京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20990号 / 医博第4336号 / 新制||医||1027(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 武田 俊一, 教授 山田 亮, 教授 藤渕 航 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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Cell cycle control genes in the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe have been studied in detail in the past few years. The cdc25+ and wee1+ genes of S. pombe play key roles in the commitment to division. Genes homologous to the mitotic inducer cdc25+ and the mitotic inhibitor wee1+ of Schizosaccharomyces pombe were searched for in Saccharomyces cerevisiae using DNA cross-hybridization as the method of detection. Such homologs were not found in Saccharomyces cerevisiae by this method. Attention was therefore turned towards sequencing and partially characterizing a previously cloned gene, WHI1+, and its mutant form WHI1-1 (now call CLN3+ and CLN3-1 respectively). Sequence analysis showed that CLN3+ is a cyclin homolog. Cyclins are probably present in all eukaryotes and play an important role in controlling the onset of mitosis. However, unlike these mitotic cyclins, CLN3+ functions in G1. CLN3-1 cells enter a new round of the cell cycle at an aberrantly small cell size and are α-factor resistant. Sequence analysis showed that the CLN3-1 protein was a truncated form of CLN3+ caused by a nonsense mutation in the CLN3+ gene. Cells overexpressing CLN3+ had the same phenotype as CLN3-1 cells, suggesting that the truncated CLN3-l protein was a hyperactive form ofthe wild-type protein. CLN3+ and CLN3-1 were placed downstream of the yeast GAL1 promoter in a shuttle plasmid. Cells transformed with these plasmids and grown in the presence of galactose and the absence of glucose produced CLN3+ or CLN3-1 in large amounts. Cell size was reduced in such cells. These cells were also α-factor resistant. / Thesis / Master of Science (MSc)

Molecular recognition is at the core of all biological processes whereby protein-protein interactions (PPI) relay messages to drive signaling events. However, many regulatory responses are driven by weak or transient PPI. These interactions are difficult to study using structural biology techniques because they are labile and result in heterogeneous populations. Moreover, interactions reconstituted using peptides are difficult to interpret because they lack context. In this thesis, I characterized key signaling complexes implicated in the replication checkpoint response (Dbf4-Rad53-Cdc7 complex), mitosis (Dbf4-Cdc5 complex), and DNA mismatch repair (clamp-MutL complex). I solved the crystal structures of the Dbf4-Rad53 and clamp-MutL weak complexes by generating fusions of the binding partners. The structures revealed that Dbf4 and MutL undergo subtle conformational movements upon engaging their binding partners, which were sufficient to alter both interfaces. Overall, the structures offer insight as to how Rad53 could inhibit Dbf4-Cdc7 during the replication checkpoint and how the clamp could activate MutL during mismatch repair. Acquiring the Dbf4-Cdc5 co-crystal structure required optimization the Dbf4 peptide. The Dbf4-Cdc5 and Dbf4-Rad53 complexes were relatable because both interactions were phosphorylation-independent even though Rad53 and Cdc5 are known to recognize phosphorylated targets. Dbf4 engaged a binding site on Cdc5 located opposite to the phosphoepitope binding pocket, which is reminiscent to its interaction with Rad53. Collectively, the structures of Dbf4 and its binding partners reveal that Rad53 and Cdc5 function beyond phosphoepitope recognition whereby they utilize additional binding surfaces to engage substrates. / Thesis / Doctor of Philosophy (PhD)

The cell cycle of the budding yeast, Saccharomyces cerevisiae, is regulated by a complex network of chemical reactions controlling the activity of the cyclin-dependent kinases (CDKs), a family of protein kinases that drive the major events of the cell cycle. A previous mathematical model by Chen et al. (2000) described a molecular mechanism for the Start transition (passage from G1 phase to S/M phase) in budding yeast. In this thesis, my main goal is to extend Chen's model to include new information about the mechanism controlling Finish (passage from S/M phase to G1 phase). Using laws of biochemical kinetics, I transcribed the hypothetical molecular mechanism into a set of differential equations. Simulations of the wild-type cell cycle and the phenotypes of more than 60 mutants provide a thorough understanding of how budding yeast cells exit mitosis. / Master of Science

Protein kinases play essential roles in orchestrating almost every step during mitosis. Aberrant kinase activity often leads to errors in the cell cycle progression which consequently becomes the underlying cause for developmental defects or abnormal cell proliferation leading to cancer. Tank Binding Kinase 1 (TBK1) is overexpressed in certain cancer types and is activated on the centrosomes during mitosis. Loss of TBK1 impairs cell division resulting in growth defects and the accumulation of multinucleated cells. Therefore, proper activation and localization of TBK1 are essential for mitotic progression. Yet, the upstream regulation of TBK1 and the function of activated TBK1 on the centrosomes is unknown. Also, the cause and consequences of overexpression of TBK1 in cancers remain to be explored. Activation of TBK1 depends on its binding to an adaptor protein which induces a conformational change leading to trans autophoshorylation on serine 172 of its kinase domain. We identified that an established innate immune response protein, NAK Associated Protein1 (NAP1/AZI2), is the adaptor required for binding and activating TBK1 during mitosis. Loss of either NAP1 or TBK1 results in the accumulation of binucleated and multinucleated cells, possibly due to several mitotic and cytokinetic defects seen in these knockout (KO) cells. We establish NAP1 as a cell cycle regulated protein which colocalizes with activated TBK1 on the centrosomes during mitosis. Furthermore, by performing an unbiased quantitative phosphoproteomics analysis during mitosis, the substrates discovered reveal that TBK1 also regulates other known cell cycle regulating kinases such as Aurora A and Aurora B. TBK1 is also an established autophagy protein and since the autophagy machinery is often impaired or remodeled to facilitate rapid cell division, we evaluated the underlying connection between TBK1 activation and autophagy. The data shows that cells lacking the essential autophagy proteins FIP200 or ATG9A exhibit overactivation and mislocalization of TBK1. By using both genetic and pharmacological inhibition of autophagy processes, we found that impaired autophagy leads to a significantly higher number of micronuclei – a hallmark for tumorigenesis that correlates with defects in mitosis and cytokinesis. Taken together our work has uncovered a novel function for the NAP1-TBK1 complex during mitosis and establishes that overactivation and mislocalization of TBK1 is a direct consequence of impaired autophagy which causes micronuclei formation. / Doctor of Philosophy / Defective cell division is the underlying cause for many human health maladies such as birth defects and cancer. Investigation into the proteins that are abnormally expressed in cancer can help us identify their physiological roles in regulating the cell cycle. Tank Binding Kinase 1 (TBK1) is often overexpressed in several types of cancer such as glioblastomas, breast, and lung cancers. It has also been extensively studied in the process of removing damaged cytosolic components from cells called autophagy. During cancer progression, cells often hijack the autophagy machinery to their advantage for abnormal cell proliferation. However, we do not completely understand the role of TBK1 in cancer pathogenesis or during normal cell division. Each cell duplicates its genomic contents and divides its organelles and cytosolic components during cell division. Centrosomes organize microtubules to attach to the duplicated genomic material to equally segregate the DNA between two daughter cells. Previous studies have shown that TBK1 is active on the centrosomes during mitosis, and the loss of TBK1 leads to reduced cell proliferation. However, the function of TBK1 and what regulates its activation on the centrosomes are unknown. Using a combination of genetic, biochemical, and molecular biology techniques, we found that an immune response protein Nak Associated Protein 1 (NAP1/AZI2) binds to TBK1 and activates it on the centrosomes during cell division. Furthermore, our study demonstrates that the loss of either NAP1 or TBK1 exhibits a multitude of different types of defects in the process of cell division. We further identified TBK1 substrates in a phosphoproteomic screen indicating that TBK1 regulates the activity of other major cell division kinases. We show that defects in autophagy machinery result in the mislocalization and overactivation of TBK1 resulting in defects during chromosome segregation, and in the formation of micronuclei. Together our study shows that an established immune response protein NAP1 regulates the function of TBK1 during cell division and there exists a connection between TBK1 activity and disrupted autophagy.

Lic.-avh. (sammanfattning) Stockholm : Karol. inst., 2006. / Härtill 3 uppsatser.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2006. / Härtill 4 uppsatser.