Browsing by Subject "14-3-3 Proteins"
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Item Characterizing the Molecular Mechanisms of Axon Guidance: Activation and Regulation of the Axon Guidance Receptor Plexin A(2012-07-17) Yang, Taehong Yang; Terman, Jonathan R.Neuronal connectivity is precisely determined by axonal pathfinding during development. The navigating axons detect attractive and repulsive environmental cues by axon guidance receptors. However, the biochemical means through which multiple signaling pathways are integrated in navigating axons is poorly understood. Semaphorins are the largest family of axon guidance cues and utilize Plexin receptors to exert repulsive effects on axon extension. The intracellular region of Plexins contains a Ras GTPase activating protein (GAP) domain, which is necessary for repulsive guidance effects. Previous studies suggest that activation of Plexin RasGAP requires interactions with both Semaphorin at the extracellular region and a Rho-family GTPase at the Rho family GTPase-binding domain (RBD). Interestingly, Semaphorin repulsion can be rapidly "turned-off" by other distinct cues and signaling cascades. However, the molecular mechanisms to activate or modulate Plexin RasGAP remain unclear. First, to further understand how the Plexin RasGAP is activated, I collaborated with the Zhang lab, and following determination of the crystal structure of the intracellular region of Plexin, I examined the roles of residues interfacing with the RasGAP domain using functional mutagenesis in the Drosophila model system. Our results demonstrate that Plexin exhibits an auto-inhibited conformation, and suggest that interaction among the previously uncharacterized juxtamembrane segment, the RBD, and the RasGAP domain is critical for Plexin RasGAP activation. Second, to better understand how Semaphorin/Plexin signaling is modulated, I characterized the results of a large-scale screen to look for proteins interacting with the cytoplasmic portion of Plexin and identified the phosphoserine binding protein 14-3-3epsilon as a specific Plexin-interacting protein. My results reveal that 14-3-3epsilon is specifically required for axon guidance during development. Moreover, Protein kinase A is found to phosphorylate Plexin in the RasGAP domain and mediates the 14-3-3epsilon interaction. Plexin-14-3-3epsilon interactions prevent Plexin from interacting with its Ras-family GTPase substrate, which effectively switches Plexin-mediated axonal repulsion to Integrin-mediated adhesion. These findings uncover both a new molecular integration point between important axon guidance signaling pathways and a biochemical logic by which this guidance information is coalesced to steer the growing axon. Therefore, these new observations on activating and silencing specific signals that are repulsive to axon growth also illuminate new approaches to neutralize axonal growth inhibition and encourage axon regeneration.Item The Hippo Signaling Pathway in Organ Size Control and Regeneraton(2012-07-17) Ren, Fangfang; Jiang, JinThe Hippo (Hpo) signaling pathway controls cell growth, proliferation and apoptosis in both Drosophila and vertebrates. Our lab has previously demonstrated that Hpo signaling regulates gene expression by inhibiting a transcription complex consisting of the transcriptional coactivator Yorkie (Yki) and the TEAD/TEF family of transcription factor Scalloped (Sd) in Drosophila. The inhibition of Yki activity is through modulating its phosphorylation status and subcellular localization by upstream kinase complex. I obtained both genetic and cellular evidence that 14-3-3 proteins are involved in this process. I also identified three Serine residues (S111, S168 and S250 of Yki as essential for restricting Yki activity. I found that 14-3-3 regulates Yki subcellular localization mainly through S168 but not the other two sites. The recent identification of intestinal stem cells (ISCs) has made the Drosophila adult midgut an excellent model to study adult stem cell biology. Multiple signaling pathways have been implicated in the regulation of ISC proliferation, self-renewal and differentiation. I obtained evidence that Hpo signaling plays an essential role in regulating ISC proliferation through both cell-autonomous and non-cell-autonomous mechanisms. Cytokines of the Upd family and multiple EGFR ligands were found to be ectopically induced when Hpo signaling is inactivated in differentiated cells, which in turn activate Jak-Stat and EGFR signaling pathways in ISCs to stimulate their proliferation. I also showed that tissue damaging reagent DSS-induced ISC proliferation is dependent on Yki activity in precursor cells. Although several signaling pathways including Jak-Stat, EGFR and Hpo pathways have been implicated in damage-induced ISC proliferation, the cell intrinsic mechanisms have remained elusive. I found that the Drosophila homolog of Myc oncogene (dMyc), which encodes a transcription regulator that affects cellular growth and cell cycle progression, functions downstream of Hpo, Jak-Stat and EGFR pathways to mediate their effects on ISC proliferation. dMyc is also essential for adult midgut homeostasis as well as regeneration after exposure to damage reagents. I also demonstrated that the regulation of dMyc levels by Hipo, Jak-Stat and EGFR pathways is at the level of transcription. [Keywords: Drosphila, hippo, Yki, instestine stem cell, regeneration]Item The Study of WNT Signaling Effector POP-1/TCF in c. Elegans Early Embryos(2005-04-29) Lo, Miao-Chia; Lin, RueylingIn C. elegans embryos, the combined Wnt/MAPK pathway polarizes the founder cell of mesendoderm, EMS blastomere, such that EMS produces two daughters with distinct developmental fates. The posterior daughter E, whose fate is specified by Wnt/MAPK, generates intestinal tissues (endoderm), whereas the anterior daughter MS generates pharynx and muscle cells (mesoderm). The downstream Wnt/MAPK effector POP-1 is asymmetrically localized in the nuclei of A-P sisters including the MS/E pair, with a higher level in the anterior cells. This phenomenon is called POP-1 nuclear asymmetry. The Wnt/MAPK signaling is required for POP-1 nuclear asymmetry. It is believed that POP-1 represses endoderm fate in MS and Wnt/MAPK allows endoderm fate in E by downregulating the nuclear level of POP-1. In this study, the potential mechanisms for POP-1 nuclear asymmetry are presented. POP-1 nuclear asymmetry requires a 14-3-3 protein PAR-5 and at least three POP-1 potential phosphorylation sites for the MAPK LIT-1. LIT-1 activity is required for both POP-1/PAR-5 interaction and phosphorylation of at least two of the three potential LIT-1 sites in vivo. Nuclear export is also required for POP-1 nuclear asymmetry. The nuclear level of LIT-1 is higher in the E blastomere, which is regulated by the upstream kinase and Wnt signaling. All together, I propose that in the E blastomere, Wnt/MAPK signaling promotes PAR-5-mediated nuclear export of POP-1, thereby lowering its nuclear level. In addition to this differential nuclear export mechanism, POP-1 nuclear asymmetry may also be regulated by differential protein degradation. This study also shows that POP-1 functions to activate a Wnt/MAPK-responsive gene, sdz-23, in the E blastomere. This challenged the commonly accepted model of Wnt/MAPK-induced gene expression in E, which is based upon the alleviation of the repressive activity of POP-1. The activation of sdz-23 in E requires the ᭣atenin binding domain of POP-1 and a low nuclear level of POP-1. These results suggest that Wnt/MAPK converts the repressor POP-1 into a transcriptional activator and therefore, the non-canonical Wnt signaling in C. elegans early embryos is found to regulate its downstream effector POP-1 in a more canonical way than previously realized.