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 A Cyclin B

CDK2CDK2

G1 S G2 M

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 정지시킨다

b. DNA 손상이 세포가 수용지 못할 정도로 심하면 , p53 은 Apoptosis-inducing gene 인 Bax gene 등을 발현시켜서 세포를 apoptosis 시킨다 . 이 때 , p53-binding protein 인 ASPP 가 p53 의 apoptosis 기작을 돕는다 . ASPP 의 결여는 p53 의 변이와 함께 비정상적 세포 증식의 원인이 된다 .

◎ Cell Growth

cdk7

cyclinH

T170 CAKAKor

By MAT1 binding

cdk2

cyclinA or E

T160 TFⅡH

RNA polymerase Ⅱ

CTD

Cell cycleregulation

Transcriptionalregulation

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 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 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.