The potassium channels IK and IK1, responsible for the action potential repolarization and resting potential respectively, are altered during cardiac hypertrophy. The activation of insulin-like growth factor-I (IGF-I) during hypertrophy may affect channel activity. The aim was to examine the modulatory effects of IGF-I on IK and IK1 through mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K) pathways during hypertrophy. With the use of specific inhibitors for ERK1/2 (PD98059), p38 MAPK (SB203580) and PI3K/Akt (LY294002), Western blot and whole cell patch-clamp were conducted on sham and aorto-caval shunt-induced hypertrophy adult rat myocytes. Basal activation levels of MAPKs and Akt were increased during hypertrophy. Acute IGF-I (10-8 M) enhanced basal activation levels of these kinases in normal hearts but only those of Akt in hypertrophied ones. IK and IK1 activities were lowered by IGF-I. Inhibition of ERK1/2, p38 MAPK, or Akt reduced basal IK activity by 70, 32, or 50%, respectively, in normal cardiomyocytes vs. 53, 34, or 52% in hypertrophied ones. However, basal activity of IK1 was reduced by 45, 48, or 45% in the former vs. 63, 43, or 24% in the latter. The inhibition of either MAPKs or Akt alleviated IGF-I effects on IK and IK1. We conclude that basal IK and IK1 are positively maintained by steady-state Akt and ERK activities. K+ channels seem to be regulated in a dichotomic manner by acutely stimulated MAPKs and Akt. Eccentric cardiac hypertrophy may be associated with a change in the regulation of the steady-state basal activities of K+ channels towards MAPKs, while that of the acute IGF-I-stimulated ones toward Akt. .

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18154号 / 医博第3874号 / 新制||医||1002(附属図書館) / 31012 / 京都大学大学院医学研究科医学専攻 / (主査)教授 武藤 学, 教授 岩田 想, 教授 松田 道行 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Phafin2 is a member of the Phafin protein family. Phafins are modular with an N-terminal PH (Pleckstrin Homology) domain followed by a central FYVE (Fab1, YOTB, Vac1, and EEA1) domain. Both the Phafin2 PH and FYVE domains bind phosphatidylinositol 3-phosphate [PtdIns(3)P], a phosphoinositide mainly found in endosomal and lysosomal membranes. Phafin2 acts as a PtdIns(3)P effector for endosomal cargo trafficking, macropinocytosis, apoptosis, and autophagy. The PtdIns(3)P binding activity is critical to the localization of Phafin2 on a specific membrane and, subsequently, helps the recruitment of other binding partners to the same membrane surface. However, there are no studies on the structural basis of PtdIns(3)P binding, the PtdIns(3)P-binding properties of each domain, and the apparent redundancy of two PtdIns(3)P binding domains in Phafin proteins. In the present dissertation, different biochemical and biophysical techniques were utilized to investigate the structural features of Phafin2 and its lipid interactions. This dissertation shows that Phafin2 is a moderately elongated monomer with a predicted α/β structure and ~40% random coil content. Phafin2 binds lipid bilayer-embedded PtdIns(3)P with high affinity; its PH and FYVE domains display distinct PtdIns(3)P-binding properties. Unlike the PH domain, the Phafin2 FYVE domain binds both membrane-embedded PtdIns(3)P and water-soluble dibutanoyl PtdIns(3)P with similar affinity. An intramolecular autoinhibition mechanism is found in Phafin2, in which a conserved C-terminal aspartic acid-rich (polyD) motif inhibits the binding of Phafin2 PH domain to PtdIns(3)P. The polyD motif specifically interacts with the Phafin2 PH domain. Using negative-stain Transmission Electron Microscopy, Phafin2 was found to cause membrane tubulation in a PtdIns(3)P-dependent manner. In conclusion, this study provides the structural and functional basis of Phafin2 lipid interactions and evidence of an intramolecular autoinhibition mechanism for PtdIns(3)P binding to the Phafin2 PH domain, which is mediated by the C-terminal polyD. The distinct PtdIns(3)P binding properties of the Phafin2 PH and FYVE domains may indicate that these two domains have different functions. Considering that the Phafin2 PH domain's PtdIns(3)P binding is intramolecularly regulated, cells may employ a unique mechanism to release the Phafin2 PH domain from the conserved C-terminal motif and control the functions of Phafin2 in PtdIns(3)P- and PH domain-dependent signaling pathways. / Doctor of Philosophy / Living cells need to absorb extracellular materials to sustain their growth and achieve cellular homeostasis. When cells require an uptake of liquids, they employ pinocytosis ("cell drinking"); when cells uptake solid particles, they use phagocytosis ("cell eating"); and when cells are in nutrient starvation status, they exploit an evolutionarily conserved process to survive known as autophagy ("self-eating"). Cells coordinate these activities through complex biochemical signaling systems. In each of these activities, a specific pathway is used to transfer the extracellular materials into the intracellular compartments and regulate the intracellular communications. Protein-lipid interactions are critical to these signaling pathways. This study focuses on the interactions between Phafin2 and phosphatidylinositol 3-phosphate [PtdIns(3)P]. Phafin2 is a cytoplasmic protein involved in autophagy, and PtdIns(3)P is a transient lipid signaling molecule localized to a specific organelle. After cells trigger autophagic events, Phafin2 protein molecules are associated with PtdIns(3)P. Subsequently, Phafin2 will recruit other protein binding partners. In this research project, biochemical and biophysical approaches were employed to study the structural features and PtdIns(3)P binding properties of Phafin2. Phafin2 was found to have two distinct PtdIns(3)P-binding domains; however, one of them is intramolecularly regulated. The results of this study help us to understand why Phafin2 displays two PtdIns(3)P-binding domains with different properties and how this is regulated, information that might be instrumental to understanding the roles of Phafin2 in physiological and disease scenarios.

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Understanding how plants respond to stress is of importance, considering the increasing need to feed a growing population and supply its energy. Plants have complex systems for detecting, and responding to stresses. One stress-responsive system involves myo-inositol (Ins). Ins is a precursor for cell wall components, inositol trisphosphate (Ins(1,4,5)P3) and phosphatidylinositol phosphate signaling molecules, and an alternate ascorbic acid (AsA) synthesis pathway. The enzyme, myo-inositol oxygenase (MIOX) is encoded by four genes in Arabidopsis and catalyzes the first step of Ins catabolism producing D-glucuronic acid (DGlcA). This research focuses on MIOX metabolism of Ins during plant growth and stress responses. I have examined miox mutants for alterations in metabolism and signaling. MIOX2 and MIOX4 expression patterns correlate with miox mutant root growth in varying nutrient conditions, and changes in flowering time. In miox2 mutants, I found an increase in Ins in most tissues, which was accompanied by cold- and abscisic (ABA)- sensitivity; however, miox4 mutants are ABA- insensitive, and have a small increase of Ins in flowers. MIOX2:GFP fusion protein accumulates in the cytoplasm and MIOX4:GFP accumulates in the cytoplasm and nucleus. Overexpresser MIOX4+ plants provide a model system to examine how directing carbon from Ins into DGlcA impacts Ins levels and Ins signaling. I have examined MIOX4+ plants for alterations in MIOX4 RNA and protein, and measured Ins by gas chromatography (GC). My results indicate that MIOX4+ tissues are impacted differently by the MIOX4 transgene, with decreases in Ins after seed imbibition, and increased Ins levels later in development. Ins depletion in seedlings was correlated with a decrease in Ins(1,4,5)P3. To determine the impact of reducing Ins and Ins(1,4,5)P3 in MIOX4+ seedlings, I examined processes known to involve Ins(1,4,5)P3 signaling. MIOX4+ seed have increased seed dormancy, NaCl-sensitivity, and ABA-insensitivity. These results suggest MIOX affects Ins signaling in response to ABA. Together, these data indicate that transcriptional control of MIOX2 and MIOX4 results in distinct roles in plant growth, and that MIOX2 and MIOX4 function in metabolic and signaling processes critical for growth, nutrient sensing, and stress responses. / Ph. D.

Oomycetes, such as Phytophthora sojae, are plant pathogens that employ protein effectors that enter host cells to facilitate infection. Plants may overcome infection by recognizing pathogen effectors via intracellular receptors (R proteins) that form part of their defense system. Entry of some effector proteins into plant cells is mediated by conserved RxLR motifs in the effectors and phosphoinositides (PIPs) resident in the host plasma membrane such as phosphatidylinositol 3-phosphate (PtdIns(3)P). Recent reports differ regarding the regions on RxLR effector proteins involved in PIP recognition. To clarify these differences, I have structurally and functionally characterized the P. sojae effector, avirulence homolog-5 (Avh5). Using NMR spectroscopy, I demonstrate that Avh5 is helical in nature with a long N-terminal disordered region. Heteronuclear single quantum coherence titrations of Avh5 with the PtdIns(3)P head group, inositol 1,3-bisphosphate (Ins(1,3)P2), allowed us to identify a C-terminal lysine-rich helical region (helix 2) as the principal lipid-binding site in the protein, with the N-terminal RxLR (RFLR) motif playing a more minor role. Furthermore, mutations in the RFLR motif slightly affected PtdIns(3)P binding, while mutations in the basic helix almost abolished it. Avh5 exhibited moderate affinity for PtdIns(3)P, which increased the thermal stability of the protein. Mutations in the RFLR motif or in the basic region of Avh5 both significantly reduced protein entry into plant and human cells. Both regions independently mediated cell entry via a PtdIns(3)P-dependent mechanism. My findings support a model in which Avh5 transiently interacts with PtdIns(3)P by electrostatic interactions mainly through its positively charged helix 2 region, providing stability to the protein during RFLR-mediated host entry. / Ph. D.

7 Abstract Protein kinase C alpha (PKCα) is a serine/threonine protein kinase. PKCα is an important protein regulating cell polarity, protein secretion, apoptosis, cell proliferation and differentiation and tumorogenesis. Previous research has shown a role of PKCα also in a cancer cell migration and cancer cell invasion. The aim of this study was to investigate the role of protein kinase C alpha (PKCα) played in amoeboid mode of cancer cell invasion. We showed that higher expression of PKCα resulted in mesenchymal-amoeboid transition of K2 and MDA mesenchymal cancer cell lines, which was accompanied with decreased cancer cell invasive capability in 3D collage matrix. PKCα overexpression had no effect on the cell morphology of A375m2, however, the results showed a trend in increased invasive potential of A375m2 cells. Conversely, the expression of dominant-negative PKCα resulted in amoeboid-mesenchymal transition of A375m2 cells, and it was associated with decreased invasive potential of K2 and MDA cell lines. Furthermore, a linkage between PKCα and phosphatidylinositol 3-kinase (PI3K) was tested. The results revealed that increased activity of PKCα was accompanied with decreased level of active Akt in K2 cell line. To summarize, our results suggest a probable role of PKCα in regulation of amoeboid...

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