Treating fungal infections is challenging due to the emergence of drug resistance and the limited number of clinically useful antifungal drugs. To improve clinical outcome it will be necessary to develop new antifungal drugs with different mechanisms of action and to discover drugs that improve the fungicidal activity of current antifungals. This study reveals a new role for fungal protein kinase C (PKC) signaling in resistance to drugs targeting the ergosterol biosynthesis pathway in the pathogenic fungus, Candida albicans, and the model yeast, Saccharomyces cerevisiae. PKC signaling enabled survival of antifungal-induced cell membrane stress in part through the mitogen-activated protein kinase (MAPK) cascade and through cross-talk with calcineurin signaling in both species. The molecular chaperone Hsp90, which stabilizes client proteins including calcineurin, also stabilized the terminal C. albicans MAPK, Mkc1. This establishes new circuitry connecting PKC with Hsp90 and calcineurin, and suggests that inhibiting fungal Pkc1 can be a promising strategy for treating life-threatening fungal infections.

Treating fungal infections is challenging due to the emergence of drug resistance and the limited number of clinically useful antifungal drugs. To improve clinical outcome it will be necessary to develop new antifungal drugs with different mechanisms of action and to discover drugs that improve the fungicidal activity of current antifungals. This study reveals a new role for fungal protein kinase C (PKC) signaling in resistance to drugs targeting the ergosterol biosynthesis pathway in the pathogenic fungus, Candida albicans, and the model yeast, Saccharomyces cerevisiae. PKC signaling enabled survival of antifungal-induced cell membrane stress in part through the mitogen-activated protein kinase (MAPK) cascade and through cross-talk with calcineurin signaling in both species. The molecular chaperone Hsp90, which stabilizes client proteins including calcineurin, also stabilized the terminal C. albicans MAPK, Mkc1. This establishes new circuitry connecting PKC with Hsp90 and calcineurin, and suggests that inhibiting fungal Pkc1 can be a promising strategy for treating life-threatening fungal infections.

Thesis (Ph. D.)--University of Washington, 2001. / Vita. Includes bibliographical references (leaves 78-93).

Thesis (Ph. D.)--University of Washington, 2001. / Vita. Includes bibliographical references (leaves 102-114).

Each member of the protein kinase C (PKC) family activates cell signaling pathways with different and sometimes opposing cell functions, such as cell division, migration, or death. Because of the importance of these processes in human diseases and disorders like cancer, stroke, and Alzheimer’s disease, there is a need for drugs which modify the action of PKC. However, drug design is difficult due to the complicated nature of PKC regulation. To better understand the differential regulation of PKC activity, these studies probe the structure, dynamics, and reactivity of one of the domains responsible for PKC regulation, C1B. C1B binds signaling molecules and translocates PKC to membranes in order to release the kinase domain from inhibition. Mutagenesis and ligand-binding assays monitored with fluorescence and nuclear magnetic resonance (NMR) techniques show that a single variable residue in C1B dramatically affects the sensitivity to signal activators. Investigation of the domain structure and dynamics using NMR revealed the identity of this residue alters the dynamics of the activator binding loops, without changing the structure. NMR studies of the C1B variants in membrane-mimicking micelles showed this residue also changes the interaction of the regulatory domain with lipids. These results demonstrate PKC isoforms have evolved specific functions by tuning dynamics and membrane affinity. Alternatively, PKC can be activated by reactive oxygen species by a mechanism that does not require binding of signaling molecules or membrane localization. To investigate the role of C1B in this type of signaling, the regulatory domain reactivity is monitored via NMR and gel electrophoresis. These studies reveal a particular cysteine residue in C1B that is most reactive, an alternative conformation of C1B in which this residue is more exposed, and modification of C1B leads to unfolding and zinc loss. Because the regulatory domains are responsible for auto-inhibition of the kinase domain, C1B unfolding provides a plausible explanation for activation of PKC by reactive oxygen species. The relation of the intrinsic C1B properties to the activation of PKC can be used to develop drugs with a single mechanism and to better understand how closely related signaling proteins develop specific functions.

There is no abstract available for this dissertation. / School of Physical Education

Dopamine receptors belong to the G protein-coupled receptor (GPCR) superfamily and are classified into two families: D1-like (D1R and D5R) and D2-like (D2R, D3R and D4R), based on their ability to stimulate or inhibit adenylyl cyclase (AC). Classically, GPCRs (including D2R and D3R) are desensitized by the activation of the serine/threonine protein kinase C (PKC) upon phorbol-12-myristate-13-acetate (PMA) treatment. Previous studies demonstrate that while human D5R (hD5R) is also strongly desensitized upon PMA treatment, the human D1R (hD1R) undergo a robust PMA-induced sensitization. The aim of this PhD thesis was to explore how the canonical PKC- or phorbol ester-linked pathway can control the responsiveness of two similar GPCRs like hD1R and hD5R in an opposite fashion. Our data indicate that hD1R sensitization and hD5R desensitization are not mediated by a direct modulation of AC activity by PKC. Using a chimeric approach, we identified the third intracellular loop (IL3) as the key structural determinant controlling in an opposite manner the PMA-mediated regulation of hD1R and hD5R. To delineate the potential PKC phosphorylation sites, a series of mutation of serine (Ser) and threonine (Thr) located into IL3 of hD1R and hD5R were used. No hD1R mutation decreased the PMA-mediated sensitization. This suggests that hD1R phosphorylation is not required for PMA-induced sensitization. In contrast, our results indicate that PMA-mediated hD5R desensitization occurs through a hierarchical phosphorylation of Ser260, Ser261, Ser271 and Ser274. Notably, these hD5R mutants exhibited a PMA-induced sensitization, reminiscent of the PMA-induced hD1R sensitization. Additionally, using short hairpin RNAs (shRNAs), we showed that PKCε is the potentiating PKC while the desensitizing isoform is δ. Overall, our work suggests the presence or absence of specific Ser residues on IL3 of hD1-like receptors dictate if phosphorylation-dependent desensitization (through PKCδ) or phosphorylation-independent potentiation (via PKCε) will occur.

Neonatal ventral hippocampus (nVH) lesioned animals show molecular and behavioral abnormalities analogous to those described in schizophrenia. As an extension to previous studies that showed an increase in ligand binding of cortical alpha-1 adrenergic receptors (AR) and a dysfunction in alpha-1 AR regulation of mesolimbic dopamine functions in post-pubertal nVH lesioned rats, we investigated the subcellular expression and activity of protein kinase C (PKC)---a second messenger in alpha-1 AR signaling---in the prefrontal cortex (PFC) and nucleus accumbens (NAcc) of post-pubertal nVH lesioned rats. Western blot analysis of membrane and cytosolic fractions showed complex changes in lesioned animals in the expression of different PKC subtypes following saline or alpha-1 AR agonist (cirazoline i.p.) injection. Among these changes, nVH lesioned animals showed a significant increase in membrane bound PKC alpha and phospho-PKC, and a decrease in cytosolic PKC gamma and PKC betaII in the PFC in comparison to sham-lesioned controls following saline. Cirazoline increased membrane bound PKC alpha in controls but decreased it in lesioned animals. In the NAcc, lesioned animals showed an increase in membrane bound and cytosolic PKC epsilon and PKC lambda levels following saline. Following cirazoline, lesioned animals showed a decrease in membrane bound PKC epsilon and PKC lambda, while controls showed an increase in cytosolic and membrane fractions of PKC epsilon with no change in PKC lambda. In vitro PKC activity assays showed increased basal activity in PFC slices of lesioned animals compared to controls, with no difference in NAcc slices. alpha-1 AR stimulation by the agonist phenylephrine (PE) increased PKC activity in PFC of controls while decreasing activity substantially in lesioned animals. In the NAcc, high concentrations of PE increased activity in controls, but decreased activity in lesioned animals. This abnormal expression and activity of PKC in the PFC and NAcc of nVH lesioned animals may be related to abnormal alpha-1 AR functions and may modulate some of the abnormal neuronal functions in these animals, such as working memory deficits and hyper neuronal excitability of the PFC and the NAcc.

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Type 2 diabetes accounts for 85-90% of all people with diabetes and is currently estimated to affect more than 180 million people worldwide, a figure estimated to double by the year 2030. Thus understanding the basic biology of glucose homeostasis and how it is altered during disease progression is crucial to the development of safe and effective treatment regimes. The link between high dietary fat and the development of type Il diabetes is well established. Chronic treatment of pancreatic islets with the lipid palmitate induces defects in glucose stimulated insulin secretion (GSIS) akin to those seen in the development of type Il diabetes. Previous studies from our group have identified the lipid-activated kinase protein kinase C epsilon (PKCε) as a potential mediator of some of these effects. Deletion of PKCε in mice results in complete protection from high-fat diet induced glucose intolerance. This protection is associated with enhanced circulating insulin suggesting that PKCε may be involved in the regulation of insulin release from the pancreatic β-Cell. The data presented here suggests that PKCs plays an important role in the regulation of insulin secretion under both physiological and pathophysiological conditions. We demonstrate that PKCε can be activated by chronic lipid treatment and acute cholinergic stimulation. Under these conditions insulin secretion is enhanced by PKCε deletion or inhibition suggesting that PKCε is a negative regulator of insulin secretion. Mechanistically the PKCs mediated inhibition of insulin release by acute or chronic PKCε activation appears to be distinct. The effect of PKCε induced by palmitate pre-treatment appears to be distal to calcium influx. The pool of pre-docked vesicles is enhanced in palmitate pre-treated β-cells lacking PKCε suggesting that PKCε may be involved in the regulation of vesicle dynamics. In contrast, calcium dynamics induced by cholinergic stimulation are altered by PKCε deletion, suggesting an effect on either the calcium channels themselves or on the upstream signalling. Given the ability of PKCε to inhibit insulin secretion, inhibition of PKCε in the β-cells of people suffering from insulin resistance and (or) type II diabetes represents a novel target for the treatment of type II diabetes.