phosphatases and kinase in cell signalling
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  • CaMKII is enriched at glutamatergic synapses and assemble into multimers <br /> Activated CaM binds to these units, allowing them to autophosphorylate at T286 <br /> A mutation is CaMKII that prevents autophosphorylation blocks LTP (Giese et al. 1998) <br /> 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. <br />

 phosphatases and kinase in cell signalling phosphatases and kinase in cell signalling Presentation Transcript

  • 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
  • Signals are changes in homeostasis
  • To be recognized as a signal, an event must be: 1. Sudden.
  • To be recognized as a signal, an event must be: 1. Sudden. 2. Short-lived.
  • 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 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
  • The balanced activity between kinases and phosphatases provides sudden and (often) short lived modifications of proteins, resulting in effective signaling events
  • Kinase signal cascade
  • Kinases Phosphorylases
  • The human kinome
  • Kinases share a highly conserved catalytic core
  • Three classes of protein kinases 1. Serine/threonine kinases 2. Tyrosine kinases 3. Mixed kinases
  • Three classes of protein kinases 1. Serine/threonine kinases 2. Tyrosine kinases 3. Mixed kinases
  • Serine/threonine protein kinases 1. DAG/Ca2+ dependent (PKC) 2. cAMP-dependent (PKA) 3. Ca2+/Calmodulin-dependent (CaMKII)
  • 1. DAG/Ca2+ dependent (PKC) 2. cAMP-dependent (PKA) Serine/threonine protein kinases
  • G-PROTEIN-LINKED RECEPTORS Binding domain Docking domain CYTOSOL G-PROTEIN (inactive) α subunit β subunit γ subunit GDP
  • G-PROTEIN-LINKED RECEPTORS CYTOSOL G-PROTEIN (inactive) Signaling molecule
  • G-PROTEIN-LINKED RECEPTORS CYTOSOL G-PROTEIN (inactive) Signaling molecule
  • G-PROTEIN-LINKED RECEPTORS CYTOSOL G-PROTEIN (inactive) Signaling molecule GTP
  • G-PROTEIN-LINKED RECEPTORS CYTOSOL G-PROTEIN (active) Signaling molecule
  • G-PROTEIN-LINKED RECEPTORS CYTOSOL G-PROTEIN (active)
  • G-PROTEIN-LINKED RECEPTORS CYTOSOL G-PROTEIN (active) Pi
  • G-PROTEIN-LINKED RECEPTORS CYTOSOL G-PROTEIN (inactive)
  • G-PROTEIN-LINKED RECEPTORS CYTOSOL G-PROTEIN (inactive) Signaling molecule
  • G-PROTEIN-LINKED RECEPTORS CYTOSOL G-PROTEIN (inactive) Signaling molecule
  • G-PROTEIN-LINKED RECEPTORS CYTOSOL G-PROTEIN (inactive) Signaling molecule GTP
  • G-PROTEIN-LINKED RECEPTORS CYTOSOL G-PROTEIN (active) Signaling molecule
  • G-PROTEIN-LINKED RECEPTORS CYTOSOL G-PROTEIN (active)
  • G-PROTEIN-LINKED RECEPTORS CYTOSOL G-PROTEIN (active) Pi
  • G-PROTEIN-LINKED RECEPTORS CYTOSOL G-PROTEIN (inactive)
  • G-PROTEIN-LINKED RECEPTORS CYTOSOL G-PROTEIN (active)
  • G-PROTEIN-LINKED RECEPTORS CYTOSOL G-PROTEIN (active) Adenylate cyclase
  • G-PROTEIN-LINKED RECEPTORS CYTOSOL G-PROTEIN (active) Adenylate cyclase
  • G-PROTEIN-LINKED RECEPTORS CYTOSOL G-PROTEIN (active) Adenylate cyclase ATP cAMP
  • G-PROTEIN-LINKED RECEPTORS CYTOSOL G-PROTEIN (active) Adenylate cyclase ATP cAMP A-kinase
  • G-PROTEIN-LINKED RECEPTORS CYTOSOL G-PROTEIN (active) Adenylate cyclase ATP cAMP A-kinase Nucleus CREB
  • G-PROTEIN-LINKED RECEPTORS CYTOSOL G-PROTEIN (active) Adenylate cyclase ATP cAMP A-kinase Nucleus TranscriptionCREB CBP
  • G-PROTEIN-LINKED RECEPTORS CYTOSOL G-PROTEIN (active)
  • G-PROTEIN-LINKED RECEPTORS CYTOSOL G-PROTEIN (active) Phospholipase C (PLC)
  • G-PROTEIN-LINKED RECEPTORS CYTOSOL G-PROTEIN (active) Phospholipase C (PLC)
  • G-PROTEIN-LINKED RECEPTORS CYTOSOL G-PROTEIN (active) Phospholipase C (PLC) PIP2
  • G-PROTEIN-LINKED RECEPTORS CYTOSOL G-PROTEIN (active) Phospholipase C (PLC) PIP2
  • G-PROTEIN-LINKED RECEPTORS CYTOSOL G-PROTEIN (active) Phospholipase C (PLC) DAG IP3
  • G-PROTEIN-LINKED RECEPTORS CYTOSOL G-PROTEIN (active) Phospholipase C (PLC) C-Kinase
  • G-PROTEIN-LINKED RECEPTORS CYTOSOL G-PROTEIN (active) Phospholipase C (PLC) C-Kinase
  • G-PROTEIN-LINKED RECEPTORS CYTOSOL G-PROTEIN (active) Phospholipase C (PLC) C-Kinase
  • G-PROTEIN-LINKED RECEPTORS CYTOSOL G-PROTEIN (active) Phospholipase C (PLC) C-Kinase
  • Serine/threonine protein kinases 3. Ca2+/Calmodulin-dependent (CaMKII)
  • Ca2+ -sensitive proteins: A story of co-operation
  • Calmodulin A ubiquitous mediator of Ca2+ signals Target protein
  • Calmodulin A ubiquitous mediator of Ca2+ signals Target protein Increased affinity
  • Calmodulin A ubiquitous mediator of Ca2+ signals Target protein
  • Calmodulin A ubiquitous mediator of Ca2+ signals Target protein
  • Calmodulin A ubiquitous mediator of Ca2+ signals Active target protein P
  • Calmodulin A ubiquitous mediator of Ca2+ signals Active target protein P P Secondary cytosolic effects
  • Intracellular Ca2+ waves and oscillations
  • 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
  • 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.
  • 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.
  • 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
  • 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.
  • 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.
  • 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
  • time
  • time
  • time
  • time
  • time
  • time
  • time
  • time
  • time
  • time Fully active CaM Kinase
  • CaM Kinase II, a direct substrate for calmodulin, is a Ca2+ spike frequency detector
  • Three classes of protein kinases 1. Serine/threonine kinases 2. Tyrosine kinases 3. Mixed kinases
  • The receptor tyrosine kinases
  • Binding domain tyrosine kinase domain Ligands that bind to specific RTK: FGF, NGF, IGF, BDNF, ……, XXXF
  • Binding domain tyrosine kinase domain P P P P
  • P P P P P P P P Receptor autophosporylation
  • P The phophotyrosine domain acts as a docking site for proteins possessing a particular domain called SH2 SH2
  • 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
  • P SH2 P PLCγ −> PIP2/IP3/PKC PI3-K −> IP2/PIP3/PKB GAP (GEF) −> ras/MAPK IRS Multiple pathway activation
  • Inactive Ras
  • P P Active/Inactive Ras GTP/GDP GAP GNRPSH2 SH3 SH3 Sem-5 + _
  • Receptors associated with cytoplasmic tyrosine kinases JAK JAK cytosol nucleus Enzyme-linked receptors
  • Receptors associated with cytoplasmic tyrosine kinases JAK JAKP P P P STAT STAT P P cytosol nucleus SOCS Enzyme-linked receptors
  • Receptor Serine/Threonine Kinases cytosol nucleus RII RI Enzyme-linked receptors
  • Receptor Serine/Threonine Kinases cytosol nucleus RII RI P P SMAD co-SMAD Enzyme-linked receptors
  • Three classes of protein kinases 1. Serine/threonine kinases 2. Tyrosine kinases 3. Mixed kinases
  • GTP MAP KINASE MAPK KINASE MAPKK KINASE P-SER P-SER P-SER P-THR P-THR P-TYR P-TYR Active Ras
  • GTP MAP KINASE MAPK KINASE MAPKK KINASE P-SER P-SER P-SER P-THR P-THR PKC P-TYR P-TYR Active Ras
  • MAP KINASE P-SER P-THR Direct: CREB. Indirect: JNK/c-jun DNA P-TYR
  • MAP KINASE P-SER P-THR Direct: CREB. Indirect: JNK/c-jun DNA RNA Pol II P-TYR
  • To be recognized as a signal, an event must be: 1. Sudden. 2. Short-lived.
  • Who cleans up the mess?
  • Protein Phosphatases
  • 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
  • The phosphatase reaction is conserved among all phosphatases and does not require energy
  • 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
  • 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
  • Na+ Na+ Na+Na+ Ca2+ Ca2+ Ca2+ Na+ Na+ CaM Ca2+ Ca N Ca N CaMKIICaMKII Ca2+ pCREBpCREB Ca2+ Ca2+ Ca2+ Ca2+ PP1
  • Giulio Taglialatela gtaglial@utmb.edu