phosphatases and kinase in cell signalling

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  • CaMKII is enriched at glutamatergic synapses and assemble into multimers
    Activated CaM binds to these units, allowing them to autophosphorylate at T286
    A mutation is CaMKII that prevents autophosphorylation blocks LTP (Giese et al. 1998)
    Calcineurin is a phosphatase that comprises over 1% of total protein in the brain, and affects many neuronal functions. CaN has been demonstrated to regulate the activity of ion channels, neurotransmitter and hormone release, synaptic plasticity, and gene transcription (Yakel 1997). Studies where the gene is knocked out, knocked down, and overexpressed all result in impaired behavior in rodent models. This suggests that CaN is a critical protein, in moderation. If levels are too low or too high synaptic function can be compromised.
  • phosphatases and kinase in cell signalling

    1. 1. Kinases and Phosphatases in cell signaling Giulio Taglialatela, Ph.D. John Sealy Professor and Vice Chair Dept. of Neurology Director, Mitchell Center for Neurodegenerative Diseases The University of Texas Medical Branch at Galveston
    2. 2. Signals are changes in homeostasis
    3. 3. To be recognized as a signal, an event must be: 1. Sudden.
    4. 4. To be recognized as a signal, an event must be: 1. Sudden. 2. Short-lived.
    5. 5. 0 5 10 15 20 25 30 35 TIME INTENSITY Good signals 0 5 10 15 20 25 30 35 TIME INTENSITY
    6. 6. 0 5 10 15 20 25 30 35 TIME INTENSITY Good signals 0 5 10 15 20 25 30 35 TIME INTENSITY 0 5 10 15 20 25 30 35 TIME INTENSITY 0 5 10 15 20 25 30 35 TIME INTENSITY Bad signals
    7. 7. The balanced activity between kinases and phosphatases provides sudden and (often) short lived modifications of proteins, resulting in effective signaling events
    8. 8. Kinase signal cascade
    9. 9. Kinases Phosphorylases
    10. 10. The human kinome
    11. 11. Kinases share a highly conserved catalytic core
    12. 12. Three classes of protein kinases 1. Serine/threonine kinases 2. Tyrosine kinases 3. Mixed kinases
    13. 13. Three classes of protein kinases 1. Serine/threonine kinases 2. Tyrosine kinases 3. Mixed kinases
    14. 14. Serine/threonine protein kinases 1. DAG/Ca2+ dependent (PKC) 2. cAMP-dependent (PKA) 3. Ca2+/Calmodulin-dependent (CaMKII)
    15. 15. 1. DAG/Ca2+ dependent (PKC) 2. cAMP-dependent (PKA) Serine/threonine protein kinases
    16. 16. G-PROTEIN-LINKED RECEPTORS Binding domain Docking domain CYTOSOL G-PROTEIN (inactive) α subunit β subunit γ subunit GDP
    17. 17. G-PROTEIN-LINKED RECEPTORS CYTOSOL G-PROTEIN (inactive) Signaling molecule
    18. 18. G-PROTEIN-LINKED RECEPTORS CYTOSOL G-PROTEIN (inactive) Signaling molecule
    19. 19. G-PROTEIN-LINKED RECEPTORS CYTOSOL G-PROTEIN (inactive) Signaling molecule GTP
    20. 20. G-PROTEIN-LINKED RECEPTORS CYTOSOL G-PROTEIN (active) Signaling molecule
    21. 21. G-PROTEIN-LINKED RECEPTORS CYTOSOL G-PROTEIN (active)
    22. 22. G-PROTEIN-LINKED RECEPTORS CYTOSOL G-PROTEIN (active) Pi
    23. 23. G-PROTEIN-LINKED RECEPTORS CYTOSOL G-PROTEIN (inactive)
    24. 24. G-PROTEIN-LINKED RECEPTORS CYTOSOL G-PROTEIN (inactive) Signaling molecule
    25. 25. G-PROTEIN-LINKED RECEPTORS CYTOSOL G-PROTEIN (inactive) Signaling molecule
    26. 26. G-PROTEIN-LINKED RECEPTORS CYTOSOL G-PROTEIN (inactive) Signaling molecule GTP
    27. 27. G-PROTEIN-LINKED RECEPTORS CYTOSOL G-PROTEIN (active) Signaling molecule
    28. 28. G-PROTEIN-LINKED RECEPTORS CYTOSOL G-PROTEIN (active)
    29. 29. G-PROTEIN-LINKED RECEPTORS CYTOSOL G-PROTEIN (active) Pi
    30. 30. G-PROTEIN-LINKED RECEPTORS CYTOSOL G-PROTEIN (inactive)
    31. 31. G-PROTEIN-LINKED RECEPTORS CYTOSOL G-PROTEIN (active)
    32. 32. G-PROTEIN-LINKED RECEPTORS CYTOSOL G-PROTEIN (active) Adenylate cyclase
    33. 33. G-PROTEIN-LINKED RECEPTORS CYTOSOL G-PROTEIN (active) Adenylate cyclase
    34. 34. G-PROTEIN-LINKED RECEPTORS CYTOSOL G-PROTEIN (active) Adenylate cyclase ATP cAMP
    35. 35. G-PROTEIN-LINKED RECEPTORS CYTOSOL G-PROTEIN (active) Adenylate cyclase ATP cAMP A-kinase
    36. 36. G-PROTEIN-LINKED RECEPTORS CYTOSOL G-PROTEIN (active) Adenylate cyclase ATP cAMP A-kinase Nucleus CREB
    37. 37. G-PROTEIN-LINKED RECEPTORS CYTOSOL G-PROTEIN (active) Adenylate cyclase ATP cAMP A-kinase Nucleus TranscriptionCREB CBP
    38. 38. G-PROTEIN-LINKED RECEPTORS CYTOSOL G-PROTEIN (active)
    39. 39. G-PROTEIN-LINKED RECEPTORS CYTOSOL G-PROTEIN (active) Phospholipase C (PLC)
    40. 40. G-PROTEIN-LINKED RECEPTORS CYTOSOL G-PROTEIN (active) Phospholipase C (PLC)
    41. 41. G-PROTEIN-LINKED RECEPTORS CYTOSOL G-PROTEIN (active) Phospholipase C (PLC) PIP2
    42. 42. G-PROTEIN-LINKED RECEPTORS CYTOSOL G-PROTEIN (active) Phospholipase C (PLC) PIP2
    43. 43. G-PROTEIN-LINKED RECEPTORS CYTOSOL G-PROTEIN (active) Phospholipase C (PLC) DAG IP3
    44. 44. G-PROTEIN-LINKED RECEPTORS CYTOSOL G-PROTEIN (active) Phospholipase C (PLC) C-Kinase
    45. 45. G-PROTEIN-LINKED RECEPTORS CYTOSOL G-PROTEIN (active) Phospholipase C (PLC) C-Kinase
    46. 46. G-PROTEIN-LINKED RECEPTORS CYTOSOL G-PROTEIN (active) Phospholipase C (PLC) C-Kinase
    47. 47. G-PROTEIN-LINKED RECEPTORS CYTOSOL G-PROTEIN (active) Phospholipase C (PLC) C-Kinase
    48. 48. Serine/threonine protein kinases 3. Ca2+/Calmodulin-dependent (CaMKII)
    49. 49. Ca2+ -sensitive proteins: A story of co-operation
    50. 50. Calmodulin A ubiquitous mediator of Ca2+ signals Target protein
    51. 51. Calmodulin A ubiquitous mediator of Ca2+ signals Target protein Increased affinity
    52. 52. Calmodulin A ubiquitous mediator of Ca2+ signals Target protein
    53. 53. Calmodulin A ubiquitous mediator of Ca2+ signals Target protein
    54. 54. Calmodulin A ubiquitous mediator of Ca2+ signals Active target protein P
    55. 55. Calmodulin A ubiquitous mediator of Ca2+ signals Active target protein P P Secondary cytosolic effects
    56. 56. Intracellular Ca2+ waves and oscillations
    57. 57. The ability of intracellular Ca2+ stores to release Ca2+ is affected by the relative concentration of free cytosolic Ca2+ : It is increased by slightly elevated Ca2+ and decreased by very high Ca2+ concentrations. 0 2 4 6 8 10 12 Cytpsolic Ca2+ concentration ERCa2+release
    58. 58. Intracellular Ca2+ increases are characterized by: - frequency - amplitude (intensity) Consequently, cells have developed systems that will respond to either intensity or frequency (or both) of Ca2+ increases.
    59. 59. CaM Kinase II, a direct substrate for calmodulin, is a Ca2+ spike frequency detector In order for CaM Kinase to be fully active, each catalytic subunit must be autophosphorylated in a calmudulin-dependent fashion. Autophosphorylation of each subsequent subunit follows a co- operative kinetic.
    60. 60. CaM Kinase II, a direct substrate for calmodulin, is a Ca2+ spike frequency detector In order for CaM Kinase to be fully active, each catalytic subunit must be autophosphorylated in a calmudulin-dependent fashion. Autophosphorylation of each subsequent subunit follows a co- operative kinetic. calmudulin
    61. 61. CaM Kinase II, a direct substrate for calmodulin, is a Ca2+ spike frequency detector In order for CaM Kinase to be fully active, each catalytic subunit must be autophosphorylated in a calmudulin-dependent fashion. Autophosphorylation of each subsequent subunit follows a co- operative kinetic.
    62. 62. CaM Kinase II, a direct substrate for calmodulin, is a Ca2+ spike frequency detector In order for CaM Kinase to be fully active, each catalytic subunit must be autophosphorylated in a calmudulin-dependent fashion. Autophosphorylation of each subsequent subunit follows a co- operative kinetic.
    63. 63. CaM Kinase II, a direct substrate for calmodulin, is a Ca2+ spike frequency detector In order for CaM Kinase to be fully active, each catalytic subunit must be autophosphorylated in a calmudulin-dependent fashion. Autophosphorylation of each subsequent subunit follows a co- operative kinetic. Fully active CaM kinase
    64. 64. time
    65. 65. time
    66. 66. time
    67. 67. time
    68. 68. time
    69. 69. time
    70. 70. time
    71. 71. time
    72. 72. time
    73. 73. time Fully active CaM Kinase
    74. 74. CaM Kinase II, a direct substrate for calmodulin, is a Ca2+ spike frequency detector
    75. 75. Three classes of protein kinases 1. Serine/threonine kinases 2. Tyrosine kinases 3. Mixed kinases
    76. 76. The receptor tyrosine kinases
    77. 77. Binding domain tyrosine kinase domain Ligands that bind to specific RTK: FGF, NGF, IGF, BDNF, ……, XXXF
    78. 78. Binding domain tyrosine kinase domain P P P P
    79. 79. P P P P P P P P Receptor autophosporylation
    80. 80. P The phophotyrosine domain acts as a docking site for proteins possessing a particular domain called SH2 SH2
    81. 81. P SH2 The SH2-containing protein is then activated by either conformational change or by phosphorylation promoted by the receptor itself. P PLCγ PI3-K GAP (GEF) IRS
    82. 82. P SH2 P PLCγ −> PIP2/IP3/PKC PI3-K −> IP2/PIP3/PKB GAP (GEF) −> ras/MAPK IRS Multiple pathway activation
    83. 83. Inactive Ras
    84. 84. P P Active/Inactive Ras GTP/GDP GAP GNRPSH2 SH3 SH3 Sem-5 + _
    85. 85. Receptors associated with cytoplasmic tyrosine kinases JAK JAK cytosol nucleus Enzyme-linked receptors
    86. 86. Receptors associated with cytoplasmic tyrosine kinases JAK JAKP P P P STAT STAT P P cytosol nucleus SOCS Enzyme-linked receptors
    87. 87. Receptor Serine/Threonine Kinases cytosol nucleus RII RI Enzyme-linked receptors
    88. 88. Receptor Serine/Threonine Kinases cytosol nucleus RII RI P P SMAD co-SMAD Enzyme-linked receptors
    89. 89. Three classes of protein kinases 1. Serine/threonine kinases 2. Tyrosine kinases 3. Mixed kinases
    90. 90. GTP MAP KINASE MAPK KINASE MAPKK KINASE P-SER P-SER P-SER P-THR P-THR P-TYR P-TYR Active Ras
    91. 91. GTP MAP KINASE MAPK KINASE MAPKK KINASE P-SER P-SER P-SER P-THR P-THR PKC P-TYR P-TYR Active Ras
    92. 92. MAP KINASE P-SER P-THR Direct: CREB. Indirect: JNK/c-jun DNA P-TYR
    93. 93. MAP KINASE P-SER P-THR Direct: CREB. Indirect: JNK/c-jun DNA RNA Pol II P-TYR
    94. 94. To be recognized as a signal, an event must be: 1. Sudden. 2. Short-lived.
    95. 95. Who cleans up the mess?
    96. 96. Protein Phosphatases
    97. 97. Protein Phosphatases (PPT) Ser/Thr PPT Tyr PPT Mix PPT •MAPK PPT’s •CDC14’s •PTEN’s •CDC25’s •RTP’s •nonRTP’s •LMPT •Eya1-4 •PP1 •PP2A •PP2B (calcineurin) •PP2C
    98. 98. The phosphatase reaction is conserved among all phosphatases and does not require energy
    99. 99. Ser/Thr Protein Phosphatases Ca/CaM-indep. •PP2C•PP2B (calcineurin) •PP1 •PP2A Ca/CaM-dep. Mg/Mn-dep. Multimeric proteins formed by a catalytic subunit, a modulatory subunit and, in certain instances (PP2A), a scaffolding subunit
    100. 100. Both calcineurin and CaMKII are essential to modulate the function of the synapse in response to stimulatory Ca++ entry Both calcineurin and CaMKII are activated by binding to Ca++/calmodulin
    101. 101. Na+ Na+ Na+Na+ Ca2+ Ca2+ Ca2+ Na+ Na+ CaM Ca2+ Ca N Ca N CaMKIICaMKII Ca2+ pCREBpCREB Ca2+ Ca2+ Ca2+ Ca2+ PP1
    102. 102. Giulio Taglialatela gtaglial@utmb.edu

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