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APC/C
Cyc B
CDK1
Cyc B
WEE1/MIT1
APC/C
WEE1/MIT1
M
CDK1
Cyc D1
Cyc A
CDK4
p16INK4A, INK4 proteins
CDK2
G2
G1
Cyc D1
CDK4
Cyc A
WEE1/MIT1
p16INK4A, INK4 proteins
CDK2
S
Cyc A
CDK2
Cyc E
Cyc E
CDK2
Cyc D1
SCFFbx4
CDK4
p21CIP1
p27KIP1
p57KIP2
WEE1/MIT1
CDK2
p21CIP1
p27KIP1
p57KIP2
WEE1/MIT1
SCFFbw7
Figure 1.1. Temporal CDK activity controls cell cycle progression. Mitogen-dependent
expression of D-type cyclins facilitates activation of G1-phase CDK4/6. The mammalian family of
INK4 proteins specifically inhibit CDK4/6 activity by direct binding to the CDK, antagonizing CDK
activity. Nuclear cyclin D/CDK4/6 kinase activity drives cell cycle progression beyond the restriction
point, thereby committing the cell to one round of division. Following the G1/S transition, nuclear
CDK4/6 activity is terminated by ubiquitin mediated proteolysis of cyclin D1 by the SCFFbx4 E3
ubiquitin ligase. Cyclin E expression in late G1 facilitates CDK2 activation, and association with the
CIP/KIP family of CDK inhibitors inhibits its kinase activity. CDC25-dependent removal of inhibitory
threonine/tyrosine phosphorylation on CDK2 is also essential for activation. Cyclin E/CDK2 activity
drives E2F-dependent gene transcription, replication origin firing and S-phase progression, and this
activity is attenuated in early-S phase by SCFFbw7-mediated ubiquitylation and degradation of cyclin
E. Cyclin A expression also activates CDK2 in S-phase, facilitating DNA replication and inhibiting
origin re-licensing. Cyclin A/CDK2 activity persists through G2 phase, functioning in centrosome
duplication. Cyclin B is the sole mitotic cyclin, as cyclin B/CDK1 activity is essential for both
centrosome duplication and mitotic processes. Both cyclin A and cyclin B are substrates of the
G2/M phase E3 ligase, the anaphase promoting complex/cyclosome (APC/C).
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B.
A.
G1
G1/S
CycD1
CycD1
CDK4
Cyc E
CDK4
CDK2
PPP
Rb
CycD1
Late G1
p21 CDK4
p27
GSK3
S12 P
P
Fbx4 dimerization
Ligase activation
E2F
E2F Target
Genes
E2F DP
Nucleus
Cytoplasm
D.
C.
G1/S
S
Cyc E
GSK3
CDK2
T286 P
Cdc45
Cdc6 Cdt1
ORC
MCM 2-7
MCM
Licensed Replication Origin
2-7
CycD1
CDK4
Ub
K48-Ub
Nucleus
Cytoplasm
26S Proteasome
Figure 1.2. Cell cycle-dependent cyclin D1 regulation. (A) Mitogen-dependent cyclin D1
expression is required for CDK4/6 activation during G1 phase. Active, nuclear cyclin D1/CDK4
kinase promotes cell cycle progression in two ways. First, cyclin D1/CDK4 catalyzes Rb
phosphorylation, thereby triggering release of E2F transcription factors. Second, cyclin D1/CDK4
complexes titrate the CKIs p21CIP1 and p27KIP1 away from cyclin E/CDK2, facilitating CDK2
activation, full Rb inactivation, and gene transcription required for S-phase entry. (B) In late G1
phase, AKT-dependent inhibitory phosphorylation of GSK3β is alleviated, allowing GSK3β kinase
activation. In the cytoplasm, GSK3β phosphorylates Fbx4 on serine 12, creating a consensus 14-33ε docking site. 14-3-3ε binding promotes Fbx4 dimerization and ligase activation. (C) Cyclin
E/CDK2 activity facilitates entry into S-phase and DNA replication. Following the G1/S transition,
nuclear cyclin D1/CDK4 kinase activity is no longer required. At this time, GSK3β enters the
nucleus and phosphorylates cyclin D1 on T286, which triggers CRM1-mediated nuclear export. (D)
Once in the cytoplasm, phosphorylated cyclin D1 is recognized by the SCFFbx4-αB crystallin E3 ubiquitin
ligase. Cyclin D1 is polyubiquitylated and targeted for degradation by the 26S proteasome.
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A.
SCF E3 Ligases
Ub
Substrate
B.
SCF-like E3 Ligases
Ub
Ub
Substrate
C.
S-phase
CDT1
ORC
PCNA
Ub
P
MCM 2-7
MCM 2Replication
7 Origin
Ub
26S Proteasome
Figure 1.3. SCF and SCF-like E3 ubiquitin ligases. (A) Prototypical SKP-CULLIN-F-BOX (SCF)
complex composition. The core cullin scaffold (CUL1) associates with the RING-finger protein
RBX1 which is required for E2 recruitment and transfer of charged ubiquitin to the substrate. SKP1
also associates directly with CUL1 and facilitates substrate recruitment by binding the F-box
domain within an F-box protein, an adapter for substrate recognition. Interaction of substrates with
the SCF permits transfer of ubiquitin onto the substrate molecule by the E2 enzyme. (B) SCF-like
E3 ligases contain RBX1, but harbor different cullin scaffold proteins and substrate adapters. The
CUL7-based E3 ligase binds SKP1 and the FBW8 F-box protein, specifically, thereby functioning
much like the canonical SCF. The CUL4-based ligase utilizes a different set of substrate adapters,
including DDB1 and a DCAF protein that coordinates substrate recognition. (C) The CUL4DDB1/CDT2
E3 ligase maintains DNA replication fidelity. Following origin firing in S-phase, cyclin A/CDK2dependent phosphorylation of pre-RC components prevents origin re-licensing. Importantly,
chromatin-bound CDT1 is recognized by the CDT2 WD-repeat DCAF adapter, which is targeted to
chromatin through its PCNA interacting PIP box domain. CDT1 is subsequently polyubiquitylated
by the CUL4 E3 ubiquitin ligase and targeted for proteasomal degradation.
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Endometrial
Cancer
Esophageal,
Endometrial
Cancer
Breast Cancer
GSK3β
P T286
CycD1
Ub
Nuclear Export
CDK4
Nucleus
Cytoplasm
26S Proteasome
Esophageal
Cancer
14-3-3ε
S12 P
GSK3β
P
Dimerization
Active Ligase
Figure 1.4. Cyclin D1 regulatory pathways are targeted in human cancer. Cyclin D1 protein
accumulation is tightly controlled via phosphorylation-dependent proteolysis, and mutations targeting
cyclin D1 phosphorylation or degradation contribute to neoplastic transformation. Specific disruption
of T286 phosphorylation occurs in endometrial cancer, and mutations targeting Fbx4 have been
indentified in esophageal cancer. Furthermore, αB crystallin loss occurs in tumor-derived breast
carcinoma cell lines. Ultimately, impaired cyclin D1 proteolysis promotes accumulation of active
cyclin D1/CDK4 kinase, triggering DNA re-replication and subsequent genomic instability necessary
for neoplastic transformation.
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Protein
Targeted
Mutation
Consequence
Tumor Type
Cyclin D1
T286R
Constitutively Nuclear
Esophageal
Cyclin D1
Δ266-295
Constitutively Nuclear
Cyclin D1
P287A
Constitutively Nuclear
Esophageal
Tumor-derived esophageal carcinoma cell lines TE3, TE7, and
TE12
Cyclin D1
P287S/T
Constitutively Nuclear
Endometrial
Cyclin D1
Δ289-292
Constitutively Nuclear
Endometrial
αB crystallin Δ chr. 11
Tumor-derived breast cancer cell lines (MCF-7, MDA-MB 231)
Fbx4
S8R
Impaired ligase activity
Esophageal
Fbx4
S12L
Disrupts phosphorylation
“
Fbx4
P13S
Disrupts phosphorylation
“
Fbx4
L23Q
Dimerization-deficient
“
Fbx4
G30N
“
Fbx4
P76T
“
Table 1. Summary of mutations targeting cyclin D1 phosphorylation or Ub-ligase function.
Mutations disrupting GSK3β-dependent cyclin D1 T286 phosphorylation and nuclear export include
mutation of T286, P287, and deletion of residues corresponding to the CRM1 binding site. Mutations
targeting the SCFFbx4-αB crystallin E3 ubiquitin ligase result in impaired ligase activity toward cyclin D1 and
subsequent cyclin D1/CDK4 accumulation in the nucleus.
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A.
IR
Replication Fork Collapse
Intra-S Checkpoint
P
ATM
CHK2
CDC25
p53
p21
H2AX
CDK 2, CDK1
Signal Amplification, DNA Repair
DSB
UV Light
Nucleotide Depletion
Replication Stress
Rad9/Rad1/Hus1
Rad17
DNA Replication Checkpoint
P
ATR
ATRIP
CHK1
CDC25
p53
p21
CDK 2, CDK1
Stalled DNA
Replication
B.
DNA
damage
PhosphorylationIndependent
G1
PhosphorylationDependent
ATM
G1
P
APC/C
CycD1
CDK4
Ub-mediated proteolysis (APC/C)
G1 arrest
PhosphorylationDependent
CycD1
S-phase
P ERK
GSK3
P
CycD1
P
GSK3
CDK4
CDK4
Ub-mediated proteolysis (SCF)
G1 arrest
Ub-mediated proteolysis (SCF)
Accelerated Proteolysis
Intra S-Checkpoint
Figure 1.5. DNA damage checkpoint responses prevent genomic instability. (A) S-phase DNA
damage checkpoint activation. The intra-S-phase checkpoint response is mediated by ATM activation
following DSB induction in genomic regions outside of active DNA replication. Genotoxic insults such
as IR trigger DSBs and subsequent ATM activation. DSBs also result when stalled replication forks
collapse, leading to ATM activation. ATM auto-phosphorylation catalyzes its monomerization from an
inactive dimeric conformation, thereby facilitating downstream effector phosphorylation events
necessary for cell cycle arrest and DNA repair. Replication stress, on the other hand, triggers ATR
activation following accumulation of single-strand DNA (ssDNA). ssDNA is rapidly coated with
replication protein A (RPA), which promotes recruitment of the ATR kinase and its cofactor ATRIP.
Molecular adapters TopBP1 and claspin facilitate ATR-dependent Chk1 activation. Additional ATR
substrates are required for cell cycle arrest and DNA repair, as in the ATM-dependent DSB
response. (B) Cyclin D1 regulation following DNA damage. Previous reports established regulatory
mechanisms for cyclin D1 destruction following G1-phase DSB induction. Left panel: proposed
phosphorylation-independent cyclin D1 destruction by APC/C. Middle panel: proposed ERK-mediated
cyclin D1 phosphorylation and subsequent recognition by the SCFFbxo31 ubiquitin ligase. Right panel:
proposed mechanism for SCFFbx4-mediated cyclin D1 destruction following S-phase DNA damage.
This model is tested and confirmed in chapter 2 of this thesis.
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