nNOS及eNOS均存在於神經細胞中，並且可經由激酶磷酸化調控其活性。於神經細胞中表現的神經專一性活化因子p35與第五型細胞週期蛋白激酶(Cdk5)的結合被證明與退化性神經細胞死亡具有關係。本文主要針對Cdk5於eNOS及nNOS的功能性調控為主。在nNOS之部份，我們證明Cdk5會與nNOS 結合並且經由磷酸化抑制nNOS活性，使一氧化碳的產量減少。在eNOS之部份，利用體外(in vitro)與體內(in vivo)的磷酸化實驗證明Cdk5會使eNOS磷酸化在絲氨酸113之位置， 同時發現Cdk5抑制劑roscovitine可減少eNOS之磷酸化程度，此結果顯示了Cdk5磷酸化eNOS之專一性。此外，於絲氨酸113突變之eNOS並不會失去與Cdk5結合之能力。於SH-SY5Y細胞中，S113A eNOS與Cdk5/p35同時表現時的一氧化碳產量會比eNOS與Cdk5/p35同時表現高出兩倍。以上之結果顯示Cdk5會磷酸化nNOS及eNOS，並且負調控nNOS及eNOS的活性。

The activity of neuronal nitric oxide synthase (nNOS) and endothelial nitric oxide synthase (eNOS) were regulated by kinase through phosphorylation. The cyclin-dependent kinase 5 (Cdk5) by associating with its neuron-specific activator p35 has been demonstrated to be essential for neurodegenerative neuronal death. This study focuses on the functional regulation of nNOS and eNOS by Cdk5/p35 complex in a phosphorylation dependent manner. We found that nNOS associated with Cdk5 by immunoprecipitation (IP) and in vitro phosphorylated by Cdk5 by autoradiograph. Nitrite (NO2-) production was significantly reduced in Cdk5 over-expressing N18 cells, suggested that Cdk5 down-regulated nNOS enzymatic activity. In addition, Cdk5 phosphorylated eNOS both in vitro and in vivo on Ser 113, and the Cdk5 inhibitor roscovitine suppressed the phosphorylation of eNOS. Interaction of wild-type eNOS and S113A mutant eNOS with Cdk5 was observed in co-immunoprecipitation experiments. Co-expression of S113A eNOS and Cdk5/p35 resulted in 2-fold enhancement nitrite (NO2-) generation than co-expression of eNOS and Cdk5/p35 in SH-SY5Y cells. These data indicate that Cdk5 phosphorylated nNOS and eNOS, as well as down regulated nNOS and eNOS activity. Our results supposed that Cdk5 associated with and regulated the activity of nNOS and eNOS through phosphorylation.

Contents
Chinese Abstract i
English Abstract ii
Contents iii
Abbreviation v
1. Introduction 1
1.1 cyclin-dependent protein kinase-5 1
1.2 The Cdk5/p35 system: structural aspects 3
1.3 Functional organization of Cdk5 in cells 5
1.4 Cdk5 interacting proteins 7
1.5 Regulation of the Cdk5/p35 complex 9
1.6 The Cdk5 in Alzheimer pathology 10
1.7 Nitric oxide synthase 15
1.8 Three NOS isoforms 16
1.9 The NOS in Neurodegenerative disease 17
1.10 The NOS and Cdk5 in Neurodegenerative disease 17
1.11 The eNOS and Cdk5 in cancer cells 18
1.12 Specific aim 18
2. Materials and Methods 19
3. Results 23
3.1 nNOS and eNOS associate with Cdk5 in rat Brain lysate 23
3.2 Cdk5 Phosphorylate nNOS 24
3.3 Cdk5 regulated nNOS activity 24
3.4 Cdk5 Phosphorylated eNOS 24
3.5 eNOS(WT) and eNOS(S113A) Associates with Cdk5 26
3.7 Cdk5 regulated eNOS activity 27
4. Discussion 28
5. References 43
6. Appendix 58

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