Browsing by Subject "Mitogen-Activated Protein Kinase 1"
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Item Identification and Characterization of the Multifunctional Epigenetic Regulator CFP1 as an ERK1/2 Substrate(2014-11-21) Klein, Aileen Melanie; Sternweis, Paul C.; Cobb, Melanie H.; Goodman, Joel M.; Conrad, NicholasEpigenetic regulation of gene transcription occurs as an integration of multiple layers of signals at a genetic locus. These signals can include local chromatin structure, covalent modifications to both histone proteins and DNA, the presence of transcription factors, and modification directly to the transcriptional machinery. Our lab is interested in the control of cellular processes by the mitogen activated protein kinases ERK1/2. In a yeast two-hybrid screen with activated ERK2 (extracellular signal-regulated kinase 2) to find novel interacting partners, our lab identified CFP1 (CxxC finger protein 1), a DNA-binding protein that is a vital component of the H3K4 trimethylating Set1A/B complexes to promote gene transcription. CFP1 has also been shown to interact physically and functionally with the major maintenance DNA methyltransferase DNMT1. We are interested in defining how substrate targeting of CFP1 by ERK1/2 regulates downstream transcriptional outcomes. Interaction between ERK2 and CFP1 in cells was validated by co-immunoprecipitation from isolated mononucleosomes. Active ERK2 can phosphorylate CFP1 on multiple sites in vitro, an observation supported by studies in cells. Some of the most likely in vivo ERK1/2 phosphorylation sites include serine 224 and threonine 227. CFP1 is essential for focusing trimethylation of H3K4 at promoters, a histone modification that supports transcription from these loci. We hypothesized that phosphorylation of CFP1 by ERK1/2 during mitogenic signaling may support trimethylation of H3K4 and transcription of ERK1/2-regulated target genes. Introduction of CFP1 containing the mutation T227V into HeLa cells blocked global H3K4 trimethylation to a similar extent as CFP1 depletion. On the other hand, CFP1 S224A shows diminished transactivation capacity against a model transcriptional substrate. Neither of these mutants fail to interact with Set1B in a pulldown, suggesting that these sites may be important for Set1 complex targeting or activity towards chromatin. Consistently, CFP1 knockdown hinders induction of several ERK1/2-regulated immediate early gene targets in response to serum treatment. It will be of interest to test whether this is dependent on stable or inducible H3K4 trimethylation and what impact overexpression of point mutants will play in their transcription. Regulation of H3K4 trimethylation through CFP1 phosphorylation might represent a novel regulatory input to support transcription of ERK1/2-regulated genes.Item Identification of a Novel ERK 1/2-Interacting A-Kinase Anchoring Protein(2009-06-17) Jivan, Arif; Cobb, Melanie H.Initially identified in Chlamydomonas, radial spoke protein 3 (RSP3) is one of at least twenty identified radial spoke structural components of motile cilia and is required for axonemal sliding and flagellar motility. The mammalian orthologs for this and other radial spoke proteins, however, remain to be identified and fully characterized. Mammalian RSP3 was found to interact with ERK2 through a yeast two-hybrid screen designed to identify interactors that have a higher affinity for the phosphorylated, active form of ERK2. Confirming this finding, the human homolog long form, RSP3H, co-immunoprecipitates with ERK1/2 in HEK293 cells. Human RSP3, and its larger alternative start site gene product, radial spoke protein 3 homolog (RSP3H), are phosphorylated by ERK1/2 on threonine 286 in vitro and in cells. RSP3/RSP3H are also phosphorylated in vitro by cAMP-dependent protein kinase (PKA). Additionally, we showed that human RSP3H functions as an A-kinase anchoring protein (AKAP), and its ability to bind to the regulatory subunits of PKA, RII and RII, is regulated by ERK1/2 activity and phosphorylation. Interestingly, expression analysis of mRNA suggests RSP3/RSP3H are also present in cells that are thought to contain a single primary cilium but not motile cilia. Immunofluorescence staining of primary cilia-containing cells indicates that RSP3/RSP3H localize to nuclear punctae, specifically promyelocytic leukemia (PML) bodies, suggesting a non-cilia related role for RSP3/RSP3H in these cells. Functionally, RSP3/RSP3H may localize ERK1/2 to a distinct site of action within the cell and serve as a point of convergence of cAMP-dependent and PKA-mediated influence upon ERK1/2 downstream signaling or vice versa. These data are the first to establish a connection between ERK1/2 and what was once ostensibly thought to only be a ciliary component as well as to identify a novel ERK1/2-interacting AKAP.Item MAPK Signaling Pathways in Pancreatic Beta Cells: The Regulation of RAF Activation by Nutrient Stimuli(2011-02-01) Duan, Lingling; Cobb, Melanie H.In pancreatic β cells cells, ERK1 and ERK2 participate in nutrient sensing and their activities rise and fall as a function of glucose concentration over the physiologic range. Glucose metabolism triggers calcium influx and release of calcium from intracellular stores which are required for ERK1/2 activity. Calcium influx also activates the calcium-dependent phosphatase calcineurin, which is required for maximal ERK1/2 activation by glucose. Calcineurin controls insulin gene expression by ERK1/2-dependent and -independent mechanisms. This study showed that in β cells, glucose activates the ERK1/2 cascade primarily through B-Raf. Glucose also enhances dimerization of B-Raf with C-Raf. Furthermore, calcineurin up-regulates B-Raf activity and stabilizes C-Raf/B-Raf in response to glucose. Calcineurin binds to B-Raf in both unstimulated and stimulated cells. B-Raf phospho-T401 is one of the target sites that can be dephosphorylated by calcineurin. This study reveals that cross-talk between Raf and calcineurin is essential for the maximal activation of ERK1/2 in the glucose signaling pathways. [Keywords: TCF; POP-1; Wnt; embryogenesis; C. elegans]Item Nuclear Behaviors of ERK1/2 Signaling(2017-04-18) Karra, Aroon Saikumar; D'Orso, Iván; Cobb, Melanie H.; Conrad, Nicholas; Pearson, Gray W.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.Item Nucleocytoplasmic Localization of MAPKs(2007-08-08) Yazicioglu, Mustafa Naci; Cobb, Melanie H.Mitogen-activated protein kinases (MAPKs) comprise a family of protein-serine/threonine kinases, which participate in signal transduction pathways that control intracellular events. MAPKs are regulated by phosphorylation cascades, which are usually initiated by external stimuli including a variety of ligands. At least two upstream protein kinases are activated in series to lead to activation of a MAPK. The kinase that activates the MAPK is a MAPK kinase (MAP2K or MEK) and the kinase that phosphorylates the MAP2K is a MAP3K or MEK kinase (MEKK). Upon activation, MAPKs may translocate to the nucleus to phosphorylate nuclear targets. Previous findings from our laboratory showed that a constitutively active and nuclear form of the MAPK ERK2 is sufficient for transformation of immortalized fibroblasts (Robinson MJ et al,1998). However the mechanisms of nuclear localization of MAPKs are still not fully understood clearly. Although most nucleocytoplasmic localization events require carrier proteins known as karyopherins (importins and exportins), ERK2 enters the nucleus of permeabilized cells even if these carrier proteins are missing. This is explained by direct binding to proteins in the nuclear pore complex (NPC). Similar to ERK2 targets, NPC proteins also contain Phe-Xxx-Phe (FXF) motifs. My first aim in this project was to examine the roles of ERK2 residues that are crucial for FXF binding on nuclear localization of ERK2. Mutating these ERK2 residues decreased the nuclear import of ERK2 proteins in permeabilized cells. Secondly, the regulation of ERK2 nuclear export was analyzed. It was observed that ERK2 export occurs by two distinct processes; one energy-dependent and the other energy-independent. My final aim was analyzing the activation and nucleocytoplasmic trafficking of other MAPKs, JNK and p38.