The Rice Cyclin-Dependent Kinase – Activating Kinase R2 Regulates S-Phase Progression

1. The Rice Cyclin-Dependent Kinase –Activating Kinase R2 Regulates S-Phase Progression

2. ◎ Cell Cycle이란? ordered series of events leading to replication of cells - G1 phase : After mitosis, including cytokinesis which divides the cytoplasm between the two daughter cells, and before the onset of the next S phase there is an interval designated Gap1 or G1. - S phase : Synthesis phase during which the DNA is replicated - G2 phase : After the completion of DNA replication and chromosome duplication, in other S phase is completed, there is a second interval designated Gap2, or G2 before the set of mitosis. - M phase : Mitosis(nuclear division) and Cytokinesis(cytoplasmic division)

3. ◎ 세포 작동의 기본원리 Assemble mitosis spindle Complete cell division Replecate DNA

4. Cell cycle progression is regulated by protein Cyclin CDK(Cyclin-dependent kinase) - superfamily of eukaryotic serine-threonine protein kinase - required binding to a cyclin protein for activity. : CDK catalytic subunits do not act alone. - function 1) regulation of the eukaryotic cell division cycle. 2) the control of gene transcription and other process. 3) nutrient starvation and the control of transcription.

5. - CDK family ※ ex) M phase inducing protein

6. CAK INK-4 P15, p16, p18, p19 Cip/Kip P21, p27, p57 Cyclin A Cyclin D1-3 Cyclin E Cyclin A Cyclin B CDK4/6 CDK2 CDK2 Cyclin D G1 S G2 M ※ Cycling-dependent kinase inhibitors. 1) CIP/Kip family 2) INK 4 (Inhibitors of Cyclin-dependent kinase 4)

7. - degradation of mitotic cyclins polyubiquitination of mitotic cyclin- The regulation of S phase inducing protein ① E1 (ubiquitin-activating enzyme)-SH ↔ ubiquitin-COOH via thioester bond ② ubiquitin transferred to E2 (ubiquitin-conjugating enxymes) ③ E3 (ubiquitin ligase) : multiubiquitin → covalently linked via lysine → making substrate for proteosome

8. inactive p53 ↓ ←DNA damage active p53 ↓ binds to regulatory region of p21 gene ↓ p21 mRNA ↓ p21 protein (Cdk inhibitory protein) ↓ p21-S phase cyclin-S phase Cdk complex EX) How p53 arrests the cell cycle in G1. • active p53 • ↓

9. - The regulation of S phase inducing protein

10. Cyclin-CDK complex ① Inhitiates M phase ② S phase cdk-S phase cyclin

11. - The regulation of Cdks by cyclin degradation.

12. ◎ Checkpoint • Summary of some cell-cycle checkpoints.

13. ◎ To die or stop dividing a. DNA가 손상되었을 때 p53은 growth-arrest gene인 p21 gene등을 발현시켜서 세포분열을 일시적으로 정지시킨다 b. DNA 손상이 세포가 수용지 못할 정도로 심하면, p53은 Apoptosis-inducing gene인 Bax gene 등을 발현시켜서 세포를 apoptosis시킨다. 이 때, p53-binding protein인 ASPP가 p53의 apoptosis 기작을 돕는다. ASPP의 결여는 p53의 변이와 함께 비정상적 세포 증식의 원인이 된다.

14. ◎ Cell Growth

15. T170 T160 cdk7 cdk2 cyclinH cyclinA or E CAKAK or By MAT1 binding Cell cycle regulation Transcriptional regulation TFⅡH CTD RNA polymerase Ⅱ

16. CAK (CDK-activating kinase) - Phosphorylation of CDKs ata conserved threonine residue is required for their full activity - have low homology with eachother, and they differ in their enzyme characteristics. Animal and yeast - animal : human p40mo15/cdk7 - budding yeast : Cak1pciv1 Plant - rice : R2 - arabidopsis : Cak1At Rice CAK homologous kinase R2 - is expressed at higher levels in S-phase - regulated not only at the transcriptlevel but also at the protein and enzyme activity levels.

17. Figure 1. Subcellular Localization of R2.

18. Aphidicolin : slow elongation at replication forks by inhibiting DNA polymerasesα and δ Figure 2. Expression and CTD Kinase Activity of R2 in Synchronized Suspension Cells.

19. Figure 3. RNA Gel Blot and Protein Gel Blot Analysis of Wild-Type and R2- Overexpressing Rice Cells.

20. Figure 4. Effect of R2 Overexpression on Rice Suspension Cells.

21. Figure 5. Cell Cycle Phase Progression in Partially Synchronized Wild-Type Cells and in the R2-Overexpressing Transgenic Line 6.

22. Figure 6. Fresh Weight Increase in Wild-Type Cells and in the R2-Overexpressing Transgenic Line 6.

23. Figure 7. R2 Gene and Protein Expression in the Internodes of Partially Submerged Deepwater Rice Plants.

24. Figure 8. CTD Kinase Activity of R2 in the Internodes of Partially Submerged Deepwater Rice Plants.