Final Report Summary - PROMOTE (The molecular mechanism of the reversible switch between cell proliferation and migration. The regulatory function of the ERK pathway and ERK pathway scaffold RACK1) The ERK signalling cascade, comprised of protein kinases Raf, MEK and ERK, is an integral part of evolutionarily conserved signalling cascades that enable eukaryotic cells to sense and read a multitude of extracellular signals. ERK signalling converts extracellular signals into a variety of specific intracellular biological responses such as cell differentiation, cell movement, cell division, apoptosis and oncogenic transformation. Extracellular stimuli, including growth factors, hormones, neurotransmitters and adhesion, activate Raf which phosphorylates and activates MEK which in turn phosphorylates and activates ERK. ERK is a key regulator of intracellular responses as it phosphorylates and thus alters the function of cellular proteins leading to specific responses. Although a great deal is known about the biochemical steps involved in the transduction of signals through this pathway, considerably less is known about how multiple extracellular signals are implemented into specific biological responses. It is now widely appreciated that the activation of ERK is not the result of a simple linear sequence of enzyme reactions, but highly ordered process that involves tight regulation of all components in the pathway. The reversible assembly of signalling protein complexes is essential for the efficient transduction of signals within cells. These signalling complexes include both enzymatic proteins and proteins without known enzymatic activity - scaffold proteins, inhibitors and anchoring proteins. These non-enzymatic components regulate the amplitude, timing and localisation of the ERK signalling. Thus, the ERK pathway is embedded in the regulatory network that allows cells to respond to the incoming signal by appropriate response. Interfering with the function of individual regulatory nodes within this regulatory network re-configures the network in a way that cells respond by to the incoming signal by inappropriate biological outcome. Within the Marie Curie international reintegration grant, at the Institute of Microbiology in Prague, we are investigating the mechanism by which ERK signalling can be directed to specific functions by so called 'scaffold' proteins. These non-enzymatic proteins associate with and modulate functional interaction of the components of ERK pathway and couple the components of ERK pathway with upstream activator and downstream targets. The major goal of this project was the understanding the functioning of RACK1, a scaffold protein that associates with core kinases of the ERK pathway: Raf, MEK and ERK and targets active ERK to a specific intracellular location, focal adhesions. Specifically, we investigate the role RACK1 plays in the configuration of the ERK signalling network towards specific biological outcome, cell migration. Our work revealed novel unexpected function of RACK1 as we found that RACK1 induces the disruption of radial symmetry of adhering cells. This process, often referred to as symmetry breaking is crucial for defining cells front and rear and represent prelude to cell migration. The role of ERK and RACK1 in cell motility was further expanded by two closely related areas, the establishment of cell polarity in migrating cells and conversion of epithelial cells to migratory, mesenchymal-like phenotype. Identification of ERK substrates involved in these processes suggested that the specific cellular response is achieved through control of activity of different ERK substrates that are available for phosphorylation by ERK at a given moment in a particular location. Thus, it appears that ERK, through phosphorylation of distinct set of substrates, regulates different cellular subprograms. Consequently, the coordinated execution of these subprograms in time generates complex biological response such as cell migration.
