Recruitment and actions of the protein kinase ERK2 in transcription complexes at the serum response element

Most animals are made up of many cells, all of which originate from a fertilised egg. As the cells in an embryo divide again and again they also change their characteristics (differentiate) so that different types of cells develop forming muscles, nerves, bones, blood and all the other tissues in the animal's body. The processes of cell division and specialisation (or development) follow blueprints contained in the animal's genetic material, the DNA in the nucleus of each cell. For cells to survive, to divide, to differentiate and even to die, information in the DNA has to be accessed, decoded and transmitted to the rest of the cell and even to other cells, rather like information on a hard disk has to be accessed for a computer to function properly. The DNA also has to be copied and passed on to each daughter cell. This research proposal addresses the mechanism by which information in the DNA is accessed. The information is packaged into units called genes of which there are about 30,000 in human cells. Genes are read (transcribed) by enzymes called RNA polymerases and these must be brought to each gene at the right time so that the flow of information is orchestrated. Control of RNA polymerases relies on other proteins called transcription factors that can select specific genes and activate them (or repress them) in response to signals from inside or outside the cell. This is the fundamental process to be studied in the research described here. It focuses on a single transcription factor called Elk-1 and how signals transmitted through the cell activate and later down-regulate Elk-1 to switch on and off a subset of genes. It is a model system from which we have already learnt much about gene transcription in general, but Elk-1 and its closest relatives are particularly important in the control of genes involved in cell proliferation and survival as well as in nerve cell functions. By continuing to study Elk-1 we hope to understand one day in molecular terms exactly how genes are regulated and also the contribution of Elk-1 to the processes of learning and memory.

Elk-1 is a key MAPK-dependent transcription factor involved in the regulation of several immediate early and pro-survival genes. Elk-1 has two MAPK docking motifs and phosphorylation of the Elk-1 carboxy-terminal domain results in conformational changes in the protein that increase its affinity for the SRF:SRE complex and promote interactions with auxiliary transcription factors such as p300/CBP and Sur2, part of the Mediator complex. Inactive Elk-1 is also sumoylated and associates with Histone Deacetylases (HDACs). Consistent with its role in activating Elk-1, phosphorylation correlates with Elk-1 desumoylation, which contributes to the activation process. The characterisation of active transcription complexes at SRE promoters by mass spectrometry has provided us with further insights into Elk-1-mediated transcriptional control. We have found that active ERKs are recruited to SRE promoter complexes directly by Elk-1 and have obtained further data suggesting that additional components of SRE promoter complexes are ERK substrates. The primary objective now is to identify the additional kinase substrates, confirming their identity as ERK substrates and as components of active SRE promoter complexes. The ubiquitin proteasome system (UPS) determines the stability of many proteins, but ubiquitin conjugation to proteins is increasingly recognised to affect a range of other functions as well. Our recent studies have revealed Elk-1 to be modified by ubiquitylation. Moreover, ubiquitylation is preceded by phosphorylation implying that ubiquitylation may serve as a mechanism for Elk-1 down-regulation. However, persistent ERK activation also appears to protect Elk-1 from ubiquitylation, which could be mediated either by Elk-1 hyperphosphorylation or by association of ERK with Elk-1 in the SRE promoter complex. A further objective of this proposal is to elucidate how ERKs influence Elk-1 ubiquitylation and how this, in turn, impacts on SRE promoter function.