In order to build a multi-cellular organism and to regulate cellular functions, cells need to communicate with each other, as well as tightly regulate their behaviour in response to environmental changes. For these purposes all eukaryotic cells express a large number of membrane spanning receptors that either themselves contain catalytic activity or via cytoplasmic effector enzymes, function to transmit “signals” from the cell exterior to induce appropriate responses within the cell. Protein tyrosine kinases (PTKs) are important signalling molecules, represented by the transmembrane receptor tyrosine kinases (RTKs) in addition to the cytoplasmic non-receptor PTKs, which alter cell behaviour by phosphorylating target proteins. An additional requirement for proper signalling and multicellular organisation is the adhesion between cells as well as adhesion of cells to the extracellular matrix (ECM). Adhesion between cells and the ECM is mainly mediated by the integrin family of cell surface receptors, which functions as a structural link between the ECM and the actin cytoskeleton as well as important centres for signalling. Mammalian studies have implicated the cytoplasmic Focal Adhesion Kinase (FAK), as a major transmitter of signalling emanating from integrins, regulating cell migration, survival, proliferation and differentiation. In our studies of the sole FAK family member in Drosophila, Fak56, we have concluded that the deletion of Fak56 from the fruit fly genome causes no obvious defects in integrin-mediated adhesion, migration or signalling in vivo. Consequently, in contrast to the embryonic lethality observed in mouse knockouts, Fak56 mutant flies are both viable and fertile. However, we do find a clear genetic interaction between Fak56 and Drosophila integrins. Additionally, overexpression studies indeed indicate Fak56 as a negative regulator of integrin adhesion, given that excess Fak56 protein phenocopies loss of integrin function, causing phenotypes such as muscle detachment and wing blistering. In Drosophila, as well as in mammals, FAK family proteins are highly abundant in the CNS and in our studies we have identified a requirement of Fak56 in synaptic transmission at neuromuscular junctions. Lack of Fak56 causes a weakening of action potential conduction, resulting in sensitivity to high-frequency mechanical and electrical stimulation, manifested by epileptic-like seizures and paralysis in Fak56 mutants, a phenotype known as Bang Sensitivity (BS) in flies. We also show that Fak56 phosphorylation is directly modulated in response to alterations in intracellular calcium levels, supporting a role for Fak56 in neurotransmission. Fak56 is directly activated by the Drosophila Anaplastic Lymphoma Kinase, DAlk, receptor which was identified in our lab. We characterised DAlk as a novel RTK that is expressed in the embryonic CNS and mesoderm where it drives activation of the ERK/MAPK pathway. Indeed, we found DAlk to ectopically induce protein tyrosine phosphorylation and specifically phosphorylation of ERK, resulting in autonomous cell transformation and uncontrolled tissue growth. Subsequently, we identified a requirement for DAlk function during Drosophila embryogenesis, where it displays an essential role in gut development. Specifically, we identified the secreted molecule Jelly belly (Jeb) as a ligand for DAlk and showed that Jeb-DAlk interaction activates an ERK-mediated signalling pathway essential for visceral muscle specification and fusion, and consequently formation of the gut. The potent ability of PTKs to regulate cell behaviour, together with the strong linkage between RTK dysregulation and tumour formation, renders the negative regulation of kinase activity an important area of research. We have identified the Drosophila homologue of Cbl-interacting protein of 85kDa, dCIN85, an adaptor molecule which in mammalian cells has shown involvement in RTK endocytosis and downregulation, as well as in the regulation of actin cytoskeleton dynamics. In the fruit fly, dCIN85 displays essential functions, given that dCIN85 loss of function mutants display a grand-child less phenotype. Generation of a dCIN85 antibody, together with isoform-specific transgenic flies, have allowed us to observe a punctuate localization pattern of the SH3-domain containing dCIN85 variants, representing Rab5-positive endosomal structures. This, in addition to the confirmation of a direct dCIN85-dCbl interaction, indicates an evolutionary conservation of dCIN85 function. Interestingly, dCIN85 co-localises with dRICH1, a Cdc42 specific RhoGAP, in differentiated photoreceptor cells in eye imaginal discs. This may imply a role for dCIN85 in the regulation of the specialised endocytic recycling processes required for the assembly/maintenance of tight junctions and establishment of cell polarity in epithelial tissues.

Thesis (Ph. D.)--University of Chicago, Faculty of the Division of the Biological Sciences and the Pritzker School of Medicine, Commitee on Immunology, June 2001. / Includes bibliographical references. Also available on the Internet.

Thesis (Ph. D.)--West Virginia University, 2001. / Title from document title page. Document formatted into pages; contains vi, 195 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references (p. 182-190).

Hirschsprung’s disease (HSCR) is the major enteric nervous system anomaly affecting newborns with high incidence in Asians. HSCR is a congenital complex genetic disorder characterized by a lack of enteric ganglia along a variable length of the intestine. The receptor tyrosine kinase gene (RET) is the major HSCR gene and cis-elements in the promoter and intron of RET gene are crucial for RET expression. Abnormal RET expression leading to insufficient RET activity causes defective development of the enteric nervous system and is implicated in the pathogenesis of the Hirschsprung’s disease. The human homeobox B5, HOXB5, has an important role in the development of enteric neural crest cells, and perturbation of HOXB5 signaling causes reduced RET expression and HSCR phenotypes in mice. To investigate the roles of HOXB5 in the regulation of RET expression and in the aetiology of HSCR, I sought to(i) elucidate the underlying mechanisms that HOXB5 mediates RET expression, and (ii) to examine the interactions between HOXB5 and other transcription factors including SOX10 and NKX2-1 that have been implicated in RET expression and HSCR. In this study, I demonstrated that HOXB5 binds to the RET promoter and regulates RET expression. HOXB5 and NKX2-1 forma protein complex and mediate RET expression in a synergistic manner. In contrast, HOXB5 cooperates in an additive manner with SOX10in trans-activation from RET promoter. ChIP assay further revealed that HOXB5 and NKX2-1 interact with the same chromatin region proximate to the transcription start site of RET, suggesting that these two factors may interact with each other and regulate the transcription of RET. In silico analysis, EMSA and ChIP analysis showed that HOXB5 also binds to an enhancer element (MCS+9.7)in the intron 1 of RET gene, and HSCR-associated SNPs have been identified in this enhancer element. To further access the HOXB5 trans-activity onMCS+9.7, RET mini-gene was constructed by ligating the RET promoter to the 5’and MCS+9.7 to the 3’of a luciferase gene. Luciferase assay indicated that MCS+9.7 enhances the HOXB5 trans-activation from the RET promoter. In addition, previously identified HSCR-associated SNPs inintron 1 markedly reduce the HOXB5 trans-activation from the RET mini-gene. Moreover, the result of IP-LC-MS/MS indicated that HOXB5 could form protein-protein complexes with nuclear proteins involved in the transcription initiation of genes with TATA-less promoter. This evidence suggested that HOXB5 may cooperate with other activators or co-factors in the remodeling of chromatin conformation, local histone modification and recruitment of essential transcription factors for RNA Polymerase II based transcription from TATA-less promoter, such as RET. My data indicated that HOXB5 in coordination with other transcription factors mediates RET expression. Therefore, defects in cis-or trans-regulation of RET by HOXB5 could lead to a reduction of RET expression and contribute to the manifestation of the HSCR phenotype. / published_or_final_version / Surgery / Doctoral / Doctor of Philosophy

High-risk human papillomaviruses (HPVs) are associated with nearly all cases of cervical cancer and also contribute to other types of anogenital and oropharyngeal cancers. The high-risk HPV E6 oncoprotein contributes to malignant progression in part by the targeted degradation of the tumor suppressor p53. The activation of growth factor and nutrient sensing pathways including receptor protein tyrosine kinases (RPTKs) and mTORC1 may also support cellular transformation. Moreover, previous studies suggested that HPV16 E6 activates mTORC1. We are particularly interested in understanding the mechanisms by which HPV E6 activates mTORC1 and the function of mTORC1 activation in HPV infection. Here we show that high-risk HPV16 E6 activates mTORC1 signaling and increases cap dependent translation through an increase in S6K signaling and an increase in 4E-BP1 phosphorylation. Mechanistically we found that HPV16 E6 activates AKT under conditions of nutrient deprivation. The combined approach of phospho-tyrosine immunoprecipitations and Western blot identified HPV16 E6 mediated activation of a subset of receptor protein tyrosine kinases. HPV16 E6 activates RPTKs at least in part by increasing the internalization of phosphorylated and activated receptor species. The signaling adaptor protein Grb2 associates with HPV16 E6, and Grb2 knockdown abrogated HPV16 E6 mediated mTORC1 activation. We hypothesize that Grb2 may be important in relaying E6 mediated RPTK activation to downstream signaling cascades. In this dissertation we also evaluate mTORC1 signaling and cap dependent translation in cells expressing HPV16 E6 mutants and E6 proteins from other HPV types. Binding to p53 and the association with proteins that contain an LXXLL motif are important for HPV16 E6 mediated mTORC1 activation. An increase in mTORC1 activation and cap dependent translation is shared between high-and low-risk mucosal, but not cutaneous HPV E6 proteins. Association with proteins through their LXXLL binding motif is also important for low-risk mucosal HPV E6 activation of mTORC1 and cap dependent translation. Shared mucosal E6 activation of mTORC1 indicates that mTORC1 may be important for the viral lifecycle in mucosal epithelia. However, it does not rule out the possibility that together with other properties of high-risk HPV E6 proteins, mTORC1 activation may promote transformation.

published_or_final_version / abstract / Paediatrics and Adolescent Medicine / Master / Master of Philosophy

Dimorphic Leishmania donovani parasites exist as promastigotes in the sandfly vector and differentiate into amastigotes once injected into the skin of human hosts during a blood meal. The mechanisms and signals that are involved in triggering differentiation are not well understood in Leishmania. We have investigated whether tyrosine phosphorylation is a possible signalling component. Differential levels of tyrosine-phosphorylated proteins were observed in extracts from in vitro promastigote and amastigote cultures, with an overall reduction in the latter stage. Following this observation, the inhibition of tyrosine phosphorylation was examined in promastigotes using Tyrphostin AG1433, a broad-spectrum tyrosine phosphorylation inhibitor. AG1433 treated in vitro promastigote cultures differentiate into amastigote-like morphology, have reduced tyrosine phosphorylation level, and express the amastigote-specific marker A2 proteins. Our studies demonstrate that signal transduction mechanisms involving tyrosine phosphorylation/dephosphorylation events are involved in controlling L. donovani promastigote differentiation into amastigote forms.

Alcohol affects many neurochemical processes, causing long-lasting changes in both the adult and developing brain. The Jak-Stat transcriptional activation pathway plays a role in the control of neuronal proliferation, survival and differentiation, but the effects of ethanol on the system have not been fully elucidated. The goal of this project was to define the effects of acute and subchronic ethanol exposure on the expression of proteins in the Jak-Stat pathway, using cultured NG108-15 cells, and in addition, to test the hypothesis that these effects are mediated through the NMDA receptor. I found that ethanol dose-dependently decreased Jak2 and Stat3 following subchronic exposure of NG108-15 in culture. Acute ethanol exposure caused a dose-dependent decrease in Stat3 protein levels. Incubation with MK-801 or ketamine, two noncompetitive NMDA receptor antagonists, or the receptor agonist NMDA, produced dose-dependent decreases in Stat3 protein as well.

The Epstein-Barr virus (EBV) protein, Latent Membrane Protein 2A (LMP2A), is critical for maintaining viral latency and provides pro-survival and pro-migratory signals to EBV-positive B and epithelial cell malignancies. The N-terminus of LMP2A contains several protein-protein interaction motifs involved in the recruitment of cellular signalling proteins and it is through the recruitment of these proteins that LMP2A is able to initiate signalling. In B lymphocytes, LMP2A's ability to initiate signalling was originally proposed to proceed via a two step mechanism. Firstly, recruitment of the Lyn tyrosine kinase to the tyrosine phosphorylated YEEA site in LMP2A allows for tyrosine phosphorylation of the LMP2A immunoreceptor tyrosine-based activation motif (ITAM). This, in turn, facilitates the recruitment and activation of Syk tyrosine kinase which then initiates downstream signalling events. However, recent findings suggest this model may not be correct and argue that Syk recruitment to LMP2A is independent of the YEEA site. Therefore, we undertook a series of experiments to better understand the role of the YEEA motif and Lyn in the initiation of LMP2A signalling in B lymphocytes. We found that the YEEA site was not absolutely required for tyrosine phosphorylation of the LMP2A ITAM, or for LMP2A to activate Syk. Using siRNA to silence Lyn expression in LCLs. we found that reducing Lyn expression inhibited the ability of LMP2A to promote Syk tyrosine phosphorylation. In contrast, DG75 B cells or Lyn-deficient DT40 B cells transiently expressing higher levels of LMP2A did not require Lyn for LMP2A-mediated Syk phosphorylation. Furthermore, Lyn was not required for LMP2A-mediated Akt activation in DG75 B cells, but rather Akt activation was significantly enhanced in LMP2A-expressing cells where Lyn was reduced by siRNA. We propose that Lyn negatively regulates LMP2A-mediated Akt activation by phosphorylating Syk on Y323, which serves to recruit the c-Cbl E3 ubiquitin ligase to Syk and targets Syk for ubiquitin-mediated degradation. In sum, this work provides novel insight into how LMP2A uses Lyn to initiate and titre signalling in B cells and brings to light an unappreciated role for Lyn as a negative regulator of LMP2A-mediated Akt activation.

A yeast-two hybrid screen with Lyn identified a novel 130 kDa multidomain protein with a 36% identity to Actin Filament Associated Protein (AFAP) 110 and similar domains, including PH domains, potential sites of tyrosine and serine/threonine phosphorylation, a leucine-zipper domain, a potential actin binding site and multimerization site. AFAP110 has been shown to have a role in modulating actin filament integrity and induce lamellipodia formation, and is known to interact with Src family kinases. The aim of this thesis was to characterize this novel protein named Lyn-Associated Cytoskeletal Modulator (LACM) and determine any molecular interactions in order to attempt to elucidate a role for the protein in cell signaling through Lyn. LACM is encoded by a gene consisting of 18 exons and is located on human chromosome 5q33.1 and mouse chromosome 18 E1. LACM protein is expressed through a number of cell types including the R11 erythroid cell line, and mouse tissues including brain, lung, heart and embryos. LACM was shown to multimerize, and subcellular localization of the protein was observed to concentrate around the cell membrane at sites of filamentous actin in filopodia, lamellipodia and stress fibres. The carboxy-terminus of LACM was observed to localize the protein to sites at the cell membrane and through the cytoplasm. Removal of this terminal region resulted in all LACM protein localizing to the nucleus in punctuate spots. LACM protein was observed in heart muscle and potentially has a role at sites of nerve junctions on cardiac myocytes. LACM was shown to interact with the SH3 domain of Lyn at a polyproline motif on LACM. LACM was observed to co-localize and co-immunoprecipitate with Lyn and was tyrosine phosphorylated by the kinase domain of Lyn. Interestingly, the consititutively active Lyn and LACM caused transfected cells to