검색 상세

A Novel DNA-PK/PLK1/STING/IRF3 Axis Regulates Normal Mitotic Progression

초록/요약

cGAS-STING-IRF3 pathway has been known to elicit an immune response against foreign or self-DNA. It is well-known that during mitosis, cGAS is inactivated to prevent autoimmunity. However, I observed a significant increase in the basal phosphorylation of IRF3 during mitosis. Importantly, the loss of mitotic-specific IRF3 phosphorylation significantly delayed the mitotic progression which may partly stems from polar chromosomes formation during metaphase. Next, I observed that this phosphorylation was cGAS- and TBK1-independent but STING-dependent, thus clearly indicating that a novel non-canonical STING-IRF3 axis regulates normal mitotic progression. Then, I hypothesized that during mitosis apart from cGAS, an unknown upstream activator of STING maybe responsible for STING activation and subsequent IRF3 phosphorylation. I found out that either depletion or inhibition of DNA-PKcs resulted in decreased basal STING and IRF3 phosphorylation during mitosis. Moreover, I also found that PLK1, an important mitotic kinase, regulates basal IRF3 phosphorylation during mitosis. Further analysis revealed that either DNA-PK depletion or inhibition resulted in loss of STING phosphorylation and dimerization, indicating that DNA- PK acts upstream of STING and regulates STING activation. Whereas PLK1 inhibition specifically decreased basal IRF3 phosphorylation and subsequent dimerization. Furthermore, in vitro kinase assay showed that either DNA-PK or PLK1 can directly phosphorylate IRF3 at Ser386 residue. But only PLK1 can phosphorylate IRF3 at Ser396 residue, strongly suggesting that during mitosis PLK1 is the major kinase responsible for directly phosphorylating IRF3, that is followed by IRF3 dimerization. Surprisingly, DNA- PK complex, PLK1, STING and IRF3 interact with each other during mitosis and this interaction significantly depends on STING. Depletion of STING abolishes the interaction of DNA-PK complex and PLK1 with IRF3, indicating the role of STING as a scaffold protein for this complex of STING, IRF3, DNA-PK and PLK1. Moreover, loss of IRF3 interaction with DNA-PK/STING complex fails to recruit PLK1 to the complex. Consistently, DNA-PKcs, STING and IRF3 depleted cells commonly exhibited delayed mitotic progression in a cell-type independent manner. These data collectively suggest a novel regulatory pathway of mitotic progression comprising of STING-IRF3 axis along with DNA-PK as STING regulator and PLK1 as IRF3 regulator, and STING works as a scaffold.

more

목차

1. INTRODUCTION 1
2. MATERIALS AND METHODS 10
2.1. Cell culture 10
2.2. Antibodies 10
2.3. Synchronization and drug treatment 11
2.4. Plasmids and transfection experiments 12
2.5. Knock-down experiments 12
2.6. Immunoblotting 14
2.7. cGAMP ELISA 15
2.8. Native-PAGE 15
2.9. Time-lapse analysis 16
2.10. 2D Blue Native-PAGE 16
2.11. Immunocytochemistry 17
2.12. Immunoprecipitation 17
2.13. In-vitro kinase assay 18
2.14. Quantitative RT-PCR 19
2.15. Mitotic index 19
2.16. Statistical analysis 20
3. RESULTS 21
3.1. IRF3 is phosphorylated during mitosis in a cGAS-independent but STING-dependent manner 21
3.2. Phosphorylation of IRF3 is required for normal mitotic progression 28
3.3. STING and IRF3 dimerization is a pre-requisite during mitosis 35
3.4. DNA-PKcs activity is required for STING and IRF3 phosphorylation during mitosis 39
3.5. PLK1 is the major mitotic kinase responsible for IRF3 phosphorylation during mitosis 47
3.6. STING, IRF3, DNA-PK and PLK1 form a complex during mitosis where STING acts as a mediator 53
3.7. Recruitment of PLK1 and IRF3 to the DNA-PK/STING complex requires DNA-PK activity 62
3.8. Ku70 and Ku80 may play different roles in the activation of STING/IRF3 axis in a cell-dependent manner 66
4. DISCUSSION 71
5. REFERENCES 77

more
반출 Meta View 목록