ORIGINAL PUBLICATIONS ........................................... 1
ABBREVIATIONS ................................................... 2
ABSTRACT ........................................................ 3
INTRODUCTION .................................................... 4
REVIEW OF THE LITERATURE ........................................ 6
1 Prelude ...................................................... 6
2 a retrospective of the MAPK/JNK/SAPK signaling ............... 6
2.1 The discovery of the MAPK/JNK/SAPK protein kinases ...... 6
2.2 Elucidation of the upstream signaling cascade of JNK ... 10
3 Studies in JNK knockout mice ................................ 13
3.1 jnk1-/- mice ........................................... 14
3.2 jnk2-/- mice ........................................... 14
3.3 jnk3-/- mice ........................................... 75
3.4 Multiple JNK knockout mice ............................. 15
4 Neuronal apoptosis .......................................... 16
4.1 JNK in neuronal apoptosis .............................. 17
4.2 Dual roles for JNK in neuronal development and
apoptosis .............................................. 21
5 JNK IN DISEASE .............................................. 22
5.1 Neurodegenerative diseases ............................. 23
5.2 Ischemic stroke ........................................ 25
6 JNK SUBSTRATES INVOLVED IN REGULATION OF CELL SHAPE AND
MOVEMENT .................................................... 27
7 JNK REGULATION OF MICROTUBULES .............................. 28
7.1 Microtubule destabilizing factors stathmin and SCG10 ... 29
7.2 MAP1 Band MAP2 ......................................... 30
7.3 DCX .................................................... 33
8 JNK REGULATION OF ACTIN CYTOSKELETON ........................ 34
8.1 Actin binding proteins and regulation of cortical
actin structures ....................................... 34
8.2 Paxillin ............................................... 36
8.3 Spir ................................................... 37
8.4 MARCKS family proteins ................................. 37
OBJECTIVES ..................................................... 41
EXPERIMENTAL PROCEDURES ........................................ 42
1 Materials ................................................... 42
1.1 Plasmids ................................................ 42
1.2 Antibodies ............................................. 43
1.3 Special reagents ....................................... 44
2 METHODS ..................................................... 44
2.1 Cell culture ........................................... 44
2.2 Transfection ........................................... 45
2.3 Immunostaining ......................................... 46
2.4 Immunoblot analysis and quantification ................. 46
2.5 Preparation of recombinant proteins .................... 46
2.6 In vitro protein phosphorylation and kinetic
analysis ............................................... 47
2.7 In vivo protein phosphorylation analysis ............... 48
2.8 Tissue extract preparation ............................. 48
2.9 Protein identification by mass spectrometry ............ 49
2.10 Protein phosphorylation site analysis by mass
spectrometry ........................................... 49
2.11 Analysis of neurite outgrowth and neuronal cell
morphology ............................................. 50
2.12 FRAP ................................................... 50
2.13 Wound healing assays ................................... 57
2.14 In vitro actin polymerization and binding assay ........ 51
2.15 Analysis of filamentous actin stability in cells ....... 52
3 statistical analysis ........................................ 52
4 Kinase inhibitors ........................................... 53
RESULTS AND DISCUSSION ......................................... 54
1 THE ROLE OF JNK ISOFORMS IN WTS INDUCED NEURONAL DEATH
(STUDY I) ................................................... 54
1.1 The c-Jun stress response is suppressed in JNK
deficient neurons ...................................... 55
1.2 Neurons from jnk2-/- jnk3-/- mice are not protected
from WTS induced cell death ............................ 55
1.3 Neurons depleted of JNK1, JNK2 and JNK3 are protected
from WTS induced cell death ............................ 56
1.4 Nuclear JNK activity, but not c-Jun nor ATF2, is
critical for WTS induced neuronal death ................ 57
2 Identification of brain substrates for JNK .................. 58
3 CONSTITUTIVELY ACTIVE CYTOPLASMIC JNK1 IS A DOMINANT
REGULATOR OF DENDRITE architecture: Role of MAP2 as an
effector (Study II) ......................................... 60
3.1 JNK1 activity is high in cerebellum and is
unregulated during neuronal differentiation ............ 60
3.2 Identification of the dendrite specific protein MAP2
as a JNK substrate ..................................... 60
3.3 MAP2 dependent process outgrowth is enhanced by high
JNK activity ........................................... 63
3.4 Primary neurons exhibits shorter and more numerous
dendrites upon JNK inhibition .......................... 63
3.5 Development of compartment specific JNK inhibitors ..... 64
3.6 The cytoplasmic pool of basally active JNK regulate
neuronal cell morphology ............................... 64
3.7 JNK1 deficient cerebellar neurons display increased
dendrite complexity .................................... 65
3.8 Morphogenetic defects of JNKl knockout motor cortex .... 66
3.9 Progressions from Study II ............................. 66
4 JNK1 PHOSPHORYLATION OF SCG10 DETERMINES MICROTUBULE
DYNAMICS AND AXODENDRITIC length (Study III) ................ 67
4.1 JNK1 binds to and phosphorylates stathmin family
members ................................................ 67
4.2 Efficient JNK mediated phosphorylation of the
neuronal specific protein SCG10 ........................ 68
4.3 SCG10 serine 62/73 is phosphorylated by JNK in cells ... 69
4.4 SCG10 phospho-S73 is reduced in JNK knockout brain
and colocalizes with active JNK ........................ 70
4.5 SCG10 regulated axonal length in cortical neurons ...... 70
4.6 Microtubule turnover is decreased in neurons upon JNK
inhibition or SCG10-AA expression ...................... 71
5 MARCKS-RELATED PROTEIN INDUCED F-ACTIN BUNDLING AND CELL
MIGRATION IS REGULATED BY JNK MEDIATED PHOSPHORYLATION
(STUDY IV) .................................................. 72
5.7 Identification of MRP as a JNK phosphorylation target
in brain tissue ........................................ 72
5.2 JNK phosphorylates three distinct sites in the
C-terminal tail domain of MRP .......................... 72
5.3 JNK phosphorylated MRP has increased actin filament
bundling activity in vitro ............................. 75
5.4 Molecular modeling of MRP .............................. 76
5.5 MRP localizes to lamellipodia and induce outgrowth of
finger-like filopodial protrusions ..................... 77
5.6 JNK phosphorylated MRP reduces filamentous actin
turnover in cells ...................................... 78
5.7 JNK mediated phosphorylation of MRP leads to
increased stability ofactin structures in cells ........ 79
5.8 JNK activity blocks MRP induced cell migration and
regulates cell migration in a substrate dependent
manner ................................................. 79
5.9 A role for JNK phosphorylated MRP in neuronal
migration and actin structure stability ................ 81
5.10 Conclusions from Study IV .............................. 81
CONCLUDING REMARKS ............................................. 83
ACKNOWLEDGEMENTS ............................................... 86
REFERENCES ..................................................... 88
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