Archipelago/hCDC 4 and Endometrial Cancer

1. Archipelago/hCDC 4 and Endometrial Cancer Keji Baruwa March 20, 2003

2. SCF Ubiquitin Ligase Complex • (Budding yeast) • Triggers DNA replication by catalyzing ubiquitination of SIC1 3 components: ySKP1, CDC53 (cullin), & F-box protein CDC4 Lyapina et. al

3. Proteolytic Degradation • Proteins are marked for proteolytic degradation by attachment of multiubiqutin chains • Activated by E1 • Ubiquitin then transferred to E2 (ubiquitin conjugating enzyme) • E3 • Once substrate is multiubiquitinated, it is then recognized and degraded by 26S proteasome Lyapina et al.

4. Ubiquitination Pathway Discovered in Budding Yeast • Components of this pathway: CDC53, CDC4, & ySKP1-assemble into ubiquitin ligase complex, SCFCDC4 • SCFCDC4, collaborates with yCDC34 to catalyze ubiquitination of SIC1 Lyapina et al.

5. Multiple SCF Complexes in Yeast • Analysis has revealed that SIC1 proteolysis requires CDC4 • G1 cyclin proteolysis depends on GRR1 (distinct F-box containing protein) • SCF complexes assembled with GRR1 instead of CDC4 bind G1 cyclins but not SIC15 Lyapina et al.

6. Components of SCF Ubiquitination Highly Conserved During Evolution • Human homologs of yCDC34 and ySKP1 have been identified • F-box containing proteins like CDC4 (WD 40 repeats) and GRR1 have been reported in many eukaryotes • Potential human counterpart of GRR1 & SKP2 identified with hSKP1 as a cyclin A/CDK2-associated protein needed for S phase progression Lyapina et al.

7. Phosphorylation of SCF • SCF substrates in budding yeast must be phosphorylated before ubiquitination • Many human cell cycle regulators are targeted for ubiquitination after CDK phosphorylation. (example p27) • Cyclins E and D1 are degraded by ubiquitin-dependent pathway after phosphorylation at a specific site Lyapina et al.

8. Phosphorylation, Cont’d • SCF-bound Cyclin A/CDK2 may phosphorylate SCF subunits or potential substrates like E2F-1/DP-1, which will activate SCF-dependent ubiquitination Lyapina et al.

9. Does SCF Activity Really Exist in Animal Cells • S. cerevisiae cdc53ts mutants arrest at G1/S transition; C. elegans cul-1mutants fail to exit cycle • Ubiquitin-like proteins that are conjugated to proteins that involve E1 and E2 homologs • Human Cullin, Cul 2 assembles with von Hippel-Lindau tumor suppressor protein ElonginB/Elongin C complex Lyapina et al.

10. hCUL1 and hSKP1 interact in vivo Lyapina et al.

11. Human Cul1, hSKP1, & SKP2 • Human Cul1 can interact with human SKP1, SKP2, and Cyclin A/CDK2 • Association mediated by SKP2 Lyapina et al.

12. Human Cul1 directly interacts with hSKP1 and SKP2 • hCul1, hSKP1, and SKP2 can assemble into an SCF-like particle when co expressed in insect cells Lyapina et al.

13. Functions of Homologues of SCF Complex • Kinectochore function • S-phase progression • Exit from the cell cycle • Transcript elongation • Regulation of Hypoxia-inducible genes • Suppression of tumor genesis Lyapina et al.

14. hCUL1 • Assembles to SCF-like complexes in human cells • Associates with hSKP1 in transfected HeLa S3 cells • Assembles into complexes with both hSKP1 and F-box protein SKP2 in vitro • Complements the growth of cdc53ts mutant • Associates with ubiquitination-promoting activity in HeLa S3 cell lysate • SUBUNIT OF AN SCF-LIKE E3 COMPLEX IN HUMAN CELLS Lyapina et al.

15. Archipelago • Contains 7 WD 40 Repeats and F-box • Binds Cyclin E • Down regulates cyclin in vivo • (SCF complex might be involved in turnover of cyclin E) Mitchell

16. Fbw7 • Over expression decreases levels of cyclin E • hCDC4 and Fbw7 interact with cyclin e • This interaction depends upon cyclin e being phosphorylated at the threonine position • Mutation in cdc4 cyclin e stabiliz ed Mutations in WD 40 domain of hCDC4/Fbw7 stop phosphorylation of cyclin e Mitchell

17. hCDC4/Fbw7/Ago • Tumor suppressor • Negative regulator of cell proliferation that normally prevents cancer progression • Disruption may deregulate cell division at more than one level Schwab & Tyers

18. Cell Cycle Balancing Acts(Schwab and Tyers)

19. hCDC4 • Targets cyclin E to SCF • Mutated in at least 16% of endometrial tumors • Mutations are found either in the substrate binding domain of protein or at the amino terminus • If mutated-loss of heterozygosity • Localized to chromosome 4q32- (deleted in 30% of human tumors) Schwab & Tyers

20. hCDC4 Cont’d • Mutated in primary human tumors • May function as a tumor suppressor • Accumulation of Cyclin E may depend on ability of hCDC4 to bind substrate • 2 hit hypothesis Schwab and Tyers

21. Endometrial Cancer • Originates in the endometrial lining of the uterus • Most common gynecologic malignancy • Normally occurs in postmenopausal women • Estrogen dependent disease • Chronic exposure to estrogen without normal balance of progesterone is believed to be major risk of this cancer http://www.oncologychannel.com/endometrialcancer/