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

◎ Cell Cycle 이란 ? 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)

◎ 세포 작동의 기본원리 Complete cell divisionAssemble mitosis spindle Replecate DNA

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.

- CDK family ※ ex) M phase inducing protein

P15, p16, p18, p19 P21, p27, p57 INK-4 Cip/Kip Cyclin D1-3 CDK4/6 Cyclin E Cyclin D Cyclin A Cyclin B CDK2 G1 SG2M CAK ※ Cycling-dependent kinase inhibitors. 1) CIP/Kip family 2) INK 4 (Inhibitors of Cyclin-dependent kinase 4)

- 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

EX) How p53 arrests the cell cycle in G1. active p53 ↓ 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

- The regulation of S phase inducing protein

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

- The regulation of Cdks by cyclin degradation.

◎ Checkpoint -Summary of some cell-cycle checkpoints.

◎ To die or stop dividing a. DNA 가 손상되었을 때 p53 은 growth-arrest gene 인 p21 gene 등을 발현시켜서 세포분열을 일시적으로 정지 시킨다 b. DNA 손상이 세포가 수용지 못할 정도로 심하면, p53 은 Apoptosis-inducing gene 인 Bax gene 등을 발현시켜 서 세포를 apoptosis 시킨다. 이 때, p53-binding protein 인 ASPP 가 p53 의 apoptosis 기작을 돕는다. ASPP 의 결여 는 p53 의 변이와 함께 비정상적 세포 증식의 원인이 된다.

◎ Cell Growth

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

CAK (CDK-activating kinase) - Phosphorylation of CDKs at a conserved threonine residue is required for their full activity - have low homology with each other, and they differ in their enzyme characteristics. Animal and yeast - animal : human p40 mo15 /cdk7 - budding yeast : Cak1p civ1 Plant - rice : R2 - arabidopsis : Cak1At Rice CAK homologous kinase R2 - is expressed at higher levels in S-phase - regulated not only at the transcript level but also at the protein and enzyme activity levels.

Figure 1. Subcellular Localization of R2.

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

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

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

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

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

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

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