The Menkes protein (ATP7A; MNK) cycles via the plasma membrane both in basal and elevated extracellular copper using a C-terminal di-leucine endocytic signal.
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Menkes disease is an X-linked recessive copper deficiency disorder caused by mutations in the ATP7A ( MNK ) gene which encodes a copper transporting P-type ATPase (MNK). MNK is normally localized pre- dominantly in the trans -Golgi network (TGN); however, when cells are exposed to excessive copper it is rapidly relocalized to the plasma membrane where it functions in copper efflux. In this study, the c-myc epitope was introduced within the loop connecting the first and second transmembrane regions of MNK. This myc epitope allowed detection of the protein at the surface of living cells and provided the first experimental evidence supporting the common topological model. In cells stably expressing the tagged MNK protein (MNK-tag), extracellular antibodies were internalized to the perinuclear region, indicating that MNK-tag at the TGN constitutively cycles via the plasma membrane in basal copper conditions. Under elevated copper conditions, MNK-tag was recruited to the plasma membrane; however, internalization of MNK-tag was not inhibited and the protein continued to recycle through cyto- plasmic membrane compartments. These findings suggest that copper stimulates exocytic movement of MNK to the plasma membrane rather than reducing MNK retrieval and indicate that MNK may remove copper from the cytoplasm by transporting copper into the vesicles through which it cycles. Newly internalized MNK-tag and transferrin were found to co-localize, suggesting that MNK-tag follows a clathrin-coated pit/endosomal pathway into cells. Mutation of the di-leucine, L1487 L1488, prevented uptake of anti-myc antibodies in both basal and elevated copper conditions, thereby identifying this sequence as an endocytic signal for MNK. Analysis of the effects of the di-leucine mutation in elevated copper provided further support for copper-stimulated exocytic movement of MNK from the TGN to the plasma membrane. (+info)
Identification of a zinc finger protein whose subcellular distribution is regulated by serum and nerve growth factor.
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A subclass of zinc finger proteins containing a unique protein motif called the positive regulatory (PR) domain has been described. The members include the PRDI-BF1/Blimp-1 protein, the Caenorhabditis elegans egl-43 and EVI1 gene products, and the retinoblastoma interacting protein RIZ. Here we describe a member of this family, SC-1, that exhibits several distinctive features. First, SC-1 interacts with the p75 neurotrophin receptor and is redistributed from the cytoplasm to the nucleus after nerve growth factor (NGF) treatment of transfected COS cells. The translocation of SC-1 to the nucleus was specific for p75, as NGF binding to the TrkA receptor did not lead to nuclear localization of SC-1. Thus, SC-1 provides a downstream transducer for the effects of NGF through the p75 neurotrophin receptor. Under normal growth conditions, SC-1 was found predominantly in the cytoplasm. On serum-starvation, SC-1 also translocated into the nucleus. A direct correlation between nuclear expression of SC-1 with the loss of BrdUrd incorporation was observed. These results imply that SC-1 may be involved in events associated with growth arrest. (+info)
Agonists cause nuclear translocation of phosphatidylinositol 3-kinase gamma. A Gbetagamma-dependent pathway that requires the p110gamma amino terminus.
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In hematopoietic cells, the signals initiated by activation of the phosphoinositide 3-kinase (PI3K) family have been implicated in cell proliferation and survival, membrane and cytoskeletal reorganization, chemotaxis, and the neutrophil respiratory burst. Of the four isoforms of human PI3K that phosphorylate phosphatidylinositol 4, 5-bisphosphate, only p110gamma (or PI3Kgamma) is associated with the regulatory subunit, p101, and is stimulated by G protein betagamma heterodimers. We performed immunolocalization of transfected p110gamma in HepG2 cells and found that, under resting conditions, p110gamma was present in a diffuse cytoplasmic pattern, but translocated to the cell nucleus after serum stimulation. Serum-stimulated p110gamma translocation was inhibited by pertussis toxin and could also be induced by overexpression of Gbetagamma in the absence of serum. In addition, we found that deletion of the amino-terminal 33 residues of p110gamma had no effect on association with p101 or on its agonist-regulated translocation, but truncation of the amino-terminal 82 residues yielded a p110gamma variant that did not associate with p101 and was constitutively localized in the nucleus. This finding implies that the intracellular localization of p110gamma is regulated by p101 as well as Gbetagamma. The effect of PI3Kgamma in the nucleus is an area of active investigation. (+info)
Poly(ADP-ribose) polymerase upregulates E2F-1 promoter activity and DNA pol alpha expression during early S phase.
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E2F-1, a transcription factor implicated in the activation of genes required for S phase such as DNA pol alpha, is regulated by interactions with Rb and by cell-cycle dependent alterations in E2F-1 abundance. We have shown that depletion of poly(ADP-ribose) polymerase (PARP) by antisense RNA expression downregulates pol alpha and E2F-1 expression during early S phase. To examine the role of PARP in the regulation of pol alpha and E2F-1 gene expression, we utilized immortalized mouse fibroblasts derived from wild-type and PARP knockout (PARP-/-) mice as well as PARP-/- cells stably transfected with PARP cDNA [PARP-/-(+PARP)]. After release from serum deprivation, wild-type and PARP-/-(+PARP) cells, but not PARP-/- cells, exhibited a peak of cells in S phase by 16 h and had progressed through the cell cycle by 22 h. Whereas [3H]thymidine incorporation remained negligible in PARP-/- cells, in vivo DNA replication maximized after 18 h in wild-type and PARP-/-(+PARP) cells. To investigate the effect of PARP on E2F-1 promoter activity, a construct containing the E2F-1 gene promoter fused to a luciferase reporter gene was transiently transfected into these cells. E2F-1 promoter activity in control and PARP-/-(+PARP) cells increased eightfold after 9 h, but not in PARP-/- cells. PARP-/- cells did not show the marked induction of E2F-1 expression during early S phase apparent in control and PARP-/-(+PARP) cells. RT - PCR analysis and pol alpha activity assays revealed the presence of pol alpha transcripts and a sixfold increase in activity in both wild-type and PARP-/-(+PARP) cells after 20 h, but not in PARP-/- cells. These results suggest that PARP plays a role in the induction of E2F-1 promoter activity, which then positively regulates both E2F-1 and pol alpha expression, when quiescent cells reenter the cell cycle upon recovery from aphidicolin exposure or removal of serum. (+info)
Cripto-1 induces phosphatidylinositol 3'-kinase-dependent phosphorylation of AKT and glycogen synthase kinase 3beta in human cervical carcinoma cells.
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Cripto-1 (CR-1), a member of the epidermal growth factor-CFC peptide family, activates the ras/raf/mitogen-activated protein/extracellular signal-regulated kinase/mitogen-activated protein kinase pathway. In the present study, the role of CR-1 in the phosphatidylinositol 3'-kinase (PI3K)/AKT (protein kinase B)/glycogen synthase kinase 3beta (GSK-3beta)-dependent signaling pathway was evaluated in human SiHa cervical carcinoma cells. Our data demonstrate that CR-1 can enhance the tyrosine phosphorylation of the p85 regulatory subunit of PI3K and transiently induce the phosphorylation of AKT in a time- and dose-dependent manner. In addition, CR-1 was found to induce the phosphorylation of GSK-3beta. Phosphorylation of AKT and GSK-3beta by CR-1 can be blocked by LY294002, a specific inhibitor of PI3K, thus leading to apoptosis. Finally, the apoptotic effect of LY294002 can be partially rescued by exogenous CR-1. In summary, our data suggest that human CR-1 may function as a survival factor through a PI3K-dependent signaling pathway involving AKT and GSK-3beta. (+info)
Reduction in apolipoprotein-mediated removal of cellular lipids by immortalization of human fibroblasts and its reversion by cAMP: lack of effect with Tangier disease cells.
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High density lipoprotein (HDL) phospholipids and apolipoproteins remove cellular lipids by two distinct mechanisms, but their relative contribution to reverse cholesterol transport is unknown. Whereas phospholipid-mediated cholesterol efflux from cultured cells reflects the activity of the HDL receptor SR-BI, apolipoprotein-mediated lipid removal is regulated in response to changes in cellular cholesterol content (positive) and cell proliferation rates (negative). Here we show that immortalization of human skin fibroblast lines with the papillomavirus E6/E7 oncogenes increased their proliferation rates and selectively reduced the activity of the apolipoprotein-mediated lipid removal pathway. This reduction was accompanied by a decrease in cellular cAMP levels and was reversed by treatment with a cAMP analog. The stimulatory effect of cAMP was independent of changes in cellular phenotype or activities of cholesteryl ester cycle enzymes. The severely impaired apolipoprotein-mediated lipid removal pathway in Tangier disease fibroblasts, which persisted after immortalization, was not improved by treatment with a cAMP analog, implying that the cellular defect in Tangier disease is upstream from this cAMP-dependent signaling pathway.These results indicate that papillomavirus-induced immortalization of fibroblasts selectively reduces the activity of the apolipoprotein-mediated lipid removal pathway by a cAMP-dependent process, perhaps to prevent loss of cellular lipids needed for continual membrane synthesis. (+info)
Establishment of an optimised gene transfer protocol for human primary T lymphocytes according to clinical requirements.
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Current gene therapeutic protocols directed towards the treatment of inherited disorders (eg ADA-SCID) and viral infections (eg AIDS), as well as adoptive immunotherapy approaches are based on the use of genetically modified lymphocytes. Since only insufficient transduction of T cells is obtained using existing techniques, the development of more efficient gene transfer protocols into these cells is of great importance. We present here a protocol for the highly efficient transduction of human primary T cells at high densities (1 x 106/ml) by retroviral infection. Using retroviral vectors encoding a truncated human low-affinity nerve growth factor receptor (DeltaLNGFR) as a gene transfer marker, we obtained transduction frequencies of more than 70% of CD3+ cells after two cycles of infection. Our protocol is based on the use of FBS-free media for both the production of retrovirus-containing supernatant and the cultivation of the primary T cells. Since the protocol presented here works just as efficiently under large-scale conditions, it may be easily adapted to clinical needs and 'good manufacturing practice' (GMP) standards. (+info)
Extracellular signal-regulated kinase activation is required for up-regulation of vascular endothelial growth factor by serum starvation in human colon carcinoma cells.
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Vascular endothelial growth factor (VEGF) is a potent angiogenic factor important for colon cancer neovascularization. In previous studies, serum starvation led to induction of VEGF in human colon carcinoma cells. We investigated the possible participation of mitogen-activated protein kinases in serum starvation induction of VEGF in the HT29 human colon carcinoma cell line. The extracellular signal-regulated kinases (Erks) 1 and 2 were activated after 3-6 h of serum starvation. Using transient transfection of VEGF promoter-reporter constructs, serum starvation led to an increase in VEGF promoter activity. An inhibitor of phosphorylation of Erk-1/2 blocked the increase of VEGF expression and promoter activity induced by serum starvation. Serum starvation activates several mitogen-activated protein kinases, but activation of Erk-1/2 is critical for the up-regulation of VEGF mRNA in colon carcinoma cells. (+info)