Browsing by Subject "Nuclear Pore Complex Proteins"
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Item Development of New Photocrosslinking Approaches to Discover Binding Partners of O-GlcNAc-Modified Proteins(2015-11-20) Rodriguez, Andrea Christine; Conrad, Nicholas; Kohler, Jennifer J.; Fontoura, Beatriz; Tu, BenjaminO-linked β-N-acetylglucosamine (O-GlcNAc) is an abundant post-translational modification that is regulated by two enzymes, O-GlcNAc transferase (OGT) and O-GlcNAc hydrolase (OGA). While it is elusive how O-GlcNAc alters protein function, altered O-GlcNAc levels are associated with human diseases. To gain insight into the functional consequences of O-GlcNAc-ylation, we reported a method to incorporate the diazirine photocrosslinking group onto O-GlcNAc residues in cellular proteins. Photocrosslinking O-GlcNAc, O-GlcNDAz, yields covalent crosslinking between O-GlcNAc-ylated proteins and their binding partners, which further analysis can confirm these interactions. I applied the GlcNDAz technology to a heavily O-GlcNAc-modified nucleoporin NUP98 and NUP98 leukemogenic fusions, produced under chromosomal translocation, to gain insight into the mechanism of NUP98 fusion-mediated cell transformation in leukemia. The wild-type nucleoporins are associated with nuclear trafficking. In chapter 2, I demonstrated both NUP98 and NUP98 fusions are O-GlcNAc-modified. Additionally, evidence suggested O-GlcNAc is near the site of interaction based on crosslinking experiments. While a powerful approach, the utility of in-cell O-GlcNDAz crosslinking was restricted by several limitations. To solve this challenge, I first engineered a mutant OGA that is better able to remove GlcNDAz from proteins in order to facilitate homeostasis of O-GlcNDAz modification in cells and for potential use as an enzyme to release crosslinked material (chapter 3). Next, I constructed a mutant OGT to preferentially add GlcNDAz to proteins, in order to maximize possible crosslinking material (chapter 4). Finally, I initiated the development of a chemo-enzymatic synthesis of O-GlcNDAz-ylated peptides using three enzymes: A bifunctional enzyme consisting of Bifidobacterium longum N-acetylhexosamine 1-kinase (NahK) and E. coli N-acetylglucosamine-1-phosphate uridyltransferase (GlmU) to generate UDP-GlcNDAz, then added GlcNDAz to peptides by human OGT (chapter 5). Together, these strategies allow for the development of a complementary cell free O-GlcNDAz crosslinking approach. O-GlcNDAz-modified peptides generated from this reaction can be crosslinked to molecules from cell lysates. This technology can be used to identify binding partners of O-GlcNAc-modified proteins, including both normal NUP98 and leukemogenic NUP98 fusions and may reveal functional roles of O-GlcNAc on NUPs and NUP fusions.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.Item The Regulation of Cellular Localization of Both Active and Inactive ERK1/2(2009-01-14) Goad, Daryl Len; Cobb, Melanie H.A key question concerning the regulation and activity attributed to the extracellular signal regulated kinase1/2 (ERK1/2) cascade is how the cellular response to different ligands, cellular environment, and other cellular signals is generated. Part of the answer to the question is the ligand and context-specific spatial control of ERK1/2 in the cell. By confining activity of ERK1/2 to localized regions, cells modulate signaling output. To gain insights into the spatial control of ERK1/2 I examined nuclear import of ERK2 mutants, the interactions of ERK2 with karyopherins β1 and β2, and explored the roles of cytoskeletal elements, motor proteins and scaffolding complexes in the localization of ERK1/2. Mutation or dysregulation of the ERK1/2 signaling cascade has been identified in a host of diseases from cancer to type II diabetes [1]. The ERK1/2 proteins have well defined regions involved in protein-protein interactions. We mutated ERK2 in these regions and tested the nuclear import of the mutants. The rationale for this series of experiments was to interfere with known docking motifs on ERK2 to determine residues essential for nuclear translocation. By varying the import conditions it was possible to identify ERK2 residues that impacted nuclear import in different contexts, in particular upon activation by phosphorylation and in the presence of energy. Mutation of certain residues only affected import of phosphorylated ERK2 in the presence of energy. From these data we hypothesize there are at least two mechanisms for nuclear entry of phosphorylated ERK1/2. Since our mutational analysis of ERK2 demonstrated an energy-dependent means of ERK2 nuclear import, we examined the potential roles of karyopherins in this process. Solution binding assays showed binding of both active and inactive ERK2 to the karyopherins β1 and β2. Both forms of ERK2 were released from the karyopherins in the presence of RanGTP. Based on these and other studies, we suggest that multiple karyopherins are involved in the energy-dependent transport of active ERK1/2. In addition to their interactions with upstream activators and downstream ligands, ERK1/2 interact with scaffolds and other regulatory proteins, such as kinase suppressor of Ras 1 (KSR1) and phosphoprotein enriched in astrocytes of 15 kDa (PEA15), a protein overexpressed in type II diabetes. In this study I mutated two serine residues on PEA15. Phosphorylation of these residues is known to affect ERK1/2 binding and interaction. Knock down of PEA15 by RNAi (RNA interference) caused an increase in cellular motility. Using a variety of cell types and immunofluorescence microscopy I was able to show the heterogeneity of the endogenous total ERK1/2 pools and the active ERK1/2 pools within the cell. Additionally, I showed distinct control of nuclear localization of endogenous ERK1/2 following treatment of cells with microtubule and actin filament destabilizing drugs.Item Toxic PRn Poly-Dipeptides Encoded by the C9orf72 Repeat Expansion Block Nuclear Import and Export(2017-03-02) Shi, Kevin; Nijhawan, Deepak; McKnight, Steven L.; Rizo-Rey, José; Rosen, Michael K.Expansion of the (GGGGCC)n hexanucleotide repeat within the first intron of the C9orf72 gene is the mutation that leads to the most prevalent heritable form of amyotrophic lateral sclerosis (ALS). The expanded repeat is aberrantly transcribed from both sense and anti-sense strands relative to the C9orf72 gene, and both transcripts are translated in an ATG-independent manner to yield five distinct poly-dipeptides. Expression of either the glycine:arginine (GRn) or proline:arginine (PRn) poly-dipeptide in Drosophila leads to neurodegneration of the eye, and when introduced into culturing medium, synthetic forms of both peptides lead to the death of human cells. We recently identified PRn poly-dipeptide interacting partners in vivo using an unbiased proteomics approach, demonstrating that the nuclear pore complex (NPC) as a major binding target. The biological significance of the PRn peptide-NPC interaction became apparent when we observed major defects in both mature mRNA export to the cytoplasm and protein import into the nucleus after cells were treated with PRn. The functional nucleocytoplasmic transport defect caused by PRn peptide was due to binding of the peptide to the central channel of the NPC as visualized by super-resolution microscopy. The NPC central channel is comprised of phenylalanine-glycine (FG) domain nucleoporins, and these proteins are essential for the NPC's active transport and passive size-exclusion permeability barrier functions. Purified FG domains of Nup54 and Nup98, both identified in our proteomics study as PRn targets, polymerized into structurally labile, cross-β sheet fibers under physiological conditions. Several lines of evidence suggested that the polymerized state of FG domains is relevant to intact NPC. First, PRn peptides only bound to polymerized Nup54 and Nup98 and not to the soluble versions of either protein, potentially representing how PRn binds to the FG-rich central channel. Second, the aliphatic alcohol 1,6-hexanediol (HD) selectively disrupted the permeability barrier of the NPC, while 2,5-hexanediol had no effect. Similarly, 1,6-HD can effectively solubilize Nup FG polymers in vitro, while 2,5-HD had no effect on polymer stability. Finally, PRn binding to the NPC abrogated the disruption of the permeability barrier by 1,6-HD. Similarly, PRn also protected the Nup FG polymers from solubilization by 1,6-HD. Our study has elucidated a major mechanism by which the C9orf72 expansion associated PRn poly-dipeptide inhibits the transport of macromolecules in and out of the nucleus, leading to a major disruption of cellular physiology. Our results support a model in which the FG domains of the NPC exist in equilibrium between the polymerized and unpolymerized states. By binding to polymerized FG domains and stabilizing them, the PRn peptide is understood to shift the equilibrium toward the polymerized state, with consequent blockage of nuclear transport. The aliphatic alcohol 1,6-hexanediol has the opposite effect, shifting the equilibrium toward the unstructured state of FG domains and disrupting the permeability barrier. The effects of the PRn peptide and aliphatic alcohols on nuclear pore function represent extreme conditions. More subtle changes in the equilibrium between structured and unstructured FG domains may regulate nuclear pore function in living cells.