Download Mitogen activated protein kinase

Protein phosphorylation
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Biochemistry & diversity
MAPKs
PKB
Take-home lessons
– Conformational changes
– Phosphorylation-dependent kinases
– Specificity through complex formation
Kinase architecture
• Kinase domain
• Regulatory domain
– Pseudosubstrate
– Allosteric
– External subunit
• Targeting domain
– Phospho-tyrosine binding (SH2/SH3)
– Phospholipid binding (PH, C2)
– Protein
Kinase diversity
• Serine/Threonine
– AGC kinases – allosteric regulation
– CaMK – CaM regulation
– MAP kinases – regulation by phosphorylation
• Tyrosine
– RTKs – Ligand regulation
– Non-receptor tyrosine kinases
• Cofactor
• PH cascade
Phosphorylation
• PO4- group charge alters the electron
balance of power
• Phosphorylatable residues
– Polar OH group
Serine
H O
N C C
H CH2
OH
Threonine
H O
N C C
H CH CH3
Tyrosine
H O
N C C
H CH2
OH
OH
Conformational change
• ~45 kJ from ATP hydrolysis
• ~10 kJ per ionic bond
• ~5 kJ per H-bond
Base substitution
• Experimentally modify residues to mimic
phosphorylated/non-phosphorylated form
Serine
Phosphoserine
• Serine
H O
N C C
H CH2
OH
Alanine
H O
N C C
H CH3
H O
C C
N
H CH2
OPO3Glutamic Acid
H O
N C C
H CH2
C
O
O-
Experimental Manipulation
• Residue substitution in 4EBP1
Wild type mRNA:
UCA ACC
Wild type protein:-riiydrkflmecrnspvtktModified protein:-riiydrkflmecrnApvAktGCA GCC
Modified mRNA:
Neutral=binds eIF4E
Modified protein:-riiydrkflmecrnEpvEktModified mRNA:
GAG GAG
Acidic=releases eIF4E
• Growth assay
• Immunoprecipitation
MAP kinases
• Ubiquitous S/T kinases
– Common effectors for many signals
• Diverse family
– ERK
– SAPK (JNK)
– p38
• Cascade
– MAPK
– MAPKK (MEK)
– MAPKKK (MEKK)
MAP kinases
• Activated by extracellular stimulus
– Mitogen
– Chemical stressor
– Physical stressor
• Receptor mediated
– Adapter protein
MAPK common structure
• Nucleotide binding pocket
• Substrate docking site
– Docking domain
– Separate substrate domain
p38MAPK
ATP pocket
Active site
Docking domain
Callaway et al 2005
MAPK subfamilies
• Extracellular signal regulated kinase ERK
– Raf-MEK 1/2-ERK ½
– Growth factor stimulated
• c-Jun N-Terminal Kinase JNK (SAPK)
– MEKK 1-4 – MKK 4/7 – JNK 1/2/3
– Stress (UV light, oxidative, heat) stimulated
• p38
– TAK/ASK – MKK 3/6 – p38 a/b/g/d
– Stress/cytokine stimulated
MAPK families
Stimulus
GF
Cytokines
Stress, cytokines
MAPKKK
Raf
MEKK1/2/3/4
MEKK1/2/3/4, TAK
MAPKK
MEK 1/2
MKK 4/7
MKK 3/6
MAPK
ERK 1/2
JNK 1/2
P38, SAPK
Effectors
Elk1,AP-1,
MNK, RSK,
PLA2
AP-1, NFAT,
IRS-1, HSP,
BIM/Bcl2
CREB, SRF,
MEF2, MNK,
HSP, PLA2
ERK 1/2
Widman et al. 1999
ERK 1/2
• Transcription factor activation
– Elk1, cMyc, cFos, cJun (C-term), AP-1
– Immediate/early gene upregulation
• Cell proliferation
– cdk2, stimulate G1-S transition
– G2-M transition
• Cellular function
– Protein synthesis-MNK
– Cytoskeletal remodeling-MAPK-APK
eg: Myoblast proliferation
• Muscle growth/hypertrophy requires
satellite cells
– Adult, muscle-resident stem cells
– Activate, proliferate, and differentiate to allow
growth
Satellite Cell
Basal Lamina
Cell membrane
Differentiate & fuse
Myonucleus
Proliferate
ERK1/2 control of proliferation
• Myoblasts are dependent of FGF for growth
• FGF required for S-phase entry (Clegg et al., 1987)
Remove FGF at t=0
Grow cells in FGF-rich media
Keep a few in FGF,
just to be sure
Count cells in S-phase by
nucleotide incorporation
Without FGF, cells
finish their S-phase
and don’t start
another
FGF-mediated ERK signaling
• FGFERKc-MycCyclins A,B,E; CDK2
• c-Myc inhibitor (MadMyc) forces cell cycle
withdrawal (Marampon et al., 2006)
• FGF-ERK represses myogenin expression (Tortorella, 2001)
Myc Inhibitor
Reduces cell #
Increases
differentiation
myogenin expression
250.0
FGF
IGF-1
200.0
150.0
100.0
50.0
0.0
0
Increase
myogenin
50
100
150
200
250
ERK activity
Inhibit ERK
JNK
• Cell stress response
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Heat shock
DNA damage
Reactive oxygen
UV light
• Cytokines
– Tumor Necrosis Factor (TNF) receptor
– Receptor tyrosine kinase
– GPCR
JNK
• Transcription factors
– cJun (N-term), NFAT, ATF-2, AP-1, forkhead
• Receptor modification
– IRS-1 inhibition
• Cellular function
– Apoptosis- BCL2, FasL
• Immune cell activation, cell motility
P38
• Stress response
– Heat, oxidative, UV
– Osmotic shock
– Cytokines
• Transcription factors
– MEF2, Elk-1, cFos, cJun, CREB
• Cellular function
– Apoptosis/hypertrophy
– HSP27, MNK-1
Crosstalk
• MEKK
– Most can be activated by RTK, Gq, Gi, PKC…
– Substrate preference
• MEK
– Most can be phosphorylated by any MEKK
– Substrate specificity varies with isoform
• MAPK
– Common substrates (Elk-1, cMyc, cFos, cJun)
– Unique substrates (Cellular, NFAT, HSP)
Regulatory processes
• Scaffold/complex formation provides
specificity
• MAPK negative feedback
– Upstream inactivation
– Downstream phosphatases
• Transport and localization
MAPK complexes
• ERK
– HSP90 (90 kD heat shock protein)
– 14-3-3
• JNK
– IKAP (inhibitor of kappa-B complex associated
protein)
– JIP (JNK interacting peptide)
Scaffolding
• MAPK (ERK)
– Caveolin
– 14-3-3
– KSR (kinase suppressor of ras)
• JNK
– JIP
– Ste5p
• Assembly
• Inhibition
Scaffolding
Classical
Cascade
Isoform
Specification
Activation
Specification
Scaffold differentiation of MAPK
• MAPKs are mass produced cogs in complex
molecular machines
• Function derives from complex more than cog
Multiple, independent Ste20 dependent behaviors in yeast
Elion 2001
Negative feedback
• MKP
– MAP kinase phosphatase
– Inducible, nuclear/constitutive, cytoplasmic
• DSP-dual specificity phosphatase
Transport and localization
• Nuclear import/export
– Rho/rac
– Nuclear scaffolds/nuclear anchoring proteins
• Cytoplasmic anchor
– Some b-arrestin/GPCR complexes
– MAPK dimers
• Subcellular localization
– JIP scaffold with kinesin
– JNK transport to growth cone
PI-3K-akt/PKB cascade
• Phosphatidylinositol 3’ kinase
• Phosphoinositide dependent kinase
Receptor
– PIP3 mediated membrane recruitment
PI3K
• Protein kinase B (akt)
PDK1
– PIP3 mediated membrane recruitment
– PDK1-dependent phosphorylation
akt
• Mammalian Target of Rapamycin
mTOR
– Translational effectors
– Signal integrator – GSK3, TSC, AMPK
p70S6k
4EBP
eIF2
mTOR
KEGG database http://www.genome.jp