Development of a Genetically Encodable Fluorescent Biosensor to Examine the Spatiotemporal Regulation of NEK Kinases in Living Cells

Abstract

<b>Abstract ID 26685</b> <b>Poster Board 548</b> Never in mitosis A-related kinase (NEK) family members are an understudied, yet important, family of 11 mitotic kinases that play a critical role in cell division and the cellular response to replication stress and DNA damage. The pleiotropic relationship between this kinase family and human disease is widely recognized, yet poorly understood, particularly with respect to the spatial and temporal regulation of NEK family members within the cell. Characterizing NEK kinase activity within the endogenous cellular environment requires the development of molecular tools, such as genetically targetable fluorescent biosensors, that can track changes in the activity profiles of NEK family members in living cells with high spatiotemporal resolution. Here, we describe the development and initial characterization of a novel excitation ratiometric indicator-based NEK activity reporter (ExRai-NEKAR). ExRai-NEKAR, which was created using a generalizable molecular cloning strategy based on Gibson Assembly, exhibited a 5.5-fold increase in its 395 nm/475 nm excitation ratio in the presence of NEK1 and ATP <i>in&nbsp;vitro</i>. In contrast, no change in the excitation ratio was observed when the non-hydrolyzable ATP analog, AMP-PNP, was substituted for ATP in the assay, suggesting that the observed changes in ExRai-NEKAR’s excitation ratio are dependent on NEK1-mediated phosphorylation and not protein-protein interactions. Interestingly, while ExRai-NEKAR was also efficiently phosphorylated by NEK5 leading to a robust response, incubation with either NEK2 or NEK9 caused only marginal changes in ExRai-NEKAR’s excitation ratio, consistent with previous studies suggesting that NEK1 and NEK5 share a similar consensus motif that is distinct from those preferred by NEK2 and NEK9. Importantly, treatment of 293T cells with 200 mM H₂O₂ led to a similar increase in ExRai-NEKAR fluorescence <i>in situ</i>, consistent with NEK1’s role in the DNA damage response (DDR). We are currently using a series of novel NEK inhibitors and other pharmacological agents to further characterize ExRai-NEKAR’s response in cells and to better understand the regulation of NEK family members within the endogenous cellular environment. Together, our data suggest that ExRai-NEKAR is efficiently phosphorylated by some NEK family members (e.g., NEK1 and NEK5) but not by others (e.g., NEK2 and NEK9). Likewise, live cell imaging experiments suggest that ExRai-NEKAR and its derivatives could be useful tools for monitoring real-time changes in the activity profiles of specific NEK family members with high spatiotemporal resolution under a variety of cellular conditions such as in response to pharmacological, toxicological, or pathological agents. This research was supported by DoD Grant #78992-DD-RTL (to RHN), NIH/NCI grant 1R01CA273095 (to DHD and RHN), NSF grant #DBI-1852319 (to RHN).