Quantitative Analysis of Signaling Pathways: Imaging and Modeling of the Terminal Patterning System of the Drosophila Embryo

Abstract

One of the key concepts of Systems Biology is that of a module, defined as a network with a function that is largely independent of its context. Based on the modular decomposition of large networks it may be possible to predict their dynamics and function from properties of constituent modules. Integral to such an approach is the ability to define individual modules and distinguish their core components from the rest of the network. While conceptually simple, this task can be highly nontrivial in reality.

In my thesis, I analyzed the mitogen activated protein kinase (MAPK) pathway, an essential regulator of cellular processes in all eukaryotes. The MAPK pathway is a three-tiered cascade of phosphorylation-dephosphorylation cycles. An input to the pathway can be provided by a cell surface receptor; its output is the activation level of MAPK, a kinase at the bottom of the cascade. MAPK is activated when it is phosphorylated by a MAPK kinase; this process is reversed by a MAPK phosphatase. Active MAPK controls cellular processes by phosphorylating its substrates. According to the current models, the dynamics of the MAPK cascade can be understood independently of MAPK substrates. This unidirectional view of MAPK signaling is consistent with a large body of work, but the reality is more complex: The enzymes that regulate MAPK and the substrates phosphorylated by MAPK have the potential to interact with the same domains on the MAPK protein. Thus, MAPK substrates can compete with each other, as well as with the MAPK regulators for binding to MAPK.

The experimental work in my dissertation demonstrates that this is indeed the case. Using the early Drosophila embryo as an experimental system, I have shown that the level of MAPK substrates can influence the activation status of this enzyme and its ability to distribute its activity among multiple substrates. In the first part of my thesis, I used a combination of genetic and imaging approaches to show that the spatial pattern of MAPK activation in the early embryo exhibits a striking asymmetry (Chapter 2 and Chapter 3). My subsequent genetic experiments revealed that this asymmetry is due to the spatially non-uniform distribution of the MAPK substrates that compete both among themselves and with the regulators of MAPK for access to this enzyme (Chapter 3 and Chapter 4). I have developed a chemical kinetics theory of the observed substrate competition effect and successfully tested this theory in vivo, using a large number of mutants in the MAPK pathway (Chapter 4). Finally, I was able to demonstrate that MAPK substrate competition is biologically significant and play a key role in the regulation of gene expression and cell differentiation in the embryo (Chapter 5 and Chapter 6). Going beyond the early Drosophila embryo, I propose that enzyme substrate competition is an important regulatory strategy in biomolecular networks where enzymes, such as MAPK, interact with their multiple regulators and substrates.