Phosphorylation of eukaryotic translation initiation factor 2α (eIF2α) is a conserved eukaryotic mechanism to limit protein synthesis under stress conditions. Baculovirus PK2, which resembles the C-terminal half of a protein kinase domain, inhibits eIF2α family kinases in vivo, thereby increasing viral fitness in the face of host immunological and stress responses. The mechanism by which PK2 modulates eIF2α stress response signaling remains unknown. To address this issue, a combination of biochemical, biophysical and in vivo approaches were employed to probe the mechanism of PK2 inhibition on a prototypical human eIF2α kinase, the RNA-dependent protein kinase (PKR). We discovered that PK2 inhibits PKR catalytic activity by directly binding its kinase domain. This direct interaction requires both the kinase-like C-lobe fold of PK2 and a critical 22 residue N-terminal extension that precedes it. We further show that the PK2 N-terminal extension is required but not sufficient for the ability of PK2 function.

Phosphorylation of eukaryotic translation initiation factor 2α (eIF2α) is a conserved eukaryotic mechanism to limit protein synthesis under stress conditions. Baculovirus PK2, which resembles the C-terminal half of a protein kinase domain, inhibits eIF2α family kinases in vivo, thereby increasing viral fitness in the face of host immunological and stress responses. The mechanism by which PK2 modulates eIF2α stress response signaling remains unknown. To address this issue, a combination of biochemical, biophysical and in vivo approaches were employed to probe the mechanism of PK2 inhibition on a prototypical human eIF2α kinase, the RNA-dependent protein kinase (PKR). We discovered that PK2 inhibits PKR catalytic activity by directly binding its kinase domain. This direct interaction requires both the kinase-like C-lobe fold of PK2 and a critical 22 residue N-terminal extension that precedes it. We further show that the PK2 N-terminal extension is required but not sufficient for the ability of PK2 function.

Decades of research have begun to identify oncogenic mut-drivers that are responsible for driving a large percentage of cancers. These high frequency mut-drivers have therapeutics in the clinic for patient treatment. However, there is another group of low frequency mut-drivers that fail to rise above the noise of the high frequently drivers. These low frequency drivers represent a group of genes with untapped potential for new targeted therapies. However, identifying these drivers can be difficult. This study focuses on identifying new functional phosphorylations using the phospho-docking protein 14-3-3. The family of 14-3-3 proteins have been linked to many oncogenic pathways due to the diversity in their client protein interactions. One critical problem in studying 14-3-3 interactors is uncovering the docking site on the phospho-binding partner. Our work indicates that intrinsic disorder and unbiased mass spectrometry identification rate of a given phosphorylation are important for improving the selection of a 14-3-3 docking site. Using a machine learning model, we developed a tool that combines current available 14-3-3 prediction data and our observations about disorder and phosphorylation observation to predict 14-3-3 binding sites. Our publicly available tool "14-3-3-site-finder" produces a rank order list of potential 14-3-3 docking sites that could help overcome the time-consuming process of identifying the correct site. In our efforts of identifying functional phosphorylations with 14-3-3, we have observed that 14-3-3 interacts with a non-receptor tyrosine kinase, TNK1. TNK1 is a poorly characterized kinase that has essentially nothing known about its substrates, function or regulation. TNK1 has been implicated in both tumor suppressor and oncogenic roles. Particularly, a Hodgkin Lymphoma cell line is dependent on a truncated form of TNK1 for growth. Our work uncovers the first mechanism of regulation for this kinase. We found that MARK kinase phosphorylates TNK1 within the proline rich domain allowing 14-3-3 to dock on this phosphorylation. 14-3-3 binding restrains TNK1 in the cytosol and holds TNK1 in an inactive state. Upon the release of 14-3-3, TNK1 moves to a membrane fraction where it is active. One unique feature of TNK1 is that it has a c-terminal ubiquitin association domain (UBA). In vitro ubiquitin pulldowns indicate that the TNK1 UBA has no preference for linkage type or length of ubiquitin. Further, biolayer interferometry indicates that the UBA binds ubiquitin tightly. Mutation of residues within the ubiquitin:TNK1 interface prevent ubiquitin binding and decrease TNK1 activity, preventing downstream oncogenic signaling, suggesting a UBA-centric mechanism of regulation for TNK1. Finally, we developed a small molecule inhibitor, TP-5801, that selectively targets TNK1. TP-5801 prevents downstream TNK1 phosphorylation of STAT3. Further, TP-5801 prolonged the life of mice injected with TNK1 driven Ba/F3 cells. Taken together, our data reveal the first mechanism of kinase regulation for TNK1 involving 14-3-3 binding and ubiquitin association as well as the development of a TNK1 specific therapeutic