Control of neurosecretion of neurotransmitters appears to be regulated through second messengers that change the phosphorylation state of critical enzymes and proteins. The effect of protein phosphorylation promoting agents on secretion, desensitization and Na$\sp+$/Ca$\sp{2+}$ exchange activity were investigated and protein phosphatases were identified in bovine chromaffin cells. Cells exposed to 8-Br-cAMP, PDBu or okadaic acid alone show slightly decreased rates of desensitization. Okadaic acid plus 8-Br-cAMP potentiated secretion with repeated stimulations. The protein kinase inhibitor H7 increased the desensitization rate. These phenomena are observed during secretion evoked with elevated K$\sp+$ as well as by a nicotinic agonist. Thus, the effect of phosphorylation is at a post-receptor site. Cells treated with dbcAMP, PDBu, okadaic acid or calyculin A show lowered Na$\sp+$/Ca$\sp{2+}$ exchange activity and prolong cytosolic Ca$\sp{2+}$ transients caused by depolarization. Conversely, H7 enhances Na$\sp+$/Ca$\sp{2+}$ exchange activity. Na$\sp+$/Ca$\sp{2+}$ exchange activity in isolated membrane vesicles is inhibited by PKA and PKC. The results indicate that reversible protein phosphorylation modulates Na$\sp+$/Ca$\sp{2+}$ exchange activity and suggest that modulation of the exchanger may play a role in the regulation of secretion. Four distinct peaks of phosphatase activity were observed in homogenized bovine adrenal medulla cells when fractionated using an HPLC ion exchange DEAE column. Of these phosphatases, peaks II, III and IV show preferential dephosphorylation of the $\alpha$ subunit of phosphorylase kinase relative to the $\beta$ subunit and therefore are classified as protein phosphatases type 2. These phosphatases have broad substrate specificity and distinctly different relative specific activities toward the different substrates. Phosphatase 2A is likely to be the major enzyme in bovine adrenal medulla cells. ATP induced a greater secretory response than did acetylcholine without causing preferential secretion of norepinephrine or epinephrine. These data show that the response to ATP found in cultured cells is not an artifact of cell culture, and that ATP corelease with catecholamines from the storage vesicles has a significant physiological role. Freshly isolated cells were separated on a density gradient; the lower density cells develop a much stronger response to ATP than do the higher density cells.

The epidermal growth factor receptor (EGFR) plays important roles in the control of many fundamental cellular processes including cell cycle, cell migration, cell metabolism and survival, cell proliferation and differentiation, as well as regulation of oocyte maturation and embryonic development. In the first part of this work I studied the EGFR expression and activation in cumulus cells (CCs). CCs are special cells immediately surrounding the oocyte. It has been shown that CCs in the isolated cumulus cell/oocyte complexes (COCs) exhibit both a slow rise in intracellular calcium concentration ([Ca 2+]i) and plasma membrane permeabilization in response to epidermal growth factor (EGF) stimulation. But cultured individual bovine CCs rarely showed a [Ca2+]i increase. The lack of response was confirmed to be due to a decrease of expression of endogenous EGFRs after dissociation. After CCs were reconstituted EGFR expression they showed robust, prolonged, EGF-stimulated [Ca2+] i elevations characteristic of CC responses in intact COCs followed by CC permeabilization and death. These responses were also confirmed being mediated by the IP3 signaling pathway. This EGFR activated Ca 2+ response in CCs followed by cell death may play an important role in the regulation of oocyte maturation. In the second part of this work, I identified an EGFR intramolecular regulation mechanism through study of the tyrosine phosphorylation in the EGFR regulatory domain (RD). EGFR signaling is partly controlled by tyrosine phosphorylation on the RD. There are 5 major tyrosine phosphorylation residues (992, 1068, 1086, 1148 and 1173) whose phosphorylation functions as the main platform for recruitment of downstream components. In order to understand the effect of intramolecular interactions among EGFR RD tyrosine residues, we constructed a series of single site mutant RDs. Each one replaces one major tyrosine phosphorylation residue with phenylalanine. After in vitro phosphorylation, the phosphorylation degree of each major tyrosine residue was quantitatively compared between mutant RDs and wild type RD using LC-ESI Ion Trap mass spectrometry. Our results indicate that Y1068 increases the phosphorylation of Y1148 and Y1173, Y1086 inhibits the phosphorylation of Y1068, Y1148 inhibits the phosphorylation of Y992 and Y1086, and Y1173 inhibits the phosphorylation of Y1068. Thus the EGFR exhibits extensively intramolecular interaction among its major tyrosine phosphorylation sites. Such intramolecular interaction may increase the complexity of EGFR signal transduction as well as modulate its efficacy.

Genetic, biochemical, and biophysical methods have been adopted to characterize the interactions between ligand and transmembrane receptor, between receptor and cytoplasmic proteins, and between cytoplasmic proteins in the bacterial chemosensory system in order to elucidate the signaling mechanism at the molecular level. Ligand binding to the serine receptor (Tsr) of Escherichia coli has been studied using Isothermal Titration Calorimetry (ITC) to determine the number of moles of ligand bound per mole of receptor (n), the binding constant $(K\sb{\rm a}),$ and the enthalpy of binding $(\Delta H)$ of serine to the receptor. The n-value for serine binding to octyl-$\beta$-D-glucopyranoside (OG)-solubilized Tsr (n = 0.5) was consistent with one molecule of serine binding to a receptor dimer. The values for $K\sb{\rm a}$ and $\Delta H$ were equivalent for the membrane and OG-solubilized samples and were found to be $\rm 3.6\times 10\sp4\ M\sp{-1}$ and $-18$ kcal/mol at 27$\sp\circ$C. Covalent modification of Tsr at the sites of methylation was found to have only a modest effect on serine binding. The interaction between Tsr and the methyltransferase (CheR) was also studied using ITC. Tsr bound to CheR with 1 to 1 ratio and a dissociation constant of 2 $\mu$M at 29$\sp\circ$C. Ligand-binding or modifications at the methylation sites of Tsr did not affect CheR binding. Truncation of Tar cytoplasmic-fragment at the C-terminus by 16 amino acids (aa) completely abolished CheR binding. This result led to the identification of the CheR binding site which is located at the C-terminal 5-aa segment remote from the methylation regions. The C-terminal truncated and crosslinkable Tsr mutant ($\Delta$34, D36C) was then constructed, which was found to be a poor substrate for CheR by itself. Coexpression of this binding site deleted Tsr with intact Tsr rescued its methylation activity even in the crosslinked form. The mechanism of interdimer methylation was established through this study. The autophosphorylating kinase CheA donates a phosphoryl group to either of two response regulator proteins, CheY or CheB. With ITC, it was demonstrated that CheA and a CheA fragment composed of aa residues 1 to 233 (CheA$\sb{1-233}$ bound to CheY with similar dissociation constants of 2.0 and 1.2 $\mu$M at 28$\sp\circ$C, respectively, indicating that the CheY binding site is wholly within the 1-233 aa locus. CheB bound to CheA$\sb{1-233}$ with a $K\sb{\rm d}$ of 3.2 $\mu$M, and also bound to intact CheA with the same affinity. CheY was found to compete with CheB for binding to CheA$\sb{1-233}.$ Phosphorylated CheY, in the presence of 6 mM Mg$\sp{2+},$ has a significantly lower affinity for CheA (20% of unphosphorylated form), but mutations at the active site of CheY have little effect on CheY-CheA interaction, a mutation remote from the active site (A103V) produced a 10-fold reduction in $K\sb{\rm a},$ indicating the separation of the binding site from the active site and a significant conformational change upon phosphorylation.

Fluorescent ratiometric imaging and spectrofluorometric analysis was used to study two signal transduction mechanisms in the stamen hair cells of Tradescantia virginiana. The first study determined the metabolic pathway necessary for the inactivation of Inositol 1,4,5-trisphosphate mediated calcium release from intracellular stores in the living plant cell. Tradescantia stamen hair cells, preloaded with the calcium sensitive ratiometric dye fura-2-dextran, were injected with analogs of Inositol 1,4,5-trisphosphate and cytosolic calcium levels monitored by ratiometric imaging. The injected analogs were selected due to their insensitivity to various kinases and phosphatases for which Inositol 1,4,5-trisphosphate is a substrate. We determined that the 5-phosphatase is the preferred pathway for inactivation of Inositol 1,4,5-trisphosphate in the living plant cell. The second study investigated cyclic ADP-ribose mediated calcium release in the intact plant cell and determined the presence of the metabolic machinery necessary to synthesize cyclic ADP-ribose from its precursor NAD+. Cyclic ADP-ribose was observed to cause calcium release in the stamen hair cells of Tradescantia that were preloaded with the calcium sensitive dye fura-2-dextran. Evidence of cyclic ADP-ribose synthesis was determined using two experimental techniques. Homogenates of the sea urchin Lytechnicus piclus were used as bioassays to detect cyclic ADP-ribose in extracts of Tradescantia stamen hair cells that were incubated with [special characters omitted]NAD+. Cyclic ADP-ribose synthesis was detected from fluorimetric analysis of the homogenate as the calcium sensitive dye fluo-3 was present in the homogenate to detect cyclic ADP-ribose mediated calcium release from sea urchin egg microsomes. We also determined cyclic ADP-ribose synthesis by injection of fura-2-dextran loaded stamen hair cells with [special characters omitted]NAD+ and observing a delayed calcium increase in the cytosol. These results establish the metabolic fate of inositol 1,4,5-trisphosphate in plant cells and demonstrate the biochemical capability for plant cells to synthesize cyclic ADP-ribose to mediate calcium release in plants.

This dissertation is in two parts. The first part investigates the role of lipid signalling pathways in an intracellular phenomenon i.e. the formation of the nuclear envelope. The second part is a study of lipid signalling pathways in extracellular phenomenon, i.e. HeLa cell adhesion and spreading on a gelatin substrate. The disassembly and formation of the nuclear envelope are crucial steps in the progression of mitosis. Nuclear envelope dynamics involve many steps and since vesicular transport, binding and fusion of vesicles are essential for the formation of the nuclear envelope it was our interest to explore whether there were any parallel pathways, such as found in the secretary pathways, that were also used in the formation of the nuclear envelope. There is little information on the proteins of membrane vesicles that reconstitute the nuclear envelope and practically none about their lipid composition. It was therefore important to determine their lipid structure in order to proceed with investigating whether, during the formation of the nuclear envelope, similar signalling pathways to those in the vesicle trafficking operate. Cytoplasmic membrane vesicle fractions (MVs) from sea urchin eggs which, contribute to the nuclear envelope were studied. The phospholipid composition of the membrane vesicles, MV1, MV2 and MV3 was determined using a novel approach for direct quantification of phospholipids from two-dimensional 31 P-1H nuclear magnetic resonance spectroscopy with isotropic mixing. MV2 and MV3 have similar compositions typical of the ER and the Golgi membranes. However, MV1 is mainly composed of phosphatidylinositol with phosphatidylcholine being the minor phospholipid present in MV1. Furthermore, we determined that phosphatidylinositol specific phospholipase C (PI-PLC) promoted nuclear envelope formation. However, in the absence of MV1, PI-PLC, did not induce nuclear envelope formation. Inhibition of membrane vesicle fusion in a dose dependent manner, by wortmannin (a specific inhibitor of the PI-3kinase pathway) suggested that the PI-3 kinase branch of the phosphatidylinositol pathway may be involved in the formation of the nuclear envelope. These experiments indicate that the inositol phospholipid pathways, as in constitutive membrane trafficking, are also involved in the formation of the nuclear envelope. Furthermore, it is the first time that a biological membrane, MV1, with such an unusual composition has been reported and that it has been demonstrated the phosphatidylinositol hydrolysis is involved in the formation of the nuclear envelope. In the second part the goal was to determine which lipoxygenase metabolites were involved in facilitating HeLa cell adhesion and spreading to a gelatin substrate. Reverse phase HPLC methods demonstrated that HeLa cell homogenates converted arachidonic acid into 12-, 15- and 5-hydroperoxyeicosatetraenoic (HETEs), indicating that 12, 15 and 5 lipoxygensases are active in HeLa cells. In order to investigate which lipoxygenase pathway are required to induce cell spreading the lipoxygenase pathway was inhibited by 25uM nordihydroguaiaretic acid (NDGA), a specific inhibitor of the lipoxygenases and MK866, a specific inhibitor of leukotriene biosynthesis. The effect of the different enantiomers of 12-BETE and 15-HETE on the reversal of NDGA inhibition was determined. The leukotrienes that overcome the inhibition of cell spreading by NDGA and MK866 were also determined. It can be concluded that 12,15 and 5 lipoxygenase enzymes are present and active in HeLa cells; and 12-HETE, 15-HETE, leukotrienes LTB4, LTD4 and LTC4 play an active role in HeLa cell spreading on a gelatin substrate.

The roles of the calcium binding messenger protein calmodulin and the kinesin-like calmodulin binding protein during cell division in plants were investigated employing microinjection of various probes in Tradescantia virginiana stamen hair cells. Fluorescently labeled calmodulin was found to distribute uniformly throughout the cytoplasm, and was not specifically colocalized with the microtubules of the dividing cell as has been reported earlier. Injected calmodulin redistributed during fixation as if for immunolocalization, suggesting that earlier reports were based on artifactual localizations, and consequently that the role of calmodulin in cell division should be reevaluated. Inhibition of calmodulin using a synthetic calmodulin binding peptide, prevented the actomyosin based cytoplasmic streaming and the late lateral expansion phase of cytokinesis, but did not affect the architecture of cytoplasmic strands or anaphase chromosome motion. Thus, although calmodulin is not localized specifically to the phragmoplast, it does play a regulatory role during cytokinesis. Microinjection of antibodies to the kinesin-like calmodulin binding protein (KCBP) that constitutively activate this microtubule motor protein, caused precocious nuclear envelope breakdown, metaphase arrest, and delays in cytokinesis. However, the antibodies did not inhibit chromosome motion. These results, in combination with earlier characterization of this kinesin, lead to the conclusion that KCBP is differentially involved in forming a converging bipolar spindle. Interfering with its regulation affects the dynamic transformations of the microtubule cytoskeleton between these phases. The results establish a regulatory role for calmodulin in cell division and address two possible downstream signaling pathways: inhibition of myosins involved in the lateral expansion of the phragmoplast, and inhibition of the spindle forming kinesin-like calmodulin binding protein.

Cell adhesion to the extracellular matrix initiates a cascade of intracellular signaling pathways via integrin activation. Activation of the arachidonic acid (AA) signaling pathway has been shown to be an important early signaling event in both HeLa and fibroblast cell adhesion. The purpose of this research is to study the regulation of adhesion of fibroblasts to a fibronectin substrate by AA signaling. This dissertation is divided into three specific aims: (1) To investigate how the AA signaling pathway, specifically the lipoxygenase and cyclooxygenase metabolites, leuktriene B4 and prostaglandin E2, respectively, modulate directed cell spreading and migration, using a wound healing model. It was concluded that the differential effects of arachidonic acid metabolites produced by lipoxygenase and cyclooxygenase regulate sequential aspects of fibroblast wound closure in vitro, cell spreading in to the wound, cell bridges across the wound and cell migration in to the wound. (2) To investigate how changes in intracellular calcium levels regulate AA initiated cell spreading and migration. It was concluded that the presence of extracellular calcium is necessary for intracellular calcium mobilization. Futhermore, while the epsilon isoform of protein kinase C (PKC) is sufficient to facilitate partial cell spreading in the absence of extracellular calcium, PKC alpha is activated by the influx of extracellular calcium to optimize cell spreading and enable cell migration. (3) To investigate how upstream AA signaling events are linked to downstream activation of PKC. It was concluded that phosphatidylinositol specific phospholipase C activation and diacylglycerol production are intermediary signals linking upstream AA signaling to PKC activation.

Bovine γδ T cells are activated by a cell surface antigen found on autologous monocytes in in vitro cultures. This response is known as the autologous mixed leukocyte reaction (AMLR). I found AMLR stimulation is mediated through the T cell receptor since it was blocked by Fab fragments of an anti-δ TCR mAb. Purified γδ T cells directly responded to monocyte stimulation in AMLR. We found that the stimulatory molecule on monocytes was not a peripheral membrane-associated or GPI-anchored component but an integral membrane protein. An anti-monocyte monoclonal antibody (M5) was generated in mice and shown to block the proliferation induced by monocytes in the AMLR. The ligand for mAb M5 was found on bovine and sheep monocytes, which have the capacity to stimulate AMLR, but not on human or murine macrophages that were previously shown to be non-stimulatory in the AMLR. When proliferation and cytokine production were evaluated, γδ T cells responded poorly to stimulation through the TCR alone using anti-CD3 mAb, in contrast to CD4+ and CD8+ T cells that responded well. However when stimulated with monocytes in the AMLR, 70–90% of the responding cells were γδ T cells, as assessed by IFN-γ production using flow cytometry. Stimulation through the WC1 receptor by three mAb augmented the proliferation of T cells induced by suboptimal levels of anti-CD3 or anti-δ TCR mAb and direct stimulation of WC1 by mAb produced profound IFN-γ production by γδ T cells as shown by 2-color immunofluorescence. These data strongly suggest that WC1 is functioning as a co-stimulatory molecule for γδ T cell activation. We propose a model that monocytes provide the main stimulatory signals via the M5 ligand through the γδ TCR and co-stimulatory signals by the WC1 ligand through WC1, which together result in proliferation and high IFN-γ production in the AMLR. Thus, the major immune function of bovine γδ T cells may be their rapid and strong IFN-γ production which activates macrophages.

Cell-extracellular matrix (ECM) adhesion is characterized by discrete morphological and functional stages beginning with cell-substrate attachment, followed by cell spreading and then migration. These studies defined the lipid and protein signaling pathways regulating sequential transitions of adhesion stages NIH-3T3 cells on fibronectin (FN) substrate, emphasizing the signaling pathways stimulated by release of arachidonic acid (20:4, Δ5,8,11,14 ) (AA) from membrane phospholipids by phospholipase A2 (PLA2). Initial cell attachment to FN is via the extracellular domain of beta-1 integrin receptors. The beta-1 integrin cytosolic domain is required for receptor clustering and activation of PLA2; it also acts as the assembly site for focal adhesions formation to anchor the actin cytoskeleton. AA release is mediated by the 85 kD cytosolic PLA2 and not other PLA2's. Total AA release is rate-limiting in the overall process of adhesion, and is oxidized by lipoxygenases (LOX) or cyclooxygenases (COX) to generate adhesion signaling. During adhesion two functionally and kinetically distinct phases of AA release take place. An initial transient AA release is stimulated by cell attachment, and is sufficient to signal the cell spreading stage from 5-lipoxygenase (5-LOX) oxidation. LTB4, but not the cysteinyl LTs signals cell spreading. A later sustained AA release signals migration by its oxidation by upregulated cyclooxygenase-2 (COX-2). The second AA release and COX-2 protein synthesis are regulated by ERK-1/2. Constitutive overexpression of 5-LOX enhances l spreading and increases both rate and extent of actin polymerization, but limits motility. Increased LTB4 synthesis from 5-LOX stimulates f-actin polymerization and also increases total actin and beta-1 integrin expression. Constitutive overexpression of COX-2 slows cell spreading but increases motility. Upregulated COX-2 promotes disassembly of f-actin stress fibers, and induces redistribution of f-actin to permit motility, and decreases actin and beta-1 integrin expression. These data demonstrate a bifurcation in the AA adhesion-signaling pathway, wherein oxidation by 5-LOX signals actin polymerization regulating the spreading, while ERK 1/2-induced COX-2 synthesis generating prostaglandins signaling actin depolymerization and redistribution to enable migration.

The ability of established synaptic connections to strengthen and weaken (synapse plasticity) underlies higher order behavior such as learning and memory. Several gaps remain in our understanding of the cellular and molecular changes in pre- and postsynaptic cells associated with synapse plasticity. Using the Drosophila larval neuromuscular junction as a convenient model system throughout my dissertation work, I have concentrated on understanding various molecular mechanisms that underlie this phenomenon. Specifically, I have addressed fundamental issues governing the postsynaptic cell in these processes, such as: (i) what are the crucial proteins required to scaffold the postsynaptic apparatus during plasticity? (ii) what are the molecules that are brought together at this scaffold and how are their levels regulated during plasticity? and finally (iii) what are the signals that initiate the formation of these scaffolds during plasticity? More specifically, I have uncovered a mechanism by which a novel synaptic protein, Guk-holder (Gukh), coordinates the formation of a tripartite complex at the synapse with two proteins containing multiple protein-protein interaction domains: Discs Large (DLG) and Scribble (Scrib) (Mathew et al., 2002). By bringing DLG and Scrib together at the synapse, Gukh likely coordinates the function of these two complexes. As stated above Fasciclin II (FasII) is one of several molecules that is localized to the synapse by Dig (Thomas et al., 1997a), and its levels and symmetric distribution at the synapse have been found to be important for regulating synapse plasticity (Ashley et al., 2005; Schuster et al., 1996b). I showed that FasII also undergoes fast recycling at the synaptic membrane and that this recycling is regulated by the Drosophila homolog of Amphiphysin (Amph)-(Mathew et al., 2003). Such a mechanism would be compatible with a potential activity dependent regulation of FasII levels during plasticity. To understand the initial signaling mechanisms by which synaptic scaffolds are organized during synapse formation, I further investigated the role of the Wingless receptor Dfrizzled2 (DFz2) in this process. Wg signaling has been previously implicated in synapse development and proper localization of postsynaptic proteins (Packard et al., 2002). In these studies I uncovered an unconventional mechanism involved in transducing the Wg signal in the muscle cell. I show that DFz2 is cleaved at the synaptic plasma membrane, and the c-terminus of the molecule traffics to the nucleus. This nuclear import of DFz2 is dependent upon Wg signaling and is important to transduce the downstream effects of Wg signaling at synapses (Mathew et al., 2005 in press). (Abstract shortened by UMI.)