INSIGHTS INTO THE REGULATION OF aPKC POLARITY THROUGH PROTEIN-PROTEIN INTERACTIONS
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Date
2024-03-25
Authors
Vargas, Elizabeth
Journal Title
Journal ISSN
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Publisher
University of Oregon
Abstract
Cell polarity is a crucial factor in enabling a cell to carry out its specialized functions during animal development and homeostasis. It involves the organized distribution of cellular components into distinct regions, playing a critical role in various processes like asymmetric cell division, cell migration, and epithelial morphogenesis. One critical regulator of animal cell polarity is the protein atypical Protein Kinase C (aPKC), whose catalytic activity is essential for directing the localization of downstream polarity proteins. Consequently, precise regulation of aPKC becomes imperative for the proper control of cell polarity.The regulation of aPKC's polarization and activity involves interactions with several proteins, including Par-6, Cdc42, and Par-3. However, the mechanisms through which these proteins exert their regulatory influence on aPKC have remained a subject of confusion within the cell polarity field. This dissertation investigates the intricate intermolecular interactions responsible for regulating aPKC to establish proper cell polarity.
In the first part of this work, we focus on regulation of aPKC by Par-6. Existing models suggest contradictory roles for Par-6 in either activating aPKC or relieving its autoinhibition while keeping it catalytically inactive. To address this ambiguity, we conducted structure/function analyses and in vitro binding assays and found that Par-6 may inhibit aPKC's catalytic activity through a novel interaction involving Par-6's C-terminus. More studies need to be done to address how this new interaction may be regulating aPKC’s kinase domain.
We then turn our focus onto how Par-3 interacts with aPKC and Par-6, which together form the Par complex. Previous studies have reported multiple interactions through various biochemical assays. To gain some clarity, we utilized qualitative and quantitative binding assays to understand (1) which domains within Par-3 contribute to its interaction with the Par complex and (2) the overall binding energy contributed by these interactions. Our results indicate that Par-3 PDZ2 and PDZ3 domains bind to the aPKC Kinase domain-PBM region.
Lastly, we set out to determine the mechanism behind the transition of the Par complex between its two regulators, Par-3 and Cdc42, to form two distinct complexes. While one biochemical study suggested simultaneous interaction of Par-3 and Cdc42 with the Par complex, in vivo data suggested the formation of separate complexes. Our qualitative binding assays show that Par-3 and Cdc42 negatively cooperate for binding to the Par complex. Questions remain on the detailed mechanism of competition.
In summary, this dissertation elucidates the intricacies of aPKC regulation and provides valuable insights into the mechanisms by which Par-6, Cdc42, and Par-3 contribute to the control of aPKC’s localization and activity.
This work contains both unpublished and published co-authored materials.