Doctor of Philosophy
Microbiology and Immunology
Dikeakos, Jimmy D.
PACS-1 functions are explored in three linked sections: the investigation of PACS-1 nuclear trafficking, the impact of PACS-1 on calcium homeostasis, and PACS-1 regulation of membrane protein trafficking within the regulated secretory pathway. First, our results establish PACS-1 motifs that regulate nuclear localization through the classical nuclear trafficking pathway. Moreover, we have identified a novel interaction between PACS-1 and the nucleocytoplasmic RNA-binding protein polypyrimidine tract-binding protein 1 (PTBP1), unveiling a PACS-1 nuclear trafficking function. Second, we defined a role for PACS-1 orchestrating intracellular calcium levels within corticotropic cells. We establish PACS-1 interactions with transient receptor potential cation channel 3 subfamily C (TRPC3) and extended synaptotagmin 1 (ESyt-1), demonstrating that PACS-1 controls the trafficking of these proteins to the plasma membrane. Furthermore, we identify ESyt-1 as a negative regulator of adrenocorticotropic hormone (ACTH) secretion that requires PACS-1 to complete this regulation. Third, we elucidated adaptor protein complex 1 (AP-1) residues that interact with PACS-1. We further demonstrate that PACS-1 interacts with the ACTH processing enzyme peptidylglycine alpha-amidating monooxygenase (PAM) and is required for sorting PAM into the regulated secretory pathway in corticotropic cells. In conclusion, this research significantly advances our understanding of PACS-1's diverse functions within cells. By identifying trafficking functions for PACS-1 within nucleocytoplasmic shuttling, membrane trafficking of calcium ion channels, and retaining essential prohormone processing proteins within the regulated secretory pathway, this research truly underscores the multifunctional roles PACS-1 executes to maintain cellular homeostasis.
Summary for Lay Audience
An important protein, phosphofurin acidic cluster sorting protein 1 (PACS-1), is expressed in all human cells and has multiple biological functions. My thesis is focused on PACS-1 and is divided into three sections, each exploring different roles of PACS-1 functions within cells. In the first section, I identified specific regions of PACS-1 that control its movement into the nucleus and unveiled the mechanism PACS-1 uses to shuttle in and out of the cell's nucleus. In the second section, I used a robust genetic engineering technique to remove PACS-1 from cells, studying its impact on the cell's internal calcium levels and the subsequent secretion of a specific hormone known as adrenocorticotropic hormone (ACTH). I discovered that PACS-1 interacts with two other proteins, transient receptor potential cation channel subfamily C member 3 (TRPC3) and extended synaptotagmin 1 (ESyt-1), controlling how these proteins move to the cell's surface. My data suggests that PACS-1 is essential for controlling intracellular calcium levels with TRPC3 and regulating the secretion of ACTH with ESyt-1. Lastly, in the third section, I investigated PACS-1's interactions with other proteins in the regulated secretory pathway of neuroendocrine cells. I further elucidated how the adaptor protein complex 1 protein, which aids in moving proteins around in cells with PACS-1, interacts with PACS-1. I also discovered that PACS-1 interacts with an enzyme known as peptidylglycine alpha-amidating monooxygenase (PAM), which is crucial for processing ACTH prior to hormone secretion. These findings provide novel insights into the mechanisms of how PAM is localized within cells. In conclusion, my research significantly broadens our understanding of the complex role of the PACS-1 protein in cells. The results presented in this thesis highlight how PACS-1 regulates biological processes in multiple places within the cell, demonstrating multifaceted functions in maintaining the normal function of cells.
Trothen, Steven, "Novel Roles of PACS-1 Within the Nucleus and the Regulated Secretory Pathway" (2023). Electronic Thesis and Dissertation Repository. 9789.
Available for download on Wednesday, January 01, 2025