Nuclear Behaviors of ERK1/2 Signaling

The rat sarcoma (Ras)- rapidly accelerated fibrosarcoma (Raf)- mitogen-activated protein kinase/extracellular signal-regulated protein kinase (MAPK/ERK) kinase (MEK)- ERK pathway is essential for proper development and homeostatic regulation in eukaryotic cells. Many pathway functions are carried out by ERK1 and ERK2 (ERK1/2), serine/threonine protein kinases that interact with a large number of substrates in several cell compartments. ERK1/2 are crucial for major cellular phenomena such as proliferation, differentiation, and programmed cell death. Despite a large body of knowledge about this pathway, a clear understanding of how specific signals elicit specific responses remains elusive. To dissect different modes of ERK1/2 regulation in the nucleus and on chromatin, I took a multifaceted approach to study nuclear ERK1/2 signaling. First, I investigated the direct DNA binding activity of ERK2 in response to phosphorylation and mutation of key residues based on characterization of ERK2 phosphorylated on T188, a residue proximal to the classically defined phosphorylation activation loop sites. To test the possibility that alternate phosphorylation inputs affect ERK2 in ways other than its kinase activity, I utilized phospho-mimetic mutants to assay oligonucleotide binding. I demonstrated that phosphorylation on the activation loop enhances ERK2 association with DNA and raised the notion that this understudied property of ERK2 dictates its interactions with DNA as well as substrates in the nucleus. Second, I investigated the regulatory relationship between ERK1/2 and CXXC finger protein 1 (Cfp1). Cfp1 is an epigenetic regulator that interacts with two major chromatin modifying complexes, and my studies clarified previously collected data to demonstrate that ERK2 and Cfp1 co-regulate target genes in a signal-dependent manner. Lastly, I sought to identify the extent of ERK1 and ERK2 interactions in the nucleus. To this end, I generated tools for a proximity biotinylation strategy that will not only identify transient and stable nuclear interactions of ERK1/2, but also address the long-standing question of whether ERK1 and ERK2 perform divergent functions. Through these approaches, I identified important areas of research to improve the current understanding of the nuclear behaviors of ERK1/2 signaling and discovered that ERK1/2 regulate gene expression through multiple modes of interaction with chromatin.