Investigation of protein kinase D3 (PKD3) and ARGHAP11A (MP-GAP) interaction: an epitope tag-based co-immunoprecipitation approach

Abstract

Protein interactions are fundamental to cellular signaling and regulatory pathways, driving diverse biological processes. This thesis investigates the potential interaction between protein kinase D3 (PKD3) and ARHGAP11A (MP-GAP) using an epitope tag-based co-immunoprecipitation approach. PKD3, a member of the protein kinase D family, is implicated in cell cycle regulation, cytokinesis, and cancer progression, while MP-GAP is a critical Rho GTPase-activating protein involved in cytokinesis. Given their roles, this study hypothesizes a functional relationship between PKD3 and MP-GAP in regulating cytokinesis via RhoA activity modulation. To explore this interaction, a system was developed for the expression and detection of epitope-tagged PKD3 and MP-GAP in mammalian cells. Optimized protocols for cloning, protein expression, and immunoprecipitation were established. Despite validation of constructs and experimental conditions, co-immunoprecipitation experiments failed to confirm a direct interaction. Challenges such as low expression levels, nonspecific antibody binding, and potential transient or weak interactions were addressed. These findings highlight the challenges inherent in studying transient or weak protein interactions, emphasizing the importance of leveraging complementary methods such as cross-linking or mass spectrometry to overcome experimental limitations. While no direct interaction was observed, this research provides insights into the complexities of PKD3’s regulatory functions in cytokinesis. It sets the stage for future studies aimed at unraveling its broader role in cell cycle regulation and its potential as a therapeutic target in cancer biology.