66. Apoptosis: Characteristics • Shrinkage of cell volume and nucleus • Loss of adhesion to neighbouring cells • Formation of blebs at cell surface • Dissection of chromatin into small fragments • Engulfment of the ‘corpse’ by phagocytosis
  67. 67. Apoptosis: Working Examples • Neurons (During embryonic development)  grow out of CNS to innervate organs present in the periphery of the body  usually many more neurons grow out than are needed for normal innervation  Neurons that reach their destination receive signal from the target tissue that allows them to survive  Neurons that fail to find their way to the target tissue do not receive the survival signal and are eliminated by apoptosis
  68. 68. Apoptosis: Working Example • T Lymphocytes – Cells of the immune system – Recognize and kill abnormal or pathogen infected cells – Recognizes target cells via specific receptors that are present on its surface
  69. 69. Apoptosis: T Lymphocytes – Sometimes produced during embryonic development with receptors capable of binding tightly to proteins present on surface of normal cells within the body. – T Lymphocytes that have this dangerous capability are eliminated by apoptosis.
  70. 70. Apoptosis: Medical Relevance • Apoptosis is involved in neurodegenerative diseases such as: – Alzheimers’s – Parkinson’s – Huntington’s Elimination of essential neurons during dz progression gives rise to loss of memory or decrease in motor coordination
  71. 71. Apoptosis: Triggers Apoptosis can be triggered in three ways: (a) binding of ligand to death domain receptors, (b) denial of growth factors, and (c) cell stress.
  72. 72. Central Idea “Apoptosis is important in maintaining homeostasis in multicellular organisms and failure to regulate apoptosis can result in serious damage to an organism”
  73. 73. Apoptosis is a normal occurrence! You can’t escape from it!
  74. 74. Apoptosis: A Worm’s Eye View! • First revealed in studies on nematode worm Caenorhabditis elegans »Cells can be followed with absolute precision during embryonic development »131 cells are normally destined to die by apoptosis »Worms carrying mutation in the CED- 3 gene proceed through development without losing any of their cells to apoptosis
  75. 75. Apoptosis: Caspases • A homologous gene to CED-3 found in humans • Distinctive group of cysteine proteins »i.e proteases with a key cysteine residue in their catalytic site »Activated at an early stage of apoptosis »Responsible for triggering changes observed during cell death
  76. 76. MOA: Caspases • Achieves apoptosis by cleaving a selected group of proteins • All the cells of our body contain caspases • they are normally locked in an inactive form by an integral inhibitory domain of the protein • Proteolysis cleaves the inhibitory domain off, releasing the active caspases
  77. 77. Viruses: A Hostile Take Over! • no protein synthesis is required to activate the apoptotic pathway—all the components are already present • if a virus infects a cell and takes over all protein synthesis, the cell can still commit suicide and hence prevent viral replication
  78. 78. Caspases: Targets • Focal Adhesion Kinase (FAK), PKB, and Raf 1 – Inactivation of FAK disrupts cell adhession, leading to detachment of apoptotic cell from its neighbours • Lamins – Make up inner lining of nuclear envelope – Cleavage of lamins leads to the disassembly of nuclear lamina and shrinkage of the nucleus
  79. 79. Caspases: Targets • Proteins of the Cytoskeleton – Such as those of the intermediate filaments, actin, tubulin and gelsolin – Cleavage and consequent inactivation of these proteins lead to changes in cell shape • Caspase activated Dnase (CAD) – An endonuclease – Activated following caspase cleavage of an inhibitory protein – Translocates from cytoplasm to nucleus – Attacks DNA, severing it into fragments
  80. 80. Apoptosis: What activates it? A. Internal stimuli (Intrinsic Pathway) –Abnormalities in DNA A. External stimuli (Extrinsic Pathway) – Removal of growth factors from the medium
  81. 81. The exTrinsic PaThway of aPoPTosis
  82. 82. Extrinsic Pathway of Apoptosis – Removal of growth factors from the medium – Epithelial cells of the prostate become apoptotic when deprived of the male sex hormone, testosterone » Hence prostate cancer that has spread to other tissues are often tx with drugs that interfere with testosterone production – Stimulis is carried by an extracellular messenger pr called TNF
  83. 83. Tumor Necrosis Factor • So called for its ability to kill tumor cells • Produced by cells of the immune system in response to adverse conditions, such as: • Exposure to ionizing radiation • Elevated temperature • Viral infection • Toxic chemical agents such as those used in cancer chemotherapy
  84. 84. TNF Receptor • Present in plasma membrane as a preassembled trimer • Cytoplasmic domain of each receptor subunit contains a segment of about 70 a.a called ‘death domain’ (mediates pr-pr interactions)
  85. 85. TNF: MOA • Evokes its response by binding to a transmembrane receptor, TNFR1 • Member of family related to ‘death receptor’ that mediates apoptosis • TNF binds to the trimer receptor -> change in conformation of the receptor’s death domain -> recruitment of a number of pr • Last pr to join complex are two procaspases (8 molecules)
  86. 86. TNF: MOA • Synthesis of caspases as proenzymes protects the cell from accidental proteolytic damage • When two or more procaspases are held in close association with one another, they are capable of cleaving one another’s polypeptide chain and converting the other molwcule to the fully active caspase
  87. 87. Caspase 8 • Final mature enzyme • Contains four polypeptide chains • Derived from two procaspase precursors • Described as an initiator caspase • Initiates apoptosis by cleaving and activating downstream or executioner caspases.
  88. 88. Executioner Caspases • Carry out the controlled self-destruction of the cell
  89. 89. The inTrinsic PaThway of aPoPTosis
  90. 90. Examples of Internal Stimuli • Irreparable genetic damage • Extremely high concentrations of cytosolic Ca2+ • Severe oxidative stress • Lack of survival signals (Absence of growth factors)
  91. 91. Bcl-2 Family of Proteins • Regulates activation of the intrinsic pathway • Originally identified as a tumor-causing oncogene • Subdivided into two:- – Proapoptotic : promotes apoptosis (e.g, Bad and Bax) – Antiapoptotic: protects cells from apoptosis (e.g Bcl-XL, Bcl-w, and Bcl-2)
  92. 92. Don’t Forget! “Bcl-2 acts as an oncogene by promoting survival of potential cancer cells that would otherwise die.”
  93. 93. MOA: Intrinsic Pathway • Stressful stimuli:- activates proapoptotic members of the Bcl-2 family (Bad/Bax) Translocates from the cytosol to outer mitochondrial membrane Attaches to outer mitochondrial membrane
  94. 94. MOA: Intrinsic Pathway Increases membrane permeability Promotes release of cytochrome C (which resides in the intermembrane space) Moves to cytosol Forms apoptosome (a multi protein complex that includes procaspase-9)
  95. 95. Procaspase-9 • Activated by simply joining the multiprotein complex • Does not require proteolytic cleavage • An initiator caspase; initiates executioner caspases Apoptosis
  96. 96. Do not forget! “The external pathway is receptor-mediated while the internal pathway is mitochondrial mediated! They however CONVERGE by activating the same executioner caspase, which cleaves the same cellular targets.”
  97. 97. Finally! • As cells execute the proapoptotic program they lose contact with neighbors and start to shrink • Cell disintegrates into a condensed, membrane-enclosed apoptotic body • Apoptotic bodies are recognizd by the presence of phosphatidylserine on their surface
  98. 98. Phosphatidylserine • A phospholipid that is normaly present only on the inner leaflet of the plasma membrane
  99. 99. During apoptosis, a phospholipid “scramblase” moves phosphatidylserine molecules to the outer leaflet of the plasma membrane where they are recognized as an “eat me” signal by specialized macrophages.