Archipelago/hCDC 4 and Endometrial Cancer

Keji Baruwa

March 20, 2003

SCF Ubiquitin Ligase Complex (Budding yeast) Triggers DNA replication by catalyzing ubiquitination

of SIC13 components: ySKP1, CDC53 (cullin), & F-box protein

CDC4

Lyapina et. al

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.

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.

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.

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.

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.

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.

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.

hCUL1 and hSKP1 interact in vivo

Lyapina et al.

Human Cul1, hSKP1, & SKP2

Human Cul1 can interact with human SKP1, SKP2, and Cyclin A/CDK2

Association mediated by SKP2

Lyapina et al.

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.

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.

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 CELLSLyapina et al.

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

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

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

Cell Cycle Balancing Acts(Schwab and Tyers)

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

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

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/