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Analysis of Inositol 1,4,5 trisphosphate 3-kinase A as a novel microtubule-associated protein in neuron

신경세포에서 미세소관 결합 단백질로서의 Inositol 1,4,5-trisphosphate 3-kinase A 분석

  • Dongmin Lee (이동민, 대학원 의학과 분자인체유전학전공)

초록/요약

The neuronal cytoskeleton is an essential component of neurons. It performs various functions including synaptic cargo transportation, cell migration, neuronal polarity, and neuronal morphogenesis. Specially, cytoskeleton-mediated structural plasticity has been regarded as the underlying mechanism for learning and memory. Structural plasticity is constructed by synergistic contributions of various cytoskeletal binding proteins as well as cytoskeletons themselves. In this perspective, identification and exploration of neuronal cytoskeleton binding proteins are important for understanding fundamental mechanisms for learning and memory. Inositol 1,4,5-trisphosphate 3-kinase A (IP3K-A) is a brain-specific and F-actin binding protein. However, detailed functions of IP3K-A and its regulatory mechanisms are not understood. In the present dissertation, I demonstrated that IP3K-A is a novel microtubule-associated protein (MAP) and its cAMP-dependent Protein Kinase A (PKA)-dependent phosphorylation of Ser119 leads to a significant reduction of microtubule binding affinity. PKA-dependent phosphorylation was found to contribute to the triggering mechanism for its synaptic targeting via dissociation from dendritic microtubules. Moreover, I found that IP3K-A also interacted to End-Binding protein 3 (EB3) through a microtubule-independent manner. Binding affinity of IP3K-A and EB3 was also reduced by serine 119 phosphorylation as like the interaction of IP3K-A and microtubule. EB3 is microtubule plus end tracking proteins (+Tips) which decorate plus end tips of microtubules as like an appearance of comet. EB3 functions as the central hub for cross-linker of other structures or proteins. Interestingly, interaction of IP3K-A and EB3 was more prominent in the induction of chemical long-term potentiation (cLTP), suggesting that the complex of IP3K-A and EB3 functions as an activity-dependent cross-linker for microtubule and F-actin cytoskeleton. This cross-linking effect of IP3K-A and EB3 may contribute to increase stability and frequency of dynamic microtubule invasion to dendritic spines, inducing enhanced synaptic plasticity and synaptic cargo transports. In conclusion, IP3K-A might be responsible for structural plasticity through its binding ability with cytoskeleton.

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초록/요약

신경계 세포골격 (cytoskeleton) 은 신경세포에 필요한 다양한 역할을 하며, 시냅스 단백질의 수송 (synaptic cargo transportation), 신경세포의 이동 (cell migration), 신경세포의 양극성 (neuronal polarity) 조절 및 신경세포의 형태를 만드는데 깊이 관여한다. 특히 학습과 기억의 근원적인 기전으로 생각되는 구조적 가소성 (structural plasticity) 은 세포골격의 동적 변화를 통해서 매개된다. 이러한 구조적 가소성이 일어나기 위해서는 세포골격뿐만 아니라 세포골격 결합 단백질 (cytoskeletal binding protein) 들의 도움이 필요하다. 이러한 견지에서, 신경세포 골격 결합 단백질들을 발굴하고, 그들의 기능적 역할을 연구하는 것은 학습과 기억의 기전을 이해하는데 매우 중요하다. 신경계 특이적으로 발현되는 Inositol 1,4,5-trisphosphate 3-kinase A (IP3K-A) 는 F-actin과 결합 단백질로서, 칼슘 신호전달의 핵심요소인 IP3를 인산화시킨다. 최근 IP3K-A가 세포골격의 동적 변화에 중요한 역할을 한다는 보고가 있으나, 이에 관한 자세한 기능 및 전반적인 조절에 관한 연구는 아직도 부족한 실정이다. 본 연구에서는 IP3K-A가 새로운 미세소관 결합단백질 (microtubule-associated protein) 이며, 미세소관과 IP3K-A의 결합력이 Protein kinase A (PKA) 가 매개하는 Serine 119 인산화 (phosphorylation) 에 의해서 조절됨을 알았다. 인산화에 의한 미세소관과의 결합력 감소는 IP3K-A가 가지돌기가시 (dendritic spine) 로 이동하는 기전과 밀접하게 연관되어 있으며, 이를 통해 신경세포는 IP3K-A의 시공간적 분포를 조절할 것으로 생각된다. 또한, 본 연구에서는 IP3K-A가 미세소관 말단 결합단백질 (microtubule plus end tracking proteins, +Tips) 인 EB3와도 결합하며, 둘 사이의 결합력도 역시 Serine 119의 인산화에 의해서 조절됨을 밝혔다. 이는 미세소관의 예와 동일한 것으로 상당히 흥미로운 사실이다. 또한, IP3K-A와 EB3의 결합이 cLTP 유도 시, 가지돌기가시 내에서 증가됨을 관찰하였다. 이러한 결과는 IP3K-A와 EB3 복합체가 가지돌기가시 내에서 잠재적인 세포골격 연결인자로 작용할 가능성을 시사한다. 가지돌기가시 내에서, 미세소관과 F-actin의 결합력 증가는 안정적인 시냅스 단백질의 수송을 도모하고, 이를 통하여 시냅스 가소성을 높이는 역할을 수행할 수 있을 것이다. 본 논문을 통해서, 미세소관과 IP3K-A의 결합을 최초로 규명하였으며, 이 결합을 통하여 IP3K-A의 공간적인 분포를 조절하는 역할을 수행함을 밝혔다. 또한, IP3K-A와 EB3의 결합도 최초로 발견하였으며, 이 결합은 가지돌기가시 내의 미세소관과 F-actin의 연결에 기여하는 것 같다. 궁극적으로, IP3K-A는 세포골격 조절을 통하여, 학습과 기억의 기저를 이루는 구조적 가소성의 핵심적인 축을 담당하는 것으로 생각된다.

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목차

Abstract i
Contents iii
List of TABLEs ix
List of FIGUREs x

Part 1
Inositol 1,4,5-trisphosphate 3-kinase A is a novel microtubule-associated protein: PKA-dependent phosphoregulation of microtubule binding affinity 1
1-1. Abstract 2
1-2. Introduction 3
1-3. Materials and Methods 5
1-3-1. Preparations of constructs and protein purification 5
1-3-2. GST pull-down assay 6
1-3-3. MALDI-TOF mass spectrometer analysis 6
1-3-4. Immunoblotting analysis 7
1-3-5. Microtubule co-sedimentation Assay 7
1-3-6. Immunoprecipitation 8
1-3-7. Tissue-based microtubule co-sedimentation assay 8
1-3-8. Immunocytochemistry 9
1-3-9. Immunogold labeling 10
1-3-10. Cryofixation, cryosubstitution, and low temperature embedding 11
1-3-11. Fluorescence based microtubule polymerization assay 11
1-3-12. Microtubule bundling assay using x-rhodamine tubulin 12
1-3-13. Genotyping of IP3K-A KO mouse 12
1-3-14. Transfection into HEK293 cells 13
1-3-15. Production of IP3K-A Ser119 phosphoantibody 13
1-3-16. Kinase reaction with PKA catalytic subunit 14
1-3-17. Antibodies and reagents 14
1-3-18. Cell culture 15
1-3-19. Immunoblotting densitometric analysis and image processing 16
1-3-20. Statistical analysis 16
1-4. Results 17
1-4-1. Tubulin is identified as a novel binding partner of IP3K-A. 17
1-4-2. IP3K-A binds directly to microtubules. 18
1-4-3. IP3K-A distributes along microtubule in vivo rat brain sections and primary culture neuron. 19
1-4-4. Microtubule polymerization and bundling effect of IP3K-A 20
1-4-5. IP3K-A binds to microtubule via its N-terminus region 21
1-4-6. Microtubule binding of IP3K-A is regulated by phosphorylation of IP3K-A Ser119. 22
1-4-7. PKA regulates the microtubule binding affinity of IP3K-A 23
1-4-8. Phosphorylated endogenous IP3K-A has lower binding affinity toward microtubules 25
1-5. Discussion 52
1-5-1. IP3K-A is a novel MAP protein. 52
1-5-2. PKA-dependent phosphorylation of IP3K-A and interactions between IP3K-A and microtubules 53
1-5-3. Neuronal function of PKA-dependent phosphorylation of IP3K-A and interactions between IP3K-A and microtubules 55

Part 2
Synaptic targeting of IP3K-A is mediated by PKA-dependent phosphorylation 57
2-1. Abstract 58
2-2. Introduction 59
2-3. Materials and Methods 61
2-3-1. Subcellular fractionation 61
2-3-2. Forskolin induced cLTP in dissociated hippocampal culture neurons 62
2-3-3. Forskolin induced cLTP in acute hippocampal slice preparation 63
2-3-4. Acute immobilization stress test and experimental animal management 65
2-3-5. Immunoblotting analysis 65
2-3-6. Immunocytochemistry 66
2-3-7. Statistical analysis 66
2-4. Results 67
2-4-1. Developmental expression pattern and subcellular distribution of serine 119 phosphorylated IP3K-A. 67
2-4-2. Increase of phosphorylated IP3K-A in forskolin induced cLTP 68
2-4-3. H-89, selective PKA inhibitor, disturbs synaptic targeting of IP3K-A. 70
2-4-4. Increase of serine 119 phosphorylation of IP3K-A in in vivo animal model 70
2-4-5. Synaptic accumulations of IP3K-A are induced by treatment of okadaic acid. 71
2-4-6. Rapid dephosphorylation of Ser119 IP3K-A in dendritic spines 73
2-5. Discussion 93
2-5-1. Activity-dependent targeting of synaptic proteins and new possibility of microtubules as a anchoring compartment for synaptic targeting 93
2-5-2. Synaptic targeting of IP3K-A is mediated by PKA-dependent phosphorylation. 94
2-5-3. Robust Serine 119 phosphorylation of IP3K-A by acute immobilization stress 95
2-5-4. Importance of the synaptic translocation of IP3K-A 97

Part 3
Activity-dependent interaction of IP3K-A and EB3, microtubule +end tracking protein, in dendritic spines 99
3-1. Abstract 100
3-2. Introduction 101
3-3. Material and Methods 104
3-3-1. Immunoprecipitation 104
3-3-2. Immunoblotting analysis 104
3-3-3. Immunocytochemistry 105
3-3-4. Subcloning of GST-EB3 constructs 105
3-3-5. Subcloning 10x His-EB3 106
3-3-6. Purification of His-EB3 106
3-3-7. Proximity ligation assay (PLA) for detection of protein-protein interaction 107
3-3-8. Quantification of Proximity ligation assay 108
3-3-9. Statistical analysis 108
3-4. Results 109
3-4-1. Interaction of IP3K-A and EB3, microtubule end binding protein 109
3-4-2. Identification of IP3K-A binding region within EB3 and vice versa 110
3-4-3. Binding affinity of IP3K-A and EB3 is regulated negatively by phosphorylation of IP3K-A serine 119. 111
3-4-4. Activity-dependent interaction of IP3K-A and EB3 in dendritic spine 113
3-5. Discussion 132
3-5-1. EB3 binds directly to IP3K-A via its C-terminus acidic tail. 132
3-5-2. PKA-dependent phosphorylation regulates binding affinity of IP3K-A and EB3 133
3-5-3. Physiological roles of the interaction between IP3K-A and EB3 in dendritic spine 134
4. References 138
5. 국문초록 149
6. Acknowledgement 151
7. Curriculum vitae 153

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