Date of Award

9-2014

Embargo Period

7-15-2015

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biological Sciences

Advisor(s)

Brooke M. McCartney

Abstract

Development of an embryo is a fascinating biological process that requires effective communication between neighboring cells and coordination of movement across the entire organism. At a cellular level, this is achieved by upstream signaling pathways ultimately regulating gene expression to provide cells with cues to perform certain tasks such as cell division, migration, cell rearrangements or changes in cell shape. All of these diverse tasks ultimately rely on rearrangements of the cytoskeleton. However, it is unclear what the molecular connections are between signaling and cytoskeletal dynamics. Adenomatous Polyposis Coli (APC) is a multifunctional protein that plays vital roles both in regulating the canonical Wnt signaling pathway and the cytoskeleton. Mutations of APC are associated with more than 80% of both familial and sporadic colorectal cancer cases. APC is one of few cytoskeletal proteins with direct links to cancer. However, as a multi-domain, multifunctional protein, a comprehensive understanding of APC biology has been difficult to achieve. While we have known for almost 20 years that APC proteins are essential negative regulators of Wnt signaling, the precise role they play both in regulating Wnt signaling and cytoskeletal have been unclear. In order for APC proteins to perform these diverse tasks and regulate both signaling and the cytoskeleton, it needs to be highly regulated itself. In my Ph.D. project, I approached APC proteins from many different angles, in many different developmental contexts, to gain insights into the precise role they play and how they are regulated in both the Wnt signaling and the cytoskeletal context. To better understand how APC proteins are regulated, I used Drosophila as a simpler, more tractable model. The large size of the vertebrate APCs (~300 kDa) makes it difficult to perform structure/function studies in the context of a full-length protein. Similar to vertebrates, flies have two highly conserved APC proteins (APC1 and APC2). Thus, I chose to study the fly APC homologs and mostly focused on the smaller member of the family, APC2, in my studies. To elucidate how APC proteins are regulated in the context of Wnt signaling, first we dissected the role of phosphorylation in the context of Wnt signaling. APC proteins are highly phosphorylated, and this plays a role in APCs activity in Wnt signaling. As a part of the destruction complex, APC targets the key effector of the pathway, ß-catenin for degradation. Phosphorylation of the central 20 amino acid repeats (20Rs) has received the most attention over the last decades, and has been shown to change the affinity of β-catenin binding in vitro. However, many of these in vitro models lacked an in vivo model. To test the functional significance of 20R phosphorylation in Wnt signaling, we used Drosophila APC2 and took advantage of the awesome power of genetics in this model organism. Our studies showed for the first time in an intact animal that 20R phosphorylation played an essential role. This study also suggested functional diversity among different 20Rs as well as gave us hints about the presence of macromolecular destruction complex, which we coined the term “destructosome’ (see Chapter 2). Besides the phosphorylation of the 20Rs, phosphorylation of other APC domains, such as the Axin binding SAMP repeats, had not been investigated before. Therefore, I also studied the phosphorylation of SAMP repeats and tested if it played a functionally significant role in APCs Wnt function. Similar to the 20Rs, I’ve shown that SAMP phosphorylation plays a previously uncharacterized role in APCs Wnt signaling function and proposed a novel idea of functional diversity among different SAMP repeats (see Chapter 3). As mentioned above, while studying the importance of 20R phosphorylation, I got interested in the idea of higher order destruction complex structures, or destructosome. This led me to think about the role of APC proteins in the assembly of this complex. Although it has been long appreciated that human APC can self-associate, the precise role of self-association in Wnt signaling hasn’t been explored in part due to the complexity of self-association in the vertebrate APC (vAPC) proteins. By using Drosophila APC2, I’ve identified a novel self-association domain (ASAD) and uncovered a new role for APC proteins in promoting the assembly and stability of the destructosome (see Chapter 4). I was interested in APC phosphorylation not only in the context of Wnt signaling but also in APCs cytoskeletal roles. One of the emerging themes in APCs role in regulating the actin cytoskeleton is its interaction with the formin Diaphanous (Dia). Previous work from our laboratory suggested that Drosophila APC2 and Dia cooperated during the formation of actin based structures during embryogenesis and this interaction was regulated. In order to understand this relationship further, I tested the role of phosphorylation as potential regulatory mechanism. My studies showed, in deed phosphorylation played a role in APCs activity in this context too (see Chapter 5). This study also revealed a potential cross talk between two pools of actin (linear and branched). In summary, studying APC, an exciting and highly complex protein, allowed me to think about many different biological questions from signaling to cytoskeleton in various developmental contexts. The findings from my Ph.D. research uncovered new aspects of APC biology, and showed how various regulatory mechanisms weather it’s phosphorylation or self-association, affect its functions, both during Wnt signaling and also in regulating the actin cytoskeleton. My studies will also help better understand the disease relevance of human APC proteins and provide novel insights.

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