Thesis advisor: Laura Hake / The completion of meiosis in Xenopus oocytes requires the coordinated translation of stored mRNAs. CPEB, the cytoplasmic polyadenylation element binding protein, controls the translation of developmentally important early-class maternal mRNAs. Resumption of meiosis through stimulation with progesterone leads to the phosphorylation and activation of CPEB. This results in the lengthening of the poly(A) tails and translation of mRNAs containing the cytoplasmic polyadenylation element (CPE). XGef, a putative guanine nucleotide exchange factor, binds to and is required for CPEB activation. Translation of c-mos, a MAPK kinase kinase, is controlled by CPEB, and activation of the Mos/MAPK pathway is required for meiotic maturation. In addition, the synthesis of Ringo protein, an atypical cdk binding protein and activator, is required for progesterone-induced maturation, though Ringo is able to stimulate resumption of meiosis independent of progesterone. Although much work has been done to understand the key events leading to activation of maturation promoting factor (MPF) and meiotic maturation, the events immediately following progesterone stimulation remain unclear, particularly regarding the role of XGef. The work that follows describes experiments performed to further understand the role of XGef in meiotic maturation through both Ringo and MAPK activity. It was found that XGef and Ringo interact directly and form a complex throughout early meiosis. XGef is involved in Ringo’s influence during meiosis, specifically through MEK-activation of MAPK. Notably, XGef functions in a common pathway and complex with Ringo most likely to influence CPEB phosphorylation and activation. / Thesis (BS) — Boston College, 2009. / Submitted to: Boston College. College of Arts and Sciences. / Discipline: College Honors Program. / Discipline: Biology.

The ERK-MAPK pathway is a dynamic signaling module, conserved across Eukarya, and capable of processing a myriad of environmental and cellular signals. It has been implicated in controlling important cell fate decisions in many cell types and species. In mES cells, growth factor activation of the ERK-MAPK pathway is involved in the earliest stages of lineage segregation, however very little is currently known about the mechanism by which this is accomplished. Taking a loss-of-function gene targeting approach I have reexamined the relative contribution of ERK2 activity to FGF-ERK signaling. Although ERK2 depletion results in an attenuation of the combined ERK1/2 activity, this is compensated for by the hyperactivation of the remaining ERK1 isozyme. Normal ERK1/2 function can be restored to ERK2 deficient cells by transgenic expression of either ERK1 or ERK2, indicating a degree of functional redundancy between both isoforms. When subjected to the appropriate cues, lineage commitment proceeded normally in ERK2 deficient cells, however increased self-renewal was observed under standard culture conditions. Several attempts were made to further probe ERK1/2 function by siRNA depletion, and dominant negative inhibition of ERK1 in Erk2 knockout cells, however both approaches failed to provide further insight. Furthermore, taking a candidate approach, the role of Srf, a canonical target of ERK1/2 signaling, was examined. Initial experiments indicated a role for SRK in neural differentiation, however due to issues of culture adaptation and instability in several cell lines it was not possible to conclude this line of research within the time frame of this thesis. IP-MS/MS analysis identified several proteins known to interact with ERK2 and indicated an involvement in nuclear pore function through TPR as well as transcriptional and translational regulation through RSK proteins. Moreover, this study identified DUSP6 and DUSP9 as the primary induced dual specificity phosphatases that regulate ERK2 activity in mES cells. To further probe the functional significance of the ERK:p90RSK interaction I examined a mES cell line genetically depleted for PDK1, a crucial regulator of p90RSK function. This cell line exhibits no detectable p90RSK activity, however in contrast to studies in other cell lines, p90RSK activity is dispensable for mitogen-induced cFos expression in mES cells. Subsequent experiments demonstrated a requirement for PDK1 activity in either the specification or maintenance of mES cell derived neurons. Further analysis indicated that p90RSK may be involved in a negative feedback loop regulating ERK1/2 activity, and if so may represent a point whereby ERK1/2 activity can be manipulated. To examine this I determined the effect pharmacological inhibition of p90RSK has on ERK1/2 activity and self-renewal using a novel p90Rsk inhibitor, BI-D1870. Although treatment with BI-D1870 correlated with enhanced ERK1/2 phosphorylation, the offtarget effects this molecule exhibits made it impossible to draw any firm conclusions from these experiments. Overall this study has demonstrated a degree of redundancy between ERK1/2 isozymes in mES cells. It has highlighted the complex nature of ERK1/2 regulation as well as the robustness of this pathway to perturbations in ERK dose. Furthermore, it has underscored some of the common pitfalls encountered when studying differentiation phenotypes in mES cells. Although this study failed to highlight anything more than a coincidental relationship between ERK1/2 activity and self-renewal capacity of mES cells, it has helped to highlight some important behavioral characteristics of the FGF-MAPK pathway in mES cells and provide a platform for further study.

Candida albicans is the most prevalent human fungal pathogen. The research described in this thesis has focused on the identification and characterization of the regulatory pathways in this pathogen controlling white-opaque switching, mating and biofilm formation as well as the relationship between them. White-opaque switching and mating in C. albicans are under the repression of the a1-α2 complex. Based on this, a chromatin immunoprecipitation-microarray analysis of the a1-α2 target genes was conducted to search for the master switch locus. The result identified TOS9 (WOR1) as a master regulator gene, and overexpression of TOS9 resulted in a switch en masse from white to opaque. In 2006, a novel form of communication was demonstrated between white and opaque cells in C. albicans. It was shown that minority opaque cells through the release of pheromone signaled majority white cells of the opposite mating type to become cohesive, adhesive and form enhanced biofilms. These biofilms in turn facilitated opaque cell chemotropism required for opaque cell mating. To identify the pathway regulating the white cell pheromone response, deletion mutants were generated for select genes mediating the opaque cell mating response. It was demonstrated that the pathways regulating the white and opaque cell responses to the same pheromone share the same upstream components, including receptors, heterotrimeric G protein, and mitogen-activated protein kinase cascade, but they use different downstream transcription factors that regulate the expression of genes specific to the alternative responses. This configuration, although found in higher, multicellular systems, is uncommon in fungi and suggests that it may be an antecedent to multicellularity in higher eukaryotes. In addition, it was found that a C. albicans-specific 55-amino-acid region of the first intracellular loop, IC1, of the α-pheromone receptor, is required for the α-pheromone response of white cells, but not that of opaque cells. Finally, to test the generality of the white cell pheromone response, evidence was presented that the response occurs in all tested media and in all of the 27 tested strains, including a/a and α/α strains, derivatives of the common laboratory strain SC5314, and representatives from all of the five major clades. The white cell response to pheromone, therefore, proved to be a general characteristic of MTL-homozygous strains of C. albicans.

B virus (Macacine herpesvirus 1), subfamily Alphaherpesvirinae, causes a fatal, neurovirulent infection in zoonotically infected humans. Macaques (Macaca sp.) serve as the natural host for B virus and they are frequently seropositive for B virus antibodies without showing any overt signs of disease. The global hypothesis of these studies is that B virus, a highly cytopathic virus in macaques, subverts the innate immune responses in the host (macaques) that has co-evolved with it (the virus) differently than it does the foreign host (humans). The foreign host, frequently fails to produce neutralizing antibodies early after infection and this may be due to a dysregulation or inhibition of pathways known to play a role in the innate immune response which directs the adaptive defense responses. Current knowledge is that at least five major signaling pathways can be activated after a pathogen such as B virus enters a host cell (REF). These include the IRF3 pathway, the NFkB pathway, the NFAT pathway, and the MAPK pathway. Early stimulation of one or more of these pathways leads to the induction of the proinflammatory response and subsequent induction of cytokines such as IL6, IL8 and IL10, and apoptosis. Cytokine induction and apoptosis play important roles in host-pathogen interactions, innate defense induction and subsequent adaptive immune responses. Using a primary cell model that is representative of the first target cells of B virus in the natural and foreign host, we investigated one of the key signaling pathways, the MAPK pathway, induced by B virus early after infection (Farah-Abraham and Hilliard, unpublished data). My data suggest that macaque and human cells differ in the induction kinetics of MAPK (JNK and p38) activation. These data reveal differences between foreign and natural host cells in how each controls apoptosis, and demonstrate that inhibition of p38 activation reduced and with high dose inhibition terminated B virus replication in human cells, and played a role in reduction of apoptosis-associated mediators. The importance of each component in the MAPK pathway is investigated with respect to virus replication in macaque and human cells that represent the primary target cells in acute infection. Knowledge of these events provides an understanding of how the innate immune responses can be modulated by B virus to shape the adaptive immune response to limit how the virus replicates and spreads. Further, these data may provide insight into a novel target for the design of new antivirals to inhibit this deadly zoonotic virus. This research will help us understand how the early molecular mechanisms of host-pathogen interactions result in modulation of the innate immune responses and how certain aspects of a normally defensive (protective) host response can be re-directed or modified depending on the nature of the virus:host relationship.

Monoamine oxidase-A (MAO-A) is an enzyme that has historically been linked to major depressive disorder (MDD). The prevalence of MDD among breast cancer patients is almost 25%, but realistically it is underdiagnosed within this patient population. Most breast cancer is deemed estrogen receptor positive [ER(+)] and is commonly treated with the anti-estrogenic chemotherapeutic compound tamoxifen. Resistance to tamoxifen has been associated with a paradoxical activation of the stress-associated kinase, p38(MAPK) (normally associated with cell death). Our research group has recently demonstrated that p38(MAPK) can regulate the function of MAO-A in glial cells. Taken together, MAO-A, depression and p38(MAPK) are all associated with a poor prognosis in breast cancer patients, particularly those with an ER(+) status. Several mechanisms have been proposed in each respect and we hope to further elucidate this relationship by focussing on the interaction between MAO-A and p38(MAPK) in the context of breast cancer. The hypothesis states that a functional interaction between the p38(MAPK) and MAO-A systems alters breast cancer cells in an ER-dependent manner. The proposed objectives of this project are to determine what might be influencing MAO-A function in breast cancer cells, and how changes in MAO-A function affect cell phenotype. Using pharmacological approaches (i.e. antidepressant drugs), we investigated the role of MAO-A and p38(MAPK) on selected characteristics of ER(+) (e.g. MCF-7) and ER(-) (e.g. MDA-MB-231) breast cancer cells under four treatment conditions, which include clorgyline (CLG), an antidepressant MAO-A inhibitor, and SB203580, an inhibitor of p38(MAPK). Our results indicate that the very high MAO-A activity in MDA-MB-231 (MB-231) cells was partly dependent on p38(MAPK) activity. The tumourigenic properties (e.g. anchorage-independent growth, migration) of MB-231 cells depended on both MAO-A and p38(MAPK) functions, although the effects were not additive suggesting that both inhibitors were exerting their respective effects via common signalling targets. The role of MAO-A and p38(MAPK) on MB-231 mitochondrial function and cell growth was negligible. In contrast, MAO-A and p38(MAPK) only influenced mitochondrial function in MCF-7 cells and did not affect any of the other tumourigenic properties measured. Immunocytochemical methods, supported by Western blotting, revealed an increase in E-cadherin expression in both cell lines. This suggested that MAO-A and p38(MAPK) could be influencing transitions between epithelial and mesenchymal phenotypes. Our in vitro findings suggest that MAO-A and p38(MAPK) might contribute to a common mechanism in breast cancer cell lines, but that their influence on cell phenotype is less dependent on the respective cell's ER status and perhaps more so dependent on the cell's metastatic potential. If this is the case, then the contribution of MAO-A and p38(MAPK) to [clinical] metastatic breast cancer should be duly considered. Our ongoing investigations are focussing on the influence of clinically relevant antidepressants on breast cancer cell phenotype in vitro.

This project focused on determining the effect of lithium on glucose uptake, glycogen synthesis, and insulin signaling proteins, protein kinase B (Akt1) and GSK-3, in isolated soleus muscle from female lean Zucker rats. We also investigated the role of the stress-activated p38 MAPK in the action of lithium to activate skeletal muscle glucose transport. In the absence of insulin, lithium (10 mM LiCl) increased basal glucose transport by 62% (p<0.05) and glycogen synthesis by 112%. Lithium did not alter phosphorylation of Akt ser473, but enhanced GSK-3β ser9 phosphorylation by 41%. Lithium further enhanced the effect of insulin on glucose transport (42%), glycogen synthesis (44%), and GSK-3ß phosphorylation (13%). Lithium increased phosphorylated p38 MAPK 31% without and 19% with insulin. Moreover, a selective p38 MAPK inhibitor, A304000, completely prevented the lithium-induced enhancement of glucose transport revealing the critical involvement of p38 MAPK phosphorylation in lithium-induced glucose transport in isolated skeletal muscle.

The ancient South American birds called tinamous (Tinamidae) have the smallest hearts known among birds and their cardiomyocytes have previously been shown to express significantly lower levels of the mitogen-activated protein kinase ERK compared to the more modern chicken (Gallus gallus). ERK is a well-known mediator of growth signalling in the heart, especially in hypertrophy. The aim of this project was to assess the effect of ERK knockdown on proliferation in cultured chicken cardiomyocytes. By transfecting these cells with a lipoplexed siRNA, ERK mRNA levels were knocked down to approximately half (45%, SD: 27%) compared to cells transfected with a negative control siRNA. The knockdown was coupled with a decreased proliferative response to insulin-like growth factor 1 (IGF-1) and foetal bovine serum (FBS). In conclusion, the ERK pathway was confirmed to be instrumental also in proliferative signalling. The results also support the notion that ERK itself is the rate-limiting step of this MAPK cascade. The low native expression of ERK in tinamou cardiomyocytes is expected to impose a strict limit on proliferative growth in response to various stimuli in these hearts. The genetic changes leading to higher expression levels, and with it the potential for larger hearts, in modern birds would have led to greatly increased evolutionary fitness by way of an increased aerobic scope and the ability to sustain flight.

Tumorigenesis is driven by the sequential accumulation of genetic lesions within a cell, each which confer the cell with traits that enable its abnormal growth. The result is a mass of dysregulated cells, or tumor, which, upon further mutation, may spread, or metastasize, to other organs of the body. The dissemination of tumor cells makes treatment difficult, and thus confers cancer with its associated lethality. Over the past 30 years, the RAS genes have been critical in teaching us the mechanisms underlying the molecular progression of cancer. RAS is mutated in 33% of all cancers and is often an early event in its stepwise progression. As a result, the RAS genes are widely accepted as ‘drivers’ or ‘initiators’ of human tumorigenesis. Unfortunately, efforts directed at targeting RAS in the clinic have as of yet been unsuccessful. This has triggered a need to identify genes that are required for RAS tumorigenesis that are therapeutically tractable. My research has focused on deciphering the potential role of the Rho GEF Arhgef2 in RAS-mediated tumorigenesis. I have found that Arhgef2 is a bona fide transcriptional target of RAS and is upregulated in human tumors harboring RAS mutations. Importantly, depletion of Arhgef2 in RAS-mutated cells inhibits their survival, proliferation, and tumor growth in murine models. In search of the mechanism underlying the requirement of Arhgef2 in RAS tumorigenesis, I have uncovered a novel function for Arhgef2 as a positive regulator of a central RAS pathway, the mitogen-activated protein kinase (MAPK) pathway. Thus, Arhgef2 is part of a positive feedback loop in which RAS-dependent increases in Arhgef2 expression results in the amplification of RAS signaling. Moreover, Arhgef2 confers tumor cells with properties favoring their malignant conversion, thereby implicating Arhgef2 in the formation of metastases. Together, these studies suggest that Arhgef2 plays an important role at multiple stages of tumorigenic progression and may therefore be a promising therapeutic target in RAS-mutated tumors.

Tumorigenesis is driven by the sequential accumulation of genetic lesions within a cell, each which confer the cell with traits that enable its abnormal growth. The result is a mass of dysregulated cells, or tumor, which, upon further mutation, may spread, or metastasize, to other organs of the body. The dissemination of tumor cells makes treatment difficult, and thus confers cancer with its associated lethality. Over the past 30 years, the RAS genes have been critical in teaching us the mechanisms underlying the molecular progression of cancer. RAS is mutated in 33% of all cancers and is often an early event in its stepwise progression. As a result, the RAS genes are widely accepted as ‘drivers’ or ‘initiators’ of human tumorigenesis. Unfortunately, efforts directed at targeting RAS in the clinic have as of yet been unsuccessful. This has triggered a need to identify genes that are required for RAS tumorigenesis that are therapeutically tractable. My research has focused on deciphering the potential role of the Rho GEF Arhgef2 in RAS-mediated tumorigenesis. I have found that Arhgef2 is a bona fide transcriptional target of RAS and is upregulated in human tumors harboring RAS mutations. Importantly, depletion of Arhgef2 in RAS-mutated cells inhibits their survival, proliferation, and tumor growth in murine models. In search of the mechanism underlying the requirement of Arhgef2 in RAS tumorigenesis, I have uncovered a novel function for Arhgef2 as a positive regulator of a central RAS pathway, the mitogen-activated protein kinase (MAPK) pathway. Thus, Arhgef2 is part of a positive feedback loop in which RAS-dependent increases in Arhgef2 expression results in the amplification of RAS signaling. Moreover, Arhgef2 confers tumor cells with properties favoring their malignant conversion, thereby implicating Arhgef2 in the formation of metastases. Together, these studies suggest that Arhgef2 plays an important role at multiple stages of tumorigenic progression and may therefore be a promising therapeutic target in RAS-mutated tumors.

Mammalian cells can initiate intracellular signalling pathways that activate pro-survival changes to maintain their integrity following their exposure to a range of extracellular stresses. One group of changes preserves cellular integrity through the regulation of cytoskeletal organization. Despite the recognised importance of maintaining microtubule (MT) networks, the specific mechanisms regulating cytoskeleton organisation in response to stress remain relatively poorly explored. Among the numerous proteins that regulate MT organisation, stathmin (STMN) is a key MT destabilising protein that regulates MT disassembly through its ability to bind tubulin dimers. The actions of STMN can be regulated by a number of growth factor-activated and cell cycle regulatory protein kinases. In preliminary work, our studies suggest the potential regulation of STMN by c-Jun N-terminal Kinase (JNK) in cells exposed to stress. Specifically, we observed changes in STMN phosphorylation which were coordinated with JNK activation. / This project has explored the contribution of stress-activated c-Jun N-terminal Kinase (JNK) to STMN phosphorylation observed during osmotic stress. More detailed in vitro biochemical analysis has revealed that JNK directly phosphorylates STMN. In addition, we have compared STMN phosphorylation by different MAPK family member. In particular, our results illustrated that JNK predominantly phosphorylate STMN on serine residue 38 (S38) whereas ERK most likely targeted STMN S25. By examining specifically the phosphorylation of the four regulatory serine residues in vitro, we proposed a model of hierarchical phosphorylation among STMN serine residues. Specifically, our results demonstrated that phosphorylation of S38 was a pre-requisite for S25 phosphorylation by JNK in vitro. Furthermore, our results also demonstrated the impacts of JNK binding domain (JBD) and tubulin on STMN phosphorylation in vitro. Overall, this project identified STMN as a novel JNK substrate. The results have broadened our understanding on the JNK-mediated STMN phosphorylation as the first step to provide deeper insights into the different functions of JNK in the mammalian stress response.