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Kinases and Phosphatases in cell signaling
Giulio Taglialatela, Ph.D.John Sealy Professor and Vice Chair
Dept. of NeurologyDirector, 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.
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YGood signals
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Y
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YGood signals
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Y
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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 kinases3. Mixed kinases
Three classes of protein kinases
1. Serine/threonine kinases
2. Tyrosine kinases3. 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)
subunit
subunit 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
PP
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
ER
Ca2
+ r
elea
se
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 kinases3. 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 PLC
PI3-K
GAP (GEF)
IRS
P SH2
PPLC 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
GNRPSH2SH3
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 STATP P
cytosol
nucleus
SOCS
Enzyme-linked receptors
Receptor Serine/Threonine Kinases
cytosol
nucleus
RIIRI
Enzyme-linked receptors
Receptor Serine/Threonine Kinases
cytosol
nucleus
RIIRI
P
P SMAD
co-SMAD
Enzyme-linked receptors
Three classes of protein kinases
1. Serine/threonine kinases
2. Tyrosine kinases3. Mixed kinases
GTP
MAP KINASE
MAPK KINASE
MAPKK KINASE
P-SER
P-SER
P-SER
P-THR
P-THRP-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 KINASEP-SER
P-THR
Direct: CREB. Indirect: JNK/c-jun
DNA
P-TYR
MAP KINASEP-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+
CaN
CaN
CaMKIICaMKII
Ca2+
pCREBpCREB
Ca2+
Ca2+
Ca2+
Ca2+
PP1
Giulio [email protected]