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.
| Identifer | oai:union.ndltd.org:VTETD/oai:vtechworks.lib.vt.edu:10919/116455 |
| Date | 11 October 2023 |
| Creators | Paul, Swagatika |
| Contributors | Biomedical and Veterinary Sciences, Pickrell, Alicia M., Theus, Michelle H., Morton, Paul D., Cimini, Daniela |
| Publisher | Virginia Tech |
| Source Sets | Virginia Tech Theses and Dissertation |
| Language | English |
| Detected Language | English |
| Type | Dissertation |
| Format | ETD, application/pdf |
| Rights | In Copyright, http://rightsstatements.org/vocab/InC/1.0/ |
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