Browsing by Subject "Mitogen-Activated Protein Kinase 1"
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Item Identification of a Novel ERK 1/2-Interacting A-Kinase Anchoring Protein(2009-06-17) Jivan, Arif; Cobb, MelanieInitially 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(2011-02-01T19:32:08Z) Duan, Lingling; Cobb, MelanieIn 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 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.