Author: Amber Lynn Lasek

Publisher:

Published: 2016

Total Pages: 0

ISBN-13:

Protein interactions within a living cell are complex and are necessary for transmitting signals quickly and accurately. Kinases provide an important source of regulation and communication and play important roles for triggering and maintaining signaling cascades. These processes are themselves restrained so that they occur at the correct time and place. Due to the complexity of these processes and the need for improved methods, there remain many unresolved questions regarding the interconnectedness of signaling pathways and the extent that posttranslational modifications on kinases play for regulating kinase activity and function. Many kinases are known to be activated by phosphorylation within its catalytic loop; however, these proteins have multiple sites of modification including additional sites of phosphorylation and ubiquitination. Whether and how much these sites influence the activity and function of the kinase is unresolved. Using analog-specific kinases, I have discovered that Plk1 is not only regulated by the activating phosphorylation within the catalytic loop, T210, but also requires a neighboring site, T214. Additionally, other phosphorylation sites within the catalytic domain, but not in the C-terminal localization domain, redundantly control mitotic functions of Plk1 and cell cycle progression. These results demonstrate the intricacy of signaling to control and regulate Plk1. Further analysis of the complexity of signaling within a cell is demonstrated by comparing the pathways of two proteins. For a cellular process, involved proteins can act in parallel or overlapping pathways. If they overlap, can moderately perturbing a pathway from multiple directions simultaneously cause a greater response than each individual assault? This idea was used to design a screen between differing therapeutic agents. Many of the drugs include those that directly target proteins key to signaling pathways, kinases, while others were agents that would activate signaling pathways through error induction, such as mitotic checkpoint signaling activation with nocodazole treatment. Aurora A inhibition was found to potentiate the effect of a Plk1 inhibitor, a pan-Cdk inhibitor, and DNA damage response inducer, IUdR. Overall, this work expands the knowledge of and provides insight into the way that signaling networks function within the cell.