Investigating the effects of signaling cross-talk on regimes of Erk dynamics with a high-throughput drug screen

Abstract

The Ras/Erk mitogen activated protein kinase pathway facilitates many important cellular processes, such as proliferation and differentiation. This pathway consists of a tightly controlled sequence of protein phosphorylation interactions that somehow encodes multiple cell fates. Two theories explain this plasticity by suggesting that the pathway’s activity dynamics or its interactions with other pathways determine its cell fate decision. In this thesis, I explored both theories by investigating the effects of kinase inhibitors on dynamic Erk activity and identifying pathways that combinatorially interact with Erk. I conducted a high-throughput drug screen in primary murine keratinocytes modified to express a reporter of Erk activity and a nuclear marker. I created computational pipelines to identify and analyze dynamic features of Erk activity pulses. From my analysis, I found that drug treatments pushed Erk dynamics into low-activity regimes, implying that targeted signaling pathways did not negatively regulate Ras/Erk activity. In addition, I found that frequency and length of Erk activity pulses were highly constrained and that dynamic features intercorrelated. These findings imply that cells exert tight control over pulse length and frequency and that they rigidly coordinate features of Erk activity. Finally, I analyzed drug categories that dampened Erk activity in order to identify drug targets that positively interact with the Ras/Erk pathway. In summary, I conducted a thorough exploration of regimes of Erk behavior, aiding the eventual goal to map dynamic Erk signaling to cell fate decision-making.