RESUMO
Caveolin-1 is normally localized in plasma membrane caveolae and the Golgi apparatus in mammalian cells. We found three treatments that redirected the protein to lipid storage droplets, identified by staining with the lipophilic dye Nile red and the marker protein ADRP. Caveolin-1 was targeted to the droplets when linked to the ER-retrieval sequence, KKSL, generating Cav-KKSL. Cav-DeltaN2, an internal deletion mutant, also accumulated in the droplets, as well as in a Golgi-like structure. Third, incubation of cells with brefeldin A caused caveolin-1 to accumulate in the droplets. This localization persisted after drug washout, showing that caveolin-1 was transported out of the droplets slowly or not at all. Some overexpressed caveolin-2 was also present in lipid droplets. Experimental reduction of cellular cholesteryl ester by 80% did not prevent targeting of Cav-KKSL to the droplets. Cav-KKSL expression did not grossly alter cellular triacylglyceride or cholesteryl levels, although droplet morphology was affected in some cells. These data suggest that accumulation of caveolin-1 to unusually high levels in the ER causes targeting to lipid droplets, and that mechanisms must exist to ensure the rapid exit of newly synthesized caveolin-1 from the ER to avoid this fate.
Assuntos
Caveolinas/metabolismo , Vesículas Citoplasmáticas/química , Vesículas Citoplasmáticas/metabolismo , Retículo Endoplasmático/metabolismo , Metabolismo dos Lipídeos , Sequência de Aminoácidos , Animais , Brefeldina A/farmacologia , Caveolina 1 , Caveolina 2 , Caveolinas/genética , Linhagem Celular , Ésteres do Colesterol/metabolismo , Vesículas Citoplasmáticas/efeitos dos fármacos , Retículo Endoplasmático/química , Retículo Endoplasmático/efeitos dos fármacos , Complexo de Golgi/química , Complexo de Golgi/metabolismo , Camundongos , Microscopia de Fluorescência , Modelos Biológicos , Oxazinas , Sinais Direcionadores de Proteínas/genética , Sinais Direcionadores de Proteínas/fisiologia , Transporte Proteico/efeitos dos fármacos , Ratos , Ratos Endogâmicos F344 , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Deleção de Sequência/genética , Glândula Tireoide/citologia , Triglicerídeos/metabolismoRESUMO
Recent data suggest that membrane microdomains or rafts that are rich in sphingolipids and cholesterol are important in signal transduction and membrane trafficking. Two models of raft structure have been proposed. One proposes a unique role for glycosphingolipids (GSL), suggesting that GSL-head-group interactions are essential in raft formation. The other model suggests that close packing of the long saturated acyl chains found on both GSL and sphingomyelin plays a key role and helps these lipids form liquid-ordered phase domains in the presence of cholesterol. To distinguish between these models, we compared rafts in the MEB-4 melanoma cell line and its GSL-deficient derivative, GM-95. Rafts were isolated from cell lysates as detergent-resistant membranes (DRMs). The two cell lines had very similar DRM protein profiles. The yield of DRM protein was 2-fold higher in the parental than the mutant line, possibly reflecting cytoskeletal differences. The same amount of DRM lipid was isolated from both lines, and the lipid composition was similar except for up-regulation of sphingomyelin in the mutant that compensated for the lack of GSL. DRMs from the two lines had similar fluidity as measured by fluorescence polarization of diphenylhexatriene. Methyl-beta-cyclodextrin removed cholesterol from both cell lines with the same kinetics and to the same extent, and both a raft-associated glycosyl phosphatidylinositol-anchored protein and residual cholesterol showed the same distribution between DRMs and the detergent-soluble fraction after cholesterol removal in both cell lines. Finally, a glycosyl phosphatidylinositol-anchored protein was delivered to the cell surface at similar rates in the two lines, even after cholesterol depletion with methyl-beta-cyclodextrin. We conclude that GSL are not essential for the formation of rafts and do not play a major role in determining their properties.
Assuntos
Membrana Celular/metabolismo , Glicoesfingolipídeos/metabolismo , Proteínas de Membrana/metabolismo , beta-Ciclodextrinas , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Animais , Transporte Biológico/efeitos dos fármacos , Membrana Celular/química , Membrana Celular/efeitos dos fármacos , Colesterol/metabolismo , Ciclodextrinas/farmacologia , Detergentes/farmacologia , Feminino , Glicosilfosfatidilinositóis/metabolismo , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Cinética , Melanoma/metabolismo , Melanoma/patologia , Fluidez de Membrana , Lipídeos de Membrana/química , Proteínas de Membrana/química , Proteínas de Membrana/efeitos dos fármacos , Estrutura Terciária de Proteína , Células Tumorais CultivadasRESUMO
Sphingolipid and cholesterol-rich Triton X-100-insoluble membrane fragments (detergent-resistant membranes, DRMs) containing lipids in a state similar to the liquid-ordered phase can be isolated from mammalian cells, and probably exist as discrete domains or rafts in intact membranes. We postulated that proteins with a high affinity for such an ordered lipid environment might be targeted to rafts. Saturated acyl chains should prefer an extended conformation that would fit well in rafts. In contrast, prenyl groups, which are as hydrophobic as acyl chains but have a branched and bulky structure, should be excluded from rafts. Here, we showed that at least half of the proteins in Madin-Darby canine kidney cell DRMs (other than cytoskeletal contaminants) could be labeled with [3H]palmitate. Association of influenza hemagglutinin with DRMs required all three of its palmitoylated Cys residues. Prenylated proteins, detected by [3H]mevalonate labeling or by blotting for Rap1, Rab5, Gbeta, or Ras, were excluded from DRMs. Rab5 and H-Ras each contain more than one lipid group, showing that hydrophobicity alone does not target multiply lipid-modified proteins to DRMs. Partitioning of covalently linked saturated acyl chains into liquid-ordered phase domains is likely to be an important mechanism for targeting proteins to DRMs.
Assuntos
Lipídeos/química , Proteínas de Membrana/metabolismo , Acilação , Animais , Linhagem Celular , Detergentes , Eletroforese em Gel Bidimensional , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Proteínas de Membrana/química , Ácido Palmítico/metabolismo , Prenilação de ProteínaRESUMO
GAP-43 is an abundant protein in axonal growth cones of developing and regenerating neurons. We found that GAP-43 was enriched in detergent-resistant membranes (DRMs) isolated by Triton X-100 extraction from PC12 pheochromocytoma cells and could be detected in detergent-insoluble plasma membrane remnants after extraction of cells in situ. GAP-43 is palmitoylated at Cys-3 and Cys-4. Mutation of either Cys residue prevented association with DRMs. A hybrid protein containing the first 20 amino acid residues of GAP-43 fused to beta-galactosidase was targeted to DRMs even more efficiently than GAP-43 itself. We conclude that tandem palmitoylated Cys residues can target GAP-43 to DRMs, defining a new signal for DRM targeting. We propose that tandem or closely spaced saturated fatty acyl chains partition into domains or "rafts" in the liquid-ordered phase, or a phase with similar properties, in cell membranes. These rafts are isolated as DRMs after detergent extraction. The brain-specific heterotrimeric G protein Go, which may be regulated by GAP-43 in vitro, was also enriched in DRMs from PC12 cells. Targeting of GAP-43 to rafts may function to facilitate signaling through Go. In addition, raft association may aid in sorting of GAP-43 into axonally directed vesicles in the trans-Golgi network.
Assuntos
Membrana Celular/metabolismo , Cisteína , Proteína GAP-43/metabolismo , Lipoproteínas/metabolismo , Palmitatos , Animais , Membrana Celular/efeitos dos fármacos , Detergentes/farmacologia , Proteína GAP-43/genética , Proteínas de Ligação ao GTP/análise , Glucosídeos/farmacologia , Mutação , Octoxinol/farmacologia , Células PC12 , Ligação Proteica , Processamento de Proteína Pós-Traducional , RatosRESUMO
Cytoplasmic sequestration of wild-type p53 protein occurs in a subset of primary human tumors including breast cancer, colon cancer, and neuroblastoma (NB). The sequestered p53 localizes to punctate cytoplasmic structures that represent large protein aggregates. One functional consequence of this blocked nuclear access is impairment of the p53-mediated G1 checkpoint after DNA damage. Here we show that cytoplasmic p53 from NB cells is incompetent for specific DNA binding, probably due to its sequestration. Importantly, the C-terminal domain of sequestered p53 is masked, as indicated by the failure of a C-terminally directed antibody to detect p53 in these structures. To determine (i) which domain of p53 is involved in the aggregation and (ii) whether this phenotype is potentially reversible, we generated stable NB sublines that coexpress the soluble C-terminal mouse p53 peptide DD1 (amino acids 302-390). A dramatic phenotypic reversion occurred in five of five lines. The presence of DD1 blocked the sequestration of wild-type p53 and relocated it to the nucleus, where it accumulated. The nuclear translocation is due to shuttling of wild-type p53 by heteroligomerization to DD1, as shown by coimmunoprecipitation. As expected, the nuclear heterocomplexes were functionally inactive, since DD1 is a dominant negative inhibitor of wild-type p53. In summary, we show that nuclear access of p53 can be restored in NB cells.
Assuntos
Núcleo Celular/metabolismo , DNA de Neoplasias/metabolismo , Fragmentos de Peptídeos/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Animais , Sítios de Ligação , Neoplasias Encefálicas , Neoplasias da Mama , Ciclo Celular , Linhagem Celular , Neoplasias do Colo , Citoplasma/metabolismo , Dano ao DNA , DNA de Neoplasias/química , Feminino , Humanos , Camundongos , Neuroblastoma , Oligodesoxirribonucleotídeos , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/isolamento & purificação , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/química , Proteína Supressora de Tumor p53/isolamento & purificaçãoRESUMO
Wild-type p53 protein is abnormally sequestered in the cytoplasm of a subset of primary human tumors including neuroblastomas (NB) (U. M. Moll, M. LaQuaglia, J. Benard, and G. Riou, Proc. Natl. Acad. Sci. USA 92:4407-4411, 1995; U. M. Moll, G. Riou, and A. J. Levine, Proc. Natl. Acad. Sci.USA 89:7262-7266, 1992). This may represent a nonmutational mechanism for abrogating p53 tumor suppressor function. To test this hypothesis, we established the first available in vitro model that accurately reflects the wild-type p53 sequestration found in NB tumors. We characterized a series of human NB cell lines that overexpress wild-type p53 and show that p53 is preferentially localized to discrete cytoplasmic structures, with no detectable nuclear p53. These cell lines, when challenged with a variety of DNA strand-breaking agents, all exhibit impaired p53-mediated G1 arrest. Induction analysis of p53 and p53-responsive genes show that this impairment is due to suppression of nuclear p53 accumulation. Thus, this naturally occurring translocation defect compromises the suppressor function of p53 and likely plays a role in the tumorigenesis of these tumors previously thought to be unaffected by p53 alterations.
Assuntos
Fase G1 , Neuroblastoma/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Citoplasma/metabolismo , Dano ao DNA , Humanos , Neuroblastoma/genética , Neuroblastoma/patologia , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/análiseRESUMO
Introduction of v-src or c-src527F, a transforming mutant of the c-src proto-oncogene, into the growth factor-dependent cell line FDCP-1 resulted in growth factor independence. Temperature-shift studies with cells carrying the tsLA29 mutant of v-src demonstrated that growth factor independence was oncogene-dependent; that is, the cells were growth factor-independent at the permissive temperature but became growth factor-dependent at the nonpermissive temperature. Introduction of the c-src proto-oncogene did not result in growth factor independence. The c-src2A,527F mutant, which encodes an activated tyrosine kinase but does not transform fibroblasts due to a mutation in the membrane localization sequence, induced growth factor independence. This suggests that the presence of an activated tyrosine kinase is necessary for this process but that membrane localization is not. Bioassays indicated that conditioned medium from growth factor-independent cells contained a growth factor identified by antibody neutralization studies as granulocyte-macrophage colony-stimulating factor (GM-CSF). Secretion of GM-CSF was confirmed by a quantitative enzyme-linked immunosorbent assay (ELISA) specific for GM-CSF. The presence of GM-CSF mRNA in src-infected FDCP-1 cells was demonstrated by PCR amplification of cDNAs with primers specific for GM-CSF. While GM-CSF mRNA was detected in FDCP/ts29 cells grown at 34 degrees C, it was not observed in cells infected with the tsLA29 mutant grown at the nonpermissive temperature of 39 degrees C. Transfection of v-src-infected FDCP-1 cells with a GM-CSF promoter reporter plasmid revealed src-dependent expression of luciferase; that is, while expression was observed at the permissive temperature, no expression was detected in FDCP/ts29 clone 6 cells grown at the nonpermissive temperature. No expression of the GM-CSF promoter reporter plasmid was observed in uninfected FDCP-1 cells.
Assuntos
Membrana Celular/fisiologia , Transformação Celular Viral/genética , Genes src , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Células-Tronco Hematopoéticas/fisiologia , Interleucina-3/farmacologia , Proteína Oncogênica pp60(v-src)/fisiologia , Animais , Divisão Celular/efeitos dos fármacos , DNA Recombinante/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Genes Reporter , Fator Estimulador de Colônias de Granulócitos e Macrófagos/biossíntese , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Células-Tronco Hematopoéticas/metabolismo , Camundongos , Mitomicina/farmacologia , Mutação , Proteína Oncogênica pp60(v-src)/genética , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas pp60(c-src)/genética , Proteínas Proto-Oncogênicas pp60(c-src)/fisiologia , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Temperatura , TransfecçãoRESUMO
Wild type p53 plays a crucial role in maintaining genomic stability in both normal and tumor cells in vitro. When DNA damage occurs, p53 acts as a cell cycle checkpoint and induces a cellular response that aims at restoring genomic integrity. p53 may either allow the repair of damaged DNA by inducing a transient G1 arrest or may eliminate the damaged cells by triggering apoptosis. Mutant p53 fails to mediate any of these effects. From this, a p53 status-dependent response to therapy might be expected when tumors are treated with DNA-damaging genotoxic agents: Although wild type p53-harboring tumors have an intact checkpoint that might allow them to restore genomic integrity back to a pre-exposure level, mutant p53 tumors have a corrupted checkpoint that could lead to an accelerated loss of genomic stability. Until now, no studies have been described that examine such a p53-mediated effect in vivo. The authors tested this response model in vivo comparing 32 matched biopsy pairs from patients with breast cancer before and after rigorously standardized polychemotherapy. Four of the five drugs specifically induce a wild type p53-mediated checkpoint response. Tumor tissue from matched pairs of untreated and treated biopsies of the same patient were analyzed for treatment-associated changes of p53 protein expression by immunocytochemistry and, in a few available specimens, of p53 genotype changes by polymerase chain reaction-based DNA analysis. Treatment-associated changes of the p53 immunophenotype, which the authors speculate to reflect clonal selection, occurred in 39% (12 of 31) of the specimens. One specimen was not informative. Most tumors undergoing clonal selection originally harbored mutant p53 (nine of 12), and only three of 12 tumors were wild type. This study shows that exposure to genotoxic agents is commonly associated with a change in p53 immunophenotype. Although the limited material in this cohort prevented direct analysis of genetic instability, these results suggest that tumors with altered p53 may be genomically less stable and, therefore, may be more likely to undergo treatment-induced clonal changes than wild type tumors. This study also shows that the rigorous matched sample approach, although difficult to obtain, is an important tool that allows the in vivo assessment of the tumor response to genotoxic therapy in a controlled fashion.