Wnt ligands are a family of highly conserved glycoproteins that act as morphogens to regulate development in many organisms. Drosophila Wnt-1 (Wingless) is involved in directing cell fate decisions and pattern formation during differentiation. Wnt signaling are of high interest of many developmental biologists due to their important functions, yet little is known about how these ligands function on a biochemical level. Previously it was found that Porcupine, an ER-membrane-bound acyltransferase is required for Wingless secretion. But it is unclear how a secreted morphogen requires an acyltransferase to function. Studies reported here demonstrated that Wingless undergoes a hydrophobic modification, in which a lipid moiety containing a palmitate group is covalently attached to the polypeptide through an ester linkage. And it partitions with the specialized detergent insoluble lipid raft microdomains in the plasma membrane. Porcupine is required for the modification and the raft targeting of Wingless. Blocking Wingless modification with a specific inhibitor results in the loss of rafts-association as well as loss of protein secretion. Disrupting raft microstructures by cholesterol depletion reagents also impaired Wingless secretion, indicating that the ligand secretion is dependent on its specific association with the plasma membrane. This work provided the first insight on the function of Porcupine and the important biochemical evidence on the role of specialized membrane microdomains in Wnt signaling.

The generation of site-directed mutants within the actin binding domain of the EGF receptor modulates receptor function in internalization and ligand binding. In addition, truncation of the EGFr at residue 996 results in a loss of high affinity ligand binding, inhibited internalization and reduced signaling capacity. Mutation of tyrosine 992 to phenylalanine (Y992F) and glutamate 991 to glutamine (E991Q) increases the rate at which receptors are internalized. The presence of a phenylalanine residue eliminates EGFr-mediated phosphorylation at Tyr992 while the E991Q mutation might also eliminate phosphorylation at this position due to a disruption of the kinase recognition motif. Thus, phosphorylation of Tyr992 appears to function in the regulation of receptor internalization. The mutation of tyrosine 992 to a glutamate residue (Y992E) causes a three-fold increase in receptor affinity for its ligand and demonstrates the existence of novel third and potentially fourth affinity states for the EGFr. A very high affinity EGFr state with a K$\sb{\rm d}$ of approximately 10 pM has been identified as has an intermediate state of 1.5 nM. The deletion of the C-terminal 190 amino acids of the EGFr causes a complete abolition of the previously observed high affinity state of the EGFr and also causes a significant reduction in the affinity of the low affinity state of the EGFr. Phorbol ester treatment of wild type and mutant EGFr causes a loss of the high affinity receptors, and also a decrease in the overall affinity of the receptor for its ligand which is similar to the loss seen in the deletion mutant. This suggests that control of the affinity state of the EGFr is mediated through the C-terminal 190 amino acids of the receptor. In addition, the C-terminal 190 amino acids of the receptor have been identified as containing a domain which regulates the phorbol ester induced conversion of receptor affinity. The amino acid composition in the vicinity of tyrosine 992 has been shown to play a role in the internalization of the EGF receptor and in the regulation of receptor affinity for its ligand.

Autoreactive, immature T cells (thymocytes) are deleted from the thymus during development by the process of negative selection. This mechanism occurs when the thymocyte, T cell receptor (TCR) recognizes self-antigen, causing the cell to die by an apoptotic pathway. This mechanism results in the deletion of autoreactive T cells. Thymocyte development proceeds through several stages, determined by the differential expression of the T cell co-receptor molecules CD4 and CD8. The developmental stage where negative selection occurs is one in which thymocytes are expressing a functional TCR on the cell surface along with both CD4 and CD8, termed the double positive stage. These thymocytes are in a quiescent, G0 state and make up greater than 80% of the total population. The demonstration that cell cycle progression plays a role in the apoptotic process of several quiescent cell types, as well as the requirement for mature T cells to be in the late G1 phase during activation induced cell death, suggested that thymocytes may also advance to the G1 stage of the cell cycle prior to apoptosis. It has been established that the early cell cycle genes c-fos, c-jun, and c-myc are induced in thymocytes after stimulation, indicating possible entry into the cell cycle. The hypothesis that thymocytes enter the cell cycle before undergoing apoptosis was tested by examining expression levels of the various G1 cyclins and cyclin dependent kinase inhibitors at the mRNA and protein levels. Several indications of an early G1 cell cycle transition occurring during thymocyte apoptosis were observed, such as the downregulation of p27KIP1 and p130, the upregulation of cyclin D3, and the phosphorylation of the retinoblastoma protein. Finally, the requirement for the activation of the cyclin-dependent kinase 2 (CDK2) in negative selection was examined. It was shown that the phosphorylation and expression of the TCR-mediated apoptosis-related transcription factors Nur77 and Egr1 are downstream of CDK2 activation. In addition, a protein associated with the transcription factor Egr-1 was identified as a possible target of CDK2 kinase activity.

Programmed cell death is essential for normal development and adult tissue homeostasis in almost all multicelluar organisms. Acheron gene was first isolated from the intersegmental muscles (ISMs) in Manduca sexta as a death-associated gene. Subsequently, we cloned human and mouse homolog of Acheron. Acheron encodes a novel protein that has not been previously characterized. Protein structure analysis revealed that Acheron proteins are structurally related to La proteins, but define a novel subfamily. Tissue expression analysis showed that mAcheron is widely expressed in most tissues at both the RNA and protein levels, with brain and heart displaying the highest levels. In mouse C2C12 cells, endogenous Acheron is constitutively expressed in cycling myoblasts and myotubes. Despite the presence of a putative nuclear localization site, the protein is localized predominantly in the cytoplasm. Analyses of the different Acheron transfected C2C12 cells suggested that Acheron is implicated in mediating differentiation and apoptosis in C2C12 cells by differentially regulating the expression of MyoD, Myf5 and Bcl-2. Acheron expression allows C2 C12 cells to up-regulate MyoD and differentiate into myotubes when the cells are induced to undergo differentiation. However, it does not support the myoblast self-renewal by specifically inhibiting the expression of Bcl-2, a key survival factor for ‘reserve’ cells in DM. Inhibition of Acheron activity by tAch (a putative dominant negative regulatory factor of Acheron) or antisense Acheron results in greatly increased ‘reserve’ cell population and decreased differentiation under differentiation condition. The mediation of differentiation and survival by Acheron may be achieved through its regulation on integrin—FAK signaling. To help determine how Acheron functions, we performed a yeast 2-hybrid screen with Acheron as the bait. A clone that contains partial cDNA of Ariadne was isolated from the screen. Ariadne contains RING finger domain and is known to bind to ubiquitin E2 conjugase. In vitro ubiquitination assay revealed that Ariadne has ubiquitin E3 ligase activity. We speculate that Ariadne may function as an E3 to target Acheron for ubiquitination and subsequent proteasome-dependent degradation.

During muscle differentiation in vertebrates, myoblasts initially form in somites and then migrate to proper locations in the trunk and limbs. Once there, these cells are faced with one of three choices: differentiate into myotubes, arrest as satellite cells, or initiate apoptosis and die. The molecular mechanisms that regulate the decision of myoblasts to die are poorly understood. To gain insight into this process, we have cloned death-associated genes from the intersegmental muscles of the moth Manduca sexta, a model system for developmentally regulated muscle cell death. One of the genes isolated in this screen was DALP (Death Associated LIM Only Protein), a protein that shares 52% similarity at the protein level with mammalian Hic-5. Ectopic expression of DALP in the skeletal muscles of the fruit fly Drosophila caused atrophy and disorganization of the contractile apparatus. To determine the role of DALP/Hic-5 in mammalian myogenesis, we took advantage of the mouse myoblast cell line C2C12. Ectopic expression of either DALP or Hic-5 blocked the ability of myoblasts to differentiate following serum withdrawal. These cells failed to express muscle differentiation markers such as MyoD or myosin heavy chain. In addition, these cultures displayed greatly enhanced rates of cell death. Hic-5 expression is restricted to mononucleated and apoptotic C2C12 cells in serum-depleted medium. The effects of ectopic DALP or Hic-5 expression could be prevented by contact with wild type C2C12 cells or by ectopic expression of MyoD. Gene profiling experiment demonstrated that the ectopic expression of Hic-5 results in enhanced expression of pro-apoptotic Bcl-2 family members and of polyubiquitin. Taken together, these data suggest that Hic-5 acts upstream of MyoD and functions as a negative-regulator of myoblast differentiation and may facilitate the initiation of apoptosis. In separate studies, the functional roles of another death-associated molecule, m56, were studied in Ratl fibroblasts. Our data strongly support the hypothesis that m56 is a proteasome subunit and misexpressing m56 can sensitize Ratl cells to apoptotic stimuli.

Loss of regulation of cell cycle events mediated by changes in cytosolic Ca$\sp{2+}$ ion activity has been implicated in the progression of normal cells to neoplasia. In this study, the Ca$\sp{2+}$ buffer 5,5$\sp\prime$-difluoro 1,2-bis(2-aminophenoxy) ethane-N,N,N$\sp\prime N\sp\prime$-tetra-acetic acid (5,5$\sp\prime$-dfBAPTA, abbreviated "dfB") has been used to modulate cell division in transformed and primary mouse keratinocytes. Exogenous application, via the tetra(acetoxymethyl) ester ("AM"), of 18-20 $\mu$M dfB/AM to the growth media of transformed cells inhibits cell division and DNA synthesis, without compromising the cells' viability, as shown by $\sp3$H-thymidine incorporation and flow cytometry. Bulk fluorimetry shows that cells treated with dfB/AM are able to buffer Ca$\sp{2+}$ in proportion to the concentration of dfB/AM applied. Primary cultured cells treated with 18-20 $\mu$M dfB/AM die within 3 hours of treatment. Viable dfB/AM treated cultures of transformed cells have a higher proportion of cells in the G$\sb2$ phase of the cell cycle than do controls, as shown by flow cytometry. This result, in combination with that showing reduced $\sp3$H-thymidine incorporation, suggests that 18-20 $\mu$M dfB/AM inhibits a pre- or mid-mitotic step. Light, electron, and confocal microscopies show 18-20 $\mu$M dfB/AM-treated cells to have prominent, thickened nuclear envelopes along with actin cytoskeletons that are distinguishable from controls. Upon return to medium that does not contain dfB/AM, treated transformed cells gradually resume their pre-treatment growth and division patterns.

A comparison of the structure of isolated and in situ chromatin fibers was performed using starfish (Patiria miniata) sperm nuclei. The simple protein content consisting of five major histones was found. A DNA repeat length of 222bp was calculated. Compact chromatin fibers solubilized from detergent isolated nuclei showed diameters of 38 to 44nm by electron microscopy. Chromatin fibers observed in whole cells and in mildly treated nuclear preparations revealed fiber diameters of approximately 24nm when embedded in epoxy resin. Investigation of the chromatin isolation process uncovered a mechanism where fiber integrity was lost during nuclear isolation. Following nuclease digestion and chromatin release, fiber-like structures reformed in solution. The relationship of the isolated "fibers" to the native structures is not known. Nuclear permeability has been found to be a factor in both release of cleaved chromatin from nuclei and inward diffusion of nuclease. Permeability to various sized dextrans was consistent with a mechanism where cut chromatin exits nuclei as nucleosomal chains and aggregates in solution to form the isolated chromatin state. Attempts to isolate detergent free nuclei revealed that completely isolated nuclei were susceptible to the loss of chromatin fiber integrity suggesting that the medium was incapable of maintaining fiber structure. Other buffer compositions did not improve stability of in situ fibers underscoring a general lack of understanding of the nuclear environment.

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.