Phosphate is an essential compound for life. Escherichia coli employs a signal transduction pathway that controls the expression of genes that are required for the high-affinity acquisition of phosphate and the utilization of alternate sources of phosphorous. These genes are only expressed when environmental phosphate is limiting. The seven genes for this signaling pathway encode the two-component regulatory proteins PhoB and PhoR, as well as the high-affinity phosphate transporter PstSCAB and an auxiliary protein called PhoU. As the sensor kinase PhoR has no periplasmic sensory domain, the mechanism by which these cells sense environmental phosphate is not known. This paper explores the hypothesis that it is the alternating conformations of the PstSCAB transporter which are formed as part of the normal phosphate transport cycle that signal phosphate sufficiency or phosphate limitation. We tested two variants of PstB that are predicted to lock the protein in either of two conformations for their signaling output. We observed that the pstBQ160K mutant, predicted to reside in an inward facing, open conformation signaled phosphate sufficiency whereas the pstBE179Q mutant, predicted to reside in an outward facing, closed conformation signaled phosphate starvation. Neither mutant showed phosphate transport.

Two-component systems (TCSs) are highly conserved across bacteria and are used to rapidly sense and respond to changing environmental conditions. The human pathogen Staphylococcus aureus uses the S. aureus exoprotein expression (sae) TCS to sense host signals and activate transcription of virulence factors essential to pathogenesis. Despite its importance, the mechanism by which the sae sensor kinase SaeS recognizes specific host stimuli is unknown. This thesis describes topology and mutagenesis studies of the sensing domain of SaeS, including basal expression and inducer-dependent phenotypes. Meanwhile, investigation of the sae auxiliary protein SaeP has identified a novel DNA binding function for this surface expressed lipoprotein that may be involved in fine-tuning the activity of the sae system. Overall, these structure-function studies provide insight into the sae signal transduction mechanism and raise some new questions regarding the role the sae system plays in the larger regulatory network S. aureus uses to control expression of its secreted virulence factors.

During embryonic development, cells respond to extracellular signals to establish proper tissue organization. Semaphorins (Semas) are a large class of secreted and transmembrane proteins that signal through Plexin (Plxn) receptors to guide migrating cells to their correct position and thus play critical roles in the development of various tissues including the nervous and cardiovascular systems. We have previously shown that Sema6A-PlxnA2 signaling is essential for visual system development, as decreasing endogenous Sema6A or PlxnA2 in zebrafish results in decreased cohesion of the early eye field, impaired retinal lamination, and smaller eye size. However, the molecular mechanisms governing these phenotypes are unknown. This dissertation describes the elucidation of functionally-relevant mechanisms of Sema6A-PlxnA signaling during eye development using biochemical and proteomic approaches in cell culture systems and the zebrafish as an in vivo vertebrate model of eye development. We first describe our investigations on the receptor-proximal mechanisms of Sema6A-PlxnA signaling. The Src-family tyrosine kinase Fyn was known to bind to and phosphorylate PlxnA receptors. However, the specific sites of phosphorylation and their function were unknown. Using mass spectrometry, we identified highly-conserved, Fyn-induced PlxnA tyrosine phosphorylation sites. Mutation of these tyrosines to phenylalanine nearly eliminated Fyn-dependent PlxnA phosphorylation. Furthermore, unlike mRNA encoding wild type human PlxnA2, mRNA encoding the tyrosine-to-phenylalanine mutant PlxnA2 could not rescue the smaller eye size phenotype caused by endogenous PlxnA2 knockdown in zebrafish. This suggests that Fyn-dependent PlxnA2 phosphorylation is critical for proper vertebrate eye development. Next, we report the discovery and functional characterization of a naturally-released soluble ectodomain of Sema6A (sSema6A). We show that sSema6A production is increased by PKC activity. The identification of several PKC-dependent phosphorylation sites in the intracellular region of Sema6A suggests a mechanism for PKC-dependent release of sSema6A. Importantly, we show that sSema6A is functional as it promotes the cohesion of zebrafish early eye field explants. This is the first report of a soluble ectodomain of the Sema6 class and suggests that Sema6A can have regulated, long-range signaling capacity in addition to its canonical contact-mediated functions. Finally, we present our findings characterizing the role in eye development of CRMP2, a downstream effector of Sema-Plxn signaling. CRMP2 is known to be critical for lamination of the cerebral cortex, leading us to hypothesize that CRMP2 could also be involved in the lamination of the retina. Using morpholino-based knockdown of endogenous zebrafish Crmp2, we show that Crmp2 has a critical function in visual system development. Crmp2 knockdown results in smaller eye size, impaired retinal lamination and a weakened optic tract. Together, this dissertation describes important novel Sema6A-PlxnA signaling mechanisms and places them in the context of vertebrate eye development.

CD40 is a TNF receptor superfamily (TNFRSF) member central to the development of many aspects of the adaptive immune response. CD40 signaling promotes adaptive immunity in part by inducing the expression of cytokines, chemokines, and various adhesion and co-stimulatory molecules. The family of cytoplasmic adapter proteins, the TNFR-associated factors (TRAFs), serve as major mediators of TNFRSF pathways. CD40 regulates itself in part via the signaling induced degradation of TRAF2 and TRAF3. However, the effect of CD40-induced TRAF degradation on other TRAF dependent pathways is unknown. Here I provide evidence that CD40-mediated degradation of TRAFs 2 and 3 also influences the responsiveness of immune cells to CD40-independent, TRAF2- and 3-dependent pathways. LMP1 is a functional mimic of CD40, but signals to B lymphocytes in an amplified and sustained manner. LMP1 contributes to the development of B cell lymphoma in immunosuppressed patients, and may exacerbate flares of certain autoimmune diseases. The cytoplasmic (CY) domain of LMP1 binds TRAF2 with lower avidity than the CY domain of CD40, and TRAF2 is needed for CD40-mediated degradation of TRAFs 2 and 3. LMP1 doesn't induce TRAF degradation, and employs TRAF3 as a positive mediator of cell signaling, whereas CD40 signals are inhibited by TRAF3. Here, I tested the hypothesis that relative affinity for TRAF2, and/or distinct sequence differences in the TRAF2/3 binding sites of CD40 vs. LMP1, controls the disparate ways in which CD40 and LMP1 use TRAFs 2 and 3. The results revealed that TRAF binding affinity and TRAF binding site sequence dictate a distinct subset of CD40 vs. LMP1 signaling properties. The E3 ubiquitin ligases, cIAP1 and cIAP2, have been reported to play a crucial role in CD40 signaling. Because LMP1 is a mimic of CD40 signals, I hypothesized that LMP1 requires the cIAPs for signaling. To elucidate the role of the cIAPs in CD40 and LMP1 signaling, I specifically depleted the cIAPs and found that the cIAPs are differentially utilized in CD40 and LMP1 signaling. I also sought to further the understanding of the molecular underpinnings of how CD40, but not LMP1 signaling induces TRAF2 and TRAF3 degradation upon signaling. To do this, I investigated the ability of various CD40 and LMP1 mutants to induce TRAF degradation in distinct TRAF or cIAP deficient models. I found that neither a high TRAF2 binding potential nor the presence of the cIAP molecules are required for this process. Thus, this work reveals important insights into the molecular mechanisms of and role of CD40-mediated TRAF degradation in the immune system.

In addition to contributing genes at fertilization, the sperm cell induces the oocyte to leave its arrested state and resume metabolism in the process of activation. A hallmark of oocyte activation is a release of intracellular calcium (Ca2+i) from the endoplasmic reticulum. The mediators of oocyte activation have been studied in many animal models, while little is known in the bovine model. Both Src Family Kinase (SFK) and Phospholipase C (PLC) enzymes have been reported to be involved in oocyte activation in other animal models. In this dissertation are described experiments that define the role of SFK and PLC enzymes in the pathway leading to Ca2+i and calcium induced calcium release in bovine oocyte activation. Western blotting was used to discover that SFKs Src, Hck, and Lck are present in matured bovine oocytes, and Src, Blk, and Yes are present in acrosome reacted bovine spermatozoa. The PLC δ1 and δ3 are present in both matured bovine oocytes and spermatozoa. PLC δ4, γ2, and η2 are present in matured bovine oocytes. Microinjecting a known general SFK inhibitor, PP2, significantly decreases both Ca2+i and cleavage rates. Microinjecting a 13 amino acid peptide that mimics the phosphorylated carboxyl terminal region of pp60c-src decreases both Ca2+i and cleavage rates. Microinjecting a downstream substrate of pp60c-src sequestered any signal produced by Src and decreased Ca2+i and cleavage rates. Microinjecting primary antibodies raised against PLC isotypes blocked both Ca2+i and cleavage rates, giving insight to the mechanism of calcium induced calcium release in the bovine model. The PLC isotypes δ3, δ4, and γ2 decreased Ca2+i oscillations and cleavage rates, indicating they are involved in both IP3R and RyR activation. PLC δ4 and η2 did not impact Ca2+i but did significantly decrease cleavage rates. The data presented in this dissertation increase the understanding of the pathway leading to bovine oocyte activation and further confirm that the detailed pathway differs among animal models.

The aryl hydrocarbon receptor (AHR) is a basic helix-loop-helix PER/ARNT/SIM (bHLH-PAS) transcription factor that binds ligands typified by 2,3,7,8-tetracholordibenzo-p-dioxin, translocates to the nucleus, dimerizes with the aryl hydrocarbon nuclear translocator (ARNT) and associates with specific cis xenobiotic response elements to activate transcription of genes involved with xenobiotic metabolism. AHR-mediated signal transduction has been evaluated thoroughly in the C57BL/6J mouse model system. This model system, however, may not be the most accurate model for human comparisons as the AHRb-1 allele carried by C57BL/6J contains a point mutation that prematurely truncates the receptor at 805 amino acids, while the AHRb-2, rat, and human AHR all contain an additional 42-45 amino acids at their carboxy-terminus that have 70% identity. This carboxy-terminal region could be functionally significant and the analysis of AHR-mediated signal transduction in the rat, human, or other mouse strains may better represent the physiology of the AHR pathway. ARNT is another member of the bHLH-PAS family of proteins that is essential in several distinct signal transduction pathways mediated by its dimerization with a variety of bHLH-PAS proteins. Several isoforms of ARNT have been identified in mammalian and aquatic species. While ARNT and ARNT2 exhibit >90% amino acid identity in the bHLH and PAS domains, gene knock-out of either ARNT or ARNT2 results in embryonic/perinatal lethality characterized by distinct phenotypes. This suggests that neither protein can compensate fully for the loss of the other. Since overlapping tissue specific expression of ARNT and ARNT2 does exist, but neither ARNT can compensate fully for loss of the other, this suggests that the two proteins have distinct functions in the presence of various dimerization partners. Thus, the focus of these studies is to examine the discrepancies between the rat, human, or AHRb-2 possessing the extended carboxy-terminal region and that of the AHRb-1 and also to examine the role of both ARNT and ARNT2 during AHR-mediated signal transduction.

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Abstract not available