1. 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 SCF Fbx4 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 SCF Fbw7 -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). 24 CDK2 Cyc E M S G1G2 CDK4 Cyc D1 CDK2 Cyc E CDK2 Cyc A CDK1 Cyc B p21 CIP1 p27 KIP1 p57 KIP2 WEE1/MIT1 SCF Fbx4 p21 CIP1 p27 KIP1 p57 KIP2 WEE1/MIT1 CDK2 Cyc A WEE1/MIT1 SCF Fbw7 APC/C CDK1 Cyc B APC/C CDK4 Cyc D1 p16 INK4A, INK4 proteins CDK2 Cyc A WEE1/MIT1 CDK4 Cyc D1 p16 INK4A, INK4 proteins

2. 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 p21 CIP1 and p27 KIP1 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 14-3- 3ε 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 SCF Fbx4-αB crystallin E3 ubiquitin ligase. Cyclin D1 is polyubiquitylated and targeted for degradation by the 26S proteasome. 25 Cul1 E1 Rbx1 Skp1 E2 Fbx4 BB 26S Proteasome K48-Ub Ub CDK4 CycD1 P A. G1G1 G 1 /S Nucleus Cytoplasm Rb E2F p21 p27 CDK2 Cyc E CDK4 CycD1 PPP CDK4 CycD1 DP E2F CDK4 CycD1 E2F Target Genes B. Fbx4 P GSK3  Late G1 Fbx4 P 14-3-3ε S12 Fbx4 dimerization Ligase activation GSK3  C. Nucleus Cytoplasm CDK2 Cyc E G 1 /SS MCM 2-7 Licensed Replication Origin Cdc45 ORC Cdc6 Cdt1 MCM 2-7 CDK4 CycD1 P T286 D.

3. 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 CUL4 DDB1/CDT2 E3 ligase maintains DNA replication fidelity. Following origin firing in S-phase, cyclin A/CDK2- dependent 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. 26 A. CUL1 RBX1 SKP1 E2 F-box Substrate Ub SCF E3 Ligases SCF-like E3 Ligases CUL4 E2 Ub RBX1 DDB1 Substrate DCAF B. CUL7 RBX1 SKP1 E2 FBW8 Substrate Ub C. 26S Proteasome MCM 2- 7 Replication Origin MCM 2-7 S-phase P CUL4 E2 Ub RBX1 DDB1 CDT2 CDT1 Ub CDT1 ORC PCNA

4. 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. 27 Cytoplasm 26S Proteasome Breast Cancer Nucleus GSK3β T286 Esophageal, Endometrial Cancer Nuclear Export Endometrial Cancer GSK3β Dimerization Active Ligase Esophageal Cancer DNA Re-Replication Genomic Instability Neoplastic Transformation CDK4 CycD1 P Fbx4 P P 14-3-3ε S12 CUL1 RBX1 SKP1 E2 F-box Ub CDK4 CycD1 P αBαBαBαB

5. 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 SCF Fbx4-αB crystallin E3 ubiquitin ligase result in impaired ligase activity toward cyclin D1 and subsequent cyclin D1/CDK4 accumulation in the nucleus. 28 Protein TargetedMutationConsequenceTumor Type Cyclin D1T286RConstitutively NuclearEsophageal Cyclin D1Δ266-295Constitutively NuclearEsophageal Cyclin D1P287AConstitutively Nuclear Tumor-derived esophageal carcinoma cell lines TE3, TE7, and TE12 Cyclin D1P287S/TConstitutively NuclearEndometrial Cyclin D1Δ289-292Constitutively NuclearEndometrial αB crystallinΔ chr. 11Tumor-derived breast cancer cell lines (MCF-7, MDA-MB 231) Fbx4S8RImpaired ligase activityEsophageal Fbx4S12LDisrupts phosphorylation “ Fbx4P13SDisrupts phosphorylation “ Fbx4L23QDimerization-deficient “ Fbx4G30N “ Fbx4P76T “

6. 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 SCF Fbxo31 ubiquitin ligase. Right panel: proposed mechanism for SCF Fbx4 -mediated cyclin D1 destruction following S-phase DNA damage. This model is tested and confirmed in chapter 2 of this thesis. 29 A. ERK GSK3  Fbx4 P G1G1 DNA damage APC/C CDK4 CycD1 G1G1 CDK4 CycD1 Fbx031 P ATM Phosphorylation- Independent Phosphorylation- Dependent P G1 arrest Ub-mediated proteolysis (APC/C) G1 arrest Ub-mediated proteolysis (SCF) S-phase CDK4 CycD1 Phosphorylation- Dependent P Accelerated Proteolysis Ub-mediated proteolysis (SCF) Intra S-Checkpoint GSK3  B.  IR Replication Fork Collapse DSB ATM P CHK2CDC25CDK 2, CDK1 p53 p21  H2AX Signal Amplification, DNA Repair Stalled DNA Replication UV Light Nucleotide Depletion Replication Stress RPA CHK1CDC25CDK 2, CDK1 p53 p21 ATRIP ATR P Intra-S Checkpoint DNA Replication Checkpoint Rad17 Claspin Rad9/Rad1/Hus1 TopBP1