Doctor of Philosophy

dissertationThe tumor suppressor APC is mutated in 85% of colon cancer cases. APC is necessary for normal colon homeostasis, and loss of APC function is the initiating event in colon cancer. APC is a crutial member of the β -catenin destruction complex, and it was long thought APC mutation causes disruption of this complex, removing the cell's ability to destroy excess β-catenin and activating β-catenin/Tcf/Lef-induced transcription. Recent studies, however, show that nuclear localization of β-catenin occurs only after the activation of k-ras, not during the initiation of tumorigenesis. Thus, APC must play additional roles that are disrupted when mutated to cause changes from normal colon homeostasis. In this dissertation, we utilize the apcmcr mutant zebrafish as a model to parallel the loss of APC in colon cancer to show that multiple epigenetic aberrations occur before the advent of nuclear β-catenin, showing that dysregulation of epigenetic marks correlates with the initiation of colon cancer progression. The data presented in this dissertation identify two epigenetic factors regulated by APC. We show that the histone demethylase LSD1 aberrantly represses retinoid biosynthesis, preventing terminal differentiation of the intestine. This highlights one of the epigenetic regulators that is misregulated when APC is lost and adds an epigenetic mechanism for the repression of rdh1l. We also show that in the apcmcr mutant zebrafish, retinoic acid loss causes decreased H3K27 methylation levels, adding another layer of epigenetic regulation controlled through RA. In this case, we uncover an epistatic mechanism wherein Cox-2 regulates H3K27 methylation, independent of its role in the regulation of β-catenin, when APC is mutated. We also used the developing zebrafish as a model to characterize the loss of the Ezh2, the histone methyltransferase responsible for H3K27 methylation. We show that loss of Ezh2 leads to several development phenotypes, including the loss of terminal differentiation of multiple cell types in the gut. Together, the data presented in this dissertation trace the epigenetic pathways used to both regulate retinoic acid production and used by retinoic acid to promote cellular differentiation and establish APC as a regulator of histone methylation during differentiation

Specificity for deubiquitination of monoubiquitinated FANCD2 is driven by the N-terminus of USP1

The Fanconi anemia pathway for DNA interstrand crosslink repair and the translesion synthesis pathway for DNA damage tolerance both require cycles of monoubiquitination and deubiquitination. The ubiquitin-specific protease-1 (USP1), in complex with USP1-associated factor 1, regulates multiple DNA repair pathways by deubiquitinating monoubiquitinated Fanconi anemia group D2 protein (FANCD2), Fanconi anemia group I protein (FANCI), and proliferating cell nuclear antigen (PCNA). Loss of USP1 activity gives rise to chromosomal instability. Whereas many USPs hydrolyse ubiquitin–ubiquitin linkages, USP1 targets ubiquitin–substrate conjugates at specific sites. The molecular basis of USP1's specificity for multiple substrates is poorly understood. Here, we reconstitute deubiquitination of purified monoubiquitinated FANCD2, FANCI, and PCNA and show that molecular determinants for substrate deubiquitination by USP1 reside within the highly conserved and extended N-terminus. We found that the N-terminus of USP1 harbours a FANCD2-specific binding sequence required for deubiquitination of K561 on FANCD2. In contrast, the N-terminus is not required for direct PCNA or FANCI deubiquitination. Furthermore, we show that the N-terminus of USP1 is sufficient to engineer specificity in a more promiscuous USP

Characterization of FANCL variants observed in patient cancer cells

Fanconi Anemia (FA) is a rare genetic disorder characterized by developmental defects, bone marrow failure and high predisposition to cancer. The FA DNA repair pathway is required in humans to coordinate repair of DNA interstrand crosslinks. The central event in the activation of the pathway is the monoubiquitination of FANCD2 and FANCI by the E2-E3 pair, Ube2T-FANCL, with the central UBC-RWD (URD) domain of FANCL recognising the substrates. Whole genome sequencing studies of cancer cells from patients identified point mutations in the FANCL URD domain. We analysed 17 such variants of FANCL, including known substrate binding mutants (W212A, W214A and L248A, F252A, L254A, I265A), a FA mutation (R221C) and 14 cancer-associated mutations (F110S, I136V, L149V, L154S, A192G, E215Q, E217K, R221W, T224K, M247V, F252L, N270K, V287G, E289Q) through recombinant expression analysis, thermal shift assay, interaction with FANCD2, in vitro ubiquitination activity, and cellular sensitivity to an interstrand crosslinking agent. We find that the FANCL mutations I136V, L154S, W212A and L214A, R221W, R221C, and V287G are destabilising, with N270K and E289Q destabilising the C-terminal helices of the URD domain. The hydrophobic patch mutant (L248A, F252A, L254A, I265A), along with mutations E217K, T224K, and M247V, cause defects in the catalytic function of FANCL. This highlights the C-terminal lobe of the FANCL URD domain as important for the activity and function of FANCL. These mutations which affect the fold and activity of FANCL may contribute to tumorigenesis in these non-FA cancer patients, and this implicates FA genes in general cancer progression

Identification of the Amino Acids 300–600 of IRS-2 as 14-3-3 Binding Region with the Importance of IGF-1/Insulin-Regulated Phosphorylation of Ser-573

Phosphorylation of insulin receptor substrate (IRS)-2 on tyrosine residues is a key event in IGF-1/insulin signaling and leads to activation of the PI 3-kinase and the Ras/MAPK pathway. Furthermore, phosphorylated serine/threonine residues on IRS-2 can induce 14-3-3 binding. In this study we searched IRS-2 for novel phosphorylation sites and investigated the interaction between IRS-2 and 14-3-3. Mass spectrometry identified a total of 24 serine/threonine residues on IRS-2 with 12 sites unique for IRS-2 while the other residues are conserved in IRS-1 and IRS-2. IGF-1 stimulation led to increased binding of 14-3-3 to IRS-2 in transfected HEK293 cells and this binding was prevented by inhibition of the PI 3-kinase pathway and an Akt/PKB inhibitor. Insulin-stimulated interaction between endogenous IRS-2 and 14-3-3 was observed in rat hepatoma cells and in mice liver after an acute insulin stimulus and refeeding. Using different IRS-2 fragments enabled localization of the IGF-1-dependent 14-3-3 binding region spanning amino acids 300–600. The 24 identified residues on IRS-2 included several 14-3-3 binding candidates in the region 300–600. Single alanine mutants of these candidates led to the identification of serine 573 as 14-3-3 binding site. A phospho-site specific antibody was generated to further characterize serine 573. IGF-1-dependent phosphorylation of serine 573 was reduced by inhibition of PI 3-kinase and Akt/PKB. A negative role of this phosphorylation site was implicated by the alanine mutant of serine 573 which led to enhanced phosphorylation of Akt/PKB in an IGF-1 time course experiment. To conclude, our data suggest a physiologically relevant role for IGF-1/insulin-dependent 14-3-3 binding to IRS-2 involving serine 573

Differential roles and regulation of the protein kinases PAK4, PAK5 and PAK6 in melanoma cells

The protein kinases PAK4, PAK5 and PAK6 comprise a family of ohnologues. In multiple cancers including melanomas PAK5 most frequently carries non-synonymous mutations; PAK6 and PAK4 have fewer; and PAK4 is often amplified. To help interpret these genomic data, initially we compared the cellular regulation of the sister kinases and their roles in melanoma cells. In common with many ohnologue protein kinases, PAK4, PAK5 and PAK6 each have two 14-3-3-binding phosphosites of which phosphoSer99 is conserved. PAK4 localises to the leading edge of cells in response to phorbol ester-stimulated binding of 14-3-3 to phosphoSer99 and phosphoSer181, which are phosphorylated by two different PKCs or PKDs. These phosphorylations of PAK4 are essential for its phorbol ester-stimulated phosphorylation of downstream substrates. In contrast, 14-3-3 interacts with PAK5 in response to phorbol ester-stimulated phosphorylation of Ser99 and epidermal growth factor-stimulated phosphorylation of Ser288; whereas PAK6 docks onto 14-3-3 and is prevented from localising to cell–cell junctions when Ser133 is phosphorylated in response to cAMP-elevating agents via PKA and insulin-like growth factor 1 via PKB/Akt. Silencing of PAK4 impairs viability, migration and invasive behaviour of melanoma cells carrying BRAF(V600E) or NRAS(Q61K) mutations. These defects are rescued by ectopic expression of PAK4, more so by a 14-3-3-binding deficient PAK4, and barely by PAK5 or PAK6. Together these genomic, biochemical and cellular data suggest that the oncogenic properties of PAK4 are regulated by PKC–PKD signalling in melanoma, while PAK5 and PAK6 are dispensable in this cancer

Karyomegalic interstitial nephritis and DNA damage-induced polyploidy in fan1 nuclease-defective knock-in mice

The Fan1 endonuclease is required for repair of DNA interstrand cross-links (ICLs). Mutations in human Fan1 cause karyomegalic interstitial nephritis (KIN), but it is unclear whether defective ICL repair is responsible or whether Fan1 nuclease activity is relevant. We show that Fan1 nuclease-defective (Fan1nd/nd) mice develop a mild form of KIN. The karyomegalic nuclei from Fan1nd/nd kidneys are polyploid, and fibroblasts from Fan1nd/nd mice become polyploid upon ICL induction, suggesting that defective ICL repair causes karyomegaly. Thus, Fan1 nuclease activity promotes ICL repair in a manner that controls ploidy, a role that we show is not shared by the Fanconi anemia pathway or the Slx4–Slx1 nuclease also involved in ICL repair

Fasting and systemic insulin signaling regulate phosphorylation of brain proteins that modulate cell morphology and link to neurological disorders

Diabetes is strongly associated with cognitive decline, but the molecular reasons are unknown. We found that fasting and peripheral insulin promote phosphorylation and dephosphorylation, respectively, of specific residues on brain proteins including cytoskeletal regulators such as slit-robo GTPase-activating protein 3 (srGAP3) and microtubule affinity-regulating protein kinases (MARKs), in which deficiency or dysregulation is linked to neurological disorders. Fasting activates protein kinase A (PKA) but not PKB/Akt signaling in the brain, and PKA can phosphorylate the purified srGAP3. The phosphorylation of srGAP3 and MARKs were increased when PKA signaling was activated in primary neurons. Knockdown of PKA decreased the phosphorylation of srGAP3. Furthermore, WAVE1, a protein kinase A-anchoring protein, formed a complex with srGAP3 and PKA in the brain of fasted mice to facilitate the phosphorylation of srGAP3 by PKA. Although brain cells have insulin receptors, our findings are inconsistent with the down-regulation of phosphorylation of target proteins being mediated by insulin signaling within the brain. Rather, our findings infer that systemic insulin, through a yet unknown mechanism, inhibits PKA or protein kinase(s) with similar specificity and/or activates an unknown phosphatase in the brain. Ser(858) of srGAP3 was identified as a key regulatory residue in which phosphorylation by PKA enhanced the GAP activity of srGAP3 toward its substrate, Rac1, in cells, thereby inhibiting the action of this GTPase in cytoskeletal regulation. Our findings reveal novel mechanisms linking peripheral insulin sensitivity with cytoskeletal remodeling in neurons, which may help to explain the association of diabetes with neurological disorders such as Alzheimer disease

Pim kinases phosphorylate multiple sites on Bad and promote 14-3-3 binding and dissociation from Bcl-X(L)

BACKGROUND: Pim-1, 2 and 3 are a group of enzymes related to the calcium calmodulin family of protein kinases. Over-expression of Pim-1 and Pim-2 in mice promotes the development of lymphomas, and up-regulation of Pim expression has been observed in several human cancers. RESULTS: Here we show that the pim kinases are constitutively active when expressed in HEK-293 cells and are able to phosphorylate the Bcl-2 family member Bad on three residues, Ser112, Ser136 and Ser155 in vitro and in cells. In vitro mapping showed that Pim-2 predominantly phosphorylated Ser112, while Pim-1 phosphorylated Ser112, but also Ser136 and Ser155 at a reduced rate compared to Ser112. Pim-3 was found to be the least specific for Ser112, and the most effective at phosphorylating Ser136 and Ser155. Pim-3 was also able to phosphorylate other sites in Bad in vitro, including Ser170, another potential in vivo site. Mutation of Ser136 to alanine prevented the phosphorylation of Ser112 and Ser155 by Pim kinases in HEK-293 cells, suggesting that this site must be phosphorylated first in order to make the other sites accessible. Pim phosphorylation of Bad was also found to promote the 14-3-3 binding of Bad and block its association with Bcl-X(L). CONCLUSION: All three Pim kinase family members predominantly phosphorylate Bad on Ser112 and in addition are capable of phosphorylating Bad on multiple sites associated with the inhibition of the pro-apoptotic function of Bad in HEK-293 cells. This would be consistent with the proposed function of Pim kinases in promoting cell proliferation and preventing cell death

Effects of microstructure and growth conditions on quantum emitters in gallium nitride

Single-photon emitters in gallium nitride (GaN) are gaining interest as attractive quantum systems due to the well-established techniques for growth and nanofabrication of the host material, as well as its remarkable chemical stability and optoelectronic properties. We investigate the nature of such single- photon emitters in GaN with a systematic analysis of various samples produced under different growth conditions. We explore the effect that intrinsic structural defects (dislocations and stacking faults), doping and crystal orientation in GaN have on the formation of quantum emitters. We investigate the relationship between the position of the emitters—determined via spectroscopy and photoluminescence measurements—and the location of threading dislocations—characterised both via atomic force microscopy and cathodoluminescence. We find that quantum emitters do not correlate with stacking faults or dislocations; instead, they are more likely to originate from point defects or impurities whose density is modulated by the local extended defect density