Browsing by Subject "Akt"
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Item APβ1/2 and Hip1r : insights into early and late stage clathrin adaptors in Dictyostelium discoideum(2012-05) Sosa, Ramiro Thomas; O'Halloran, Theresa; Gross, Jeffrey M.; Johnson, Arlen W.; Mehdy, Mona; Morgan, Jennifer R.Clathrin-mediated endocytosis is the process whereby specific cargoes are internalized into coated vesicles from the plasma membrane. Numerous clathrin adaptors facilitate this process by linking the coat protein clathrin to the plasma membrane by associating with PI(4,5)P2 and binding to membrane-bound cargo. Here, I investigated the role of two clathrin adaptors, APβ1/2 and Hip1r, in clathrin-mediated endocytosis. I found that Dictyostelium APβ1/2 functions in both the AP1 and AP2 complexes, unlike vertebrates, which have distinct β subunits for each AP complex. I found that APβ1/2 function is required for several clathrin-dependent processes, including cytokinesis, development and osmoregulation. I also uncovered a role for APβ1/2 in the stability other subunits of the AP1 and AP2 complexes. Finally, phenotypic comparisons of APβ1/2 mutant cells with cells missing subunits that are specific to the AP1 or AP2 complex allowed me to distinguish between endocytic defects and endosomal trafficking defects in clathrin mutants. My investigation of Hip1r centered on the known requirement for Hip1r in actin dynamics during endocytosis and a possible role for Hip1r phosphorylation in regulating actin. To determine how phosphorylation contributes to Hip1r function, I identified a specific serine residue that serves as a Hip1r phosphorylation site. I also identified a novel role for the kinase PKB in Hip1r phosphorylation. I determined that phosphorylation is not required for Hip1r localization to the plasma membrane. Similar to Hip1r, PKB is required for proper actin dynamics during endocytosis. My results support a model in which epsin recruits Hip1r to the plasma membrane during formation of clathrin-coated vesicles. Here, Hip1r functions as both a clathrin adaptor and a negative regulator of actin polymerization. I propose that phosphorylation of Hip1r by PKB triggers a reduction in the affinity of Hip1r for clathrin, which may stimulate actin polymerization and tethering of clathrin-coated vesicles with the actin cytoskeleton.Item Dietary energy balance modulates growth factor signaling during multistage epithelial carcinogenesis in mouse skin(2010-12) Moore, Tricia Wallace; DiGiovanni, John; Hursting, Stephen; Kline, Kimberly; deGraffenried, Linda; Fischer, Susan; Vasquez, KarenEnergy balance refers to the relationship between energy intake and energy expenditure. Epidemiological studies have established a clear association between energy balance and cancer, however the underlying mechanisms are unclear. The objective of the current study was to evaluate the impact of caloric consumption on epithelial carcinogenesis and identify potential mechanisms of inhibition or enhancement. Using ICR female mice, we demonstrated that positive energy balance enhanced, while negative energy balance inhibited susceptibility to multistage carcinogenesis in mouse skin. We next evaluated diet-induced changes in the epidermal proliferative response. Calorie restriction (CR) significantly reduced epidermal hyperproliferation, in the presence and absence of tumor promotion, as compared to diet-induced obesity (DIO). Additional studies were conducted to determine the impact of dietary manipulation on TPA-induced growth factor signaling. CR reduced, while DIO increased insulin like growth factor-1 receptor (IGF-1R) and epidermal growth factor receptor (EGFR) activation, which subsequently modulated signaling downstream to Akt and mTOR. These diet-induced changes in growth factor signaling were confirmed under steady-state conditions in multiple epithelial tissues (i.e., skin, liver and dorsolateral prostate) in multiple mouse strains (FVB/N, C57BL/6 and ICR). Further analyses demonstrated that caloric consumption directly correlated with levels of cell cycle progression related proteins and inversely correlated with levels of cell cycle inhibitory proteins. Genetic reduction of circulating IGF-1, liver IGF-1 deficient (LID) mouse model, inhibited two-stage skin carcinogenesis, reduced epidermal hyperproliferation and attenuated IGF-1R and EGFR growth factor signaling during tumor promotion, similar to CR, suggesting a potential for IGF-1R and EGFR crosstalk. Further studies, demonstrated that IGF-1 induced EGFR activation in cultured mouse keratinocytes, possibly due to IGF-1R and EGFR heterodimerization or IGF-1 induced changes in EGFR mRNA expression. In vivo, CR reduced, while DIO increased IGF-1R and EGFR association during tumor promotion. Furthermore, CR attenuated EGFR ligand mRNA expression both in the presence and absence of TPA treatment. Collectively, these findings suggest that dietary energy balance modulates epithelial carcinogenesis, at least in part due to diet-induced changes in levels of circulating IGF-1, which then modulate IGF-1R and EGFR crosstalk and downstream signaling to cell cycle related proteins, subsequently altering epidermal hyperproliferation.Item Gastrin-releasing peptide-mediated neuroblastoma growth: A role for the PI3K/Akt pathway(2008-12-18) Titilope Adenike Ishola; Dai H. Chung, M.D.; Tianyan Gao, Ph.D.; Kathleen O'Connor, Ph.D.; Jason M. Shohet, M.D., Ph.D.; B. Mark Evers, M.D.Neuroblastoma is the most common extracranial solid tumor in infants and children. Our lab and others have shown trophic actions of gastrin-releasing peptide (GRP), and its analogue bombesin (BBS), in neuroblastomas. Our lab also found that undifferentiated neuroblastomas express increased levels of GRP receptor (GRPR). Activation of the phosphatidylinositol-3-kinase (PI3K)/Akt pathway, a crucial regulator of cell survival, is associated with poor outcome in neuroblastomas and our lab¡¦s previous work has shown that GRPR regulates the expression of PI3K/Akt pathway components. However, the signaling mechanisms involved in this process are not clearly defined. Therefore, the objective of this project was to determine how GRP/GRPR, by way of PI3K pathway, regulates neuroblastoma growth. \r\n\r\nGRP/BBS treatment rapidly increased phosphorylation of both Akt and GSK-3ƒÒ in neuroblastoma cells. Antagonism or silencing of GRPR attenuated BBS-induced phosphorylation of Akt. PI3K inhibition also abrogated BBS-stimulated phosphorylated (p)-Akt as well as its cell cycle targets. GRP increased G1/S phase progression in SK-N-SH cells and BBS-mediated BrdU incorporation was blocked with a PI3K inhibitor. These findings identify PI3K/Akt as an important signaling pathway for GRP-mediated neuroblastoma cell growth. In order to determine the in vivo significance of GRP/GRPR, the effects of BBS treatment in nude mice with human neuroblastoma xenografts were assessed. BBS treatment significantly increased the growth and mediators of angiogenesis of SK-N-SH and BE(2)-C tumors, as well as increased p-Akt levels. A GRPR antagonist reduced BBS-stimulated tumor growth and angiogenic markers in vivo. GRP or GRPR silencing inhibited the expressions of VEGF, p-Akt, and p-mTOR in vitro. GRPR knockdown induced cell morphology changes, reduced cell size, decreased cell proliferation, and inhibited DNA synthesis, which corresponded to G2/M cell cycle arrest. Activated Akt and its downstream regulators of protein synthesis and metabolism were also significantly downregulated by GRPR silencing. GRPR knockdown upregulated the expression of PTEN, the inhibitor of the PI3K/Akt pathway. Furthermore, silencing of GRPR or GRP suppressed anchorage-independent growth; while GRPR overexpression resulted in soft agar colony formation, which was inhibited by a GRP-blocking antibody. In conclusion, these findings demonstrate that GRP/GRPR signaling regulates the PI3K/Akt pathway and promotes neuroblastoma growth, angiogenesis, and oncogenic properties.Item Mitochondrial uncoupling links lipid catabolism to Akt inhibition and blockade of skin tumorigenesis(2014-08) Nowinski, Sara Marie; Mills, Edward MichaelIn order to support rampant cell growth, tumor cells must reprogram metabolism to simultaneously drive macromolecular biosynthesis and energy production. Mitochondrial uncoupling proteins (UCPs) oppose this phenotype by inducing futile mitochondrial respiration that is disengaged from ATP synthesis. We found that uncoupling protein 3 (UCP3) was normally expressed in follicular and epidermal keratinocytes and that its levels were augmented by calcium-induced differentiation in vitro. Over-expression of a UCP3 transgene targeted to the basal epidermis by the keratin-5 promoter (K5-UCP3) led to increased differentiation of both epidermal and bulge stem cells, the progenitors of most squamous carcinomas. Consistent with this phenotype, K5-UCP3 mice were completely protected from chemically induced skin carcinogenesis. To define the mechanisms by which UCP3 conferred such strong tumor resistance, we interbred K5-UCP3 mice with a “pre-initiated” mouse model, and found that UCP3 over-expression blocked tumor promotion. Uncoupled epidermis displayed reduced proliferation after treatment with tumor promoter, along with diminished activation of Akt signaling. This effect corresponded to decreased Akt activation by epidermal growth factor (EGF) in K5-UCP3 cells, along with UCP3 overexpressing primary human keratinocytes. Mechanistic studies revealed that uncoupling drove global lipid catabolism, along with impaired recruitment of Akt to the plasma membrane. Over-expression of wild type Akt rescued tumor promoter-induced proliferation and two-stage chemical carcinogenesis in bi-transgenic mice. Collectively, these findings demonstrate that mitochondrial uncoupling is an effective strategy to limit cell proliferation and tumorigenesis through inhibition of Akt, and suggest a novel mechanism of crosstalk between mitochondrial metabolism and growth signaling.Item Role of the PI-3K/Akt and ERK pathways in phencyclidine-induced neurotoxicity in neonatal rats and the protection by lithium and BDNF(2009-06-11) Yan Xia; Kenneth M. Johnson; Xiaodong Cheng; Pramod Dash; Kelly T. Dineley; Joe GallagherPhencyclidine is an N-methyl-D-aspartate receptor (NMDAR) open channel blocker that causes schizophrenia-like symptoms in healthy humans and exacerbates psychoses in schizophrenics. In animals, PCP administration to immature rats causes wide spread neuronal death during brain development and results in behavioral deficits that resemble those observed in schizophrenic patients in later life. The purpose of this study was to investigate the mechanisms of PCP-induced neurotoxicity as well as the protection by lithium and brain-derived neurotrophic factor (BDNF). In corticostriatal slice cultures, we found that lithium and BDNF, inhibited PCP-induced caspase-3 activation and DNA fragmentation in a concentration-dependent manner, respectively. Lithium and BDNF also prevented the inhibitory action of PCP on the phosphatidylinositol-3 kinase (PI-3K)/Akt and extracellular regulated kinase (ERK) pathways and suppressed activation of the pro-apoptotic factor, glycogen synthase kinase-3â (GSK-3â), evoked by PCP. Furthermore, blocking either the PI-3K/Akt or the ERK pathway abolished the protective effects of lithium and BDNF. Western blot analysis revealed that the PI-3K/Akt and ERK pathways were stimulated by lithium and BDNF in parallel; however, inhibition of ERK and PI-3K cooperatively regulated GSK-3â activity by reducing its phosphorylation at serine 9. In vivo, acute PCP administration to rats on postnatal day (PN) 7 caused inhibition of Akt and ERK and activation of GSK-3â in the three brain regions examined (frontal cortex, striatum, and hippocampus). After the last dose of subchronic PCP administration on PN 7, 9, 11, inhibition of ERK was still found in the three regions, though it lasted for a much shorter period than after administration on PN 7 only; Inhibition of Akt was only observed in the frontal cortex; GSK-3â activity was not affected in any of the regions. Finally, subchronic PCP administration during brain development resulted in dysregulation of the PI-3K/Akt and ERK pathways upon PCP challenge in adolescence (PN 35). These studies strongly suggest that the PI-3K/Akt and ERK pathways are two important signaling transductions implicated in PCP-induced neurotoxicity in developing brains and in the protection of lithium and BDNF.