RESUMO
Apoptosis is generally accompanied by a late phase of ceramide (Cer) production, the significance of which is unknown. This study describes a previously unrecognized link between Cer accumulation and phosphatidylserine (PS) exposure at the cell surface, a characteristic of the execution phase of apoptosis resulting from a loss of plasma membrane phospholipid asymmetry. Using a fluorescent sphingomyelin (SM) analogue, N-(N-[6-[(7-nitrobenz-2-oxa-1, 3-diazol-4-yl)amino]caproyl]-sphingosylphosphorylcholine (C(6)-NBD-SM), we show that Cer is derived from SM, initially located in the outer leaflet of the plasma membrane, which gains access to a cytosolic SMase by flipping to the inner leaflet in a process of lipid scrambling paralleling PS externalization. Lipid scrambling is both necessary and sufficient for SM conversion: Ca(2+) ionophore induces both PS exposure and SM hydrolysis, whereas scrambling-deficient Raji cells do not show PS exposure or Cer formation. Cer is not required for mitochondrial or nuclear apoptotic features since these are still observed in Raji cells. SM hydrolysis facilitates cholesterol efflux to methyl-beta-cyclodextrin, which is indicative of a loss of tight SM-cholesterol interaction in the plasma membrane. We provide evidence that these biophysical alterations in the lipid bilayer are essential for apoptotic membrane blebbing/vesiculation at the cell surface: Raji cells show aberrant apoptotic morphology, whereas replenishment of hydrolyzed SM by C(6)- NBD-SM inhibits blebbing in Jurkat cells. Thus, SM hydrolysis, during the execution phase of apoptosis, results from a loss of phospholipid asymmetry and contributes to structural changes at the plasma membrane.
Assuntos
Apoptose , Membrana Celular/metabolismo , Ceramidas/biossíntese , Fosfolipídeos/metabolismo , Esfingomielinas/metabolismo , Linfócitos B/citologia , Linfócitos B/metabolismo , Linhagem Celular , Membrana Celular/ultraestrutura , Células Clonais , Humanos , Hidrólise , Líquido Intracelular/metabolismo , Metabolismo dos Lipídeos , Fosfatidilserinas/metabolismo , Linfócitos T/citologia , Linfócitos T/metabolismoRESUMO
PURPOSE: To study the relationship between cell reproductive death and exchange frequency in SW-1573 human lung tumour cells with and without incorporated iodo-deoxyuridine (IdUrd) following irradiation of plateau-phase cultures with y-rays. METHOD: Linear-quadratic (LQ) analysis was performed for the data on clonogenic survival and on the frequency of chromosomal exchanges studied with fluorescence in situ hybridization in chromosomes X and 2. RESULTS: Differences in the LQ parameters alpha and beta of both non-sensitized and sensitized chromosomes were found. In both chromosomes an increase in the number of chromosomal exchanges in IdUrd-radiosensitized cells compared with non-sensitized cells was observed. The alpha-enhancement factors of 1.7 and 1.9 for the X-chromosome and for chromosome 2, respectively, are similar. For the X-chromosome, the beta coefficient increased by a factor of 3.9 and for chromosome 2 by a factor of 1.4. After correction to a full genome equivalence, no significant difference in alpha was found between chromosomes X and 2 for both control and sensitized cells. In contrast, an almost 2.8 times higher beta was found for the sensitized X-chromosome compared to this value for chromosome 2. CONCLUSIONS: It can be concluded that the linear-quadratic analysis of dose-response relationships offers insights into the correlation between cell survival and induction of exchanges in non-sensitized and radiosensitized cells.
Assuntos
Carcinoma de Células Escamosas/patologia , Aberrações Cromossômicas , Cromossomos Humanos Par 2/efeitos da radiação , Idoxuridina/farmacologia , Neoplasias Pulmonares/patologia , Inibidores da Síntese de Ácido Nucleico/farmacologia , Radiossensibilizantes/farmacologia , Cromossomo X/efeitos da radiação , Carcinoma de Células Escamosas/radioterapia , Carcinoma de Células Escamosas/ultraestrutura , Morte Celular/efeitos da radiação , Divisão Celular/efeitos da radiação , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Terapia Combinada , Relação Dose-Resposta a Droga , Humanos , Idoxuridina/metabolismo , Hibridização in Situ Fluorescente , Neoplasias Pulmonares/radioterapia , Neoplasias Pulmonares/ultraestrutura , Inibidores da Síntese de Ácido Nucleico/metabolismo , Radiossensibilizantes/metabolismo , Timidina/metabolismo , Células Tumorais Cultivadas/efeitos dos fármacos , Células Tumorais Cultivadas/efeitos da radiaçãoRESUMO
We previously showed that ceramide (Cer) formed during the execution phase of apoptosis is derived from plasma membrane sphingomyelin (SM), most likely by a neutral sphingomyelinase activity (Tepper et al., J. Cell Biol. 150, 2000, 155-164). In this study, we investigated the involvement of a cloned putative human neutral sphingomyelinase (nSMase1) in this process. Site-directed mutagenesis of predicted catalytic residues (Glu(49), Asn(180), and His(272)) to Ala residues abolished the catalytic activity of nSMase1. Jurkat cells were retrovirally transduced with either wildtype or inactive (with all three point mutations) Myc-tagged nSMase1. Cells overexpressing wildtype nSMase1 showed dramatically elevated in vitro nSMase activity. However, nSMase1 gene transduction (wildtype or mutant) did not alter steady-state levels of SM, Cer, or glucosylceramide. Moreover, the Cer response and apoptosis sensitivity to ligation of the CD95/Fas receptor in cells overexpressing wildtype or mutant nSMase1 were identical to vector-transduced cells. We conclude that not nSMase1 but a different, yet to be identified, nSMase accounts for the generation of Cer during the execution phase of death receptor-induced apoptosis.
Assuntos
Apoptose/fisiologia , Ceramidas/biossíntese , Esfingomielina Fosfodiesterase/metabolismo , Receptor fas/metabolismo , Animais , Células COS , Domínio Catalítico/genética , Expressão Gênica , Humanos , Células Jurkat , Cinética , Mutagênese Sítio-Dirigida , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Esfingomielina Fosfodiesterase/química , Esfingomielina Fosfodiesterase/genética , Transdução GenéticaRESUMO
14-3-3 proteins may function as adapter or scaffold proteins in signal transduction pathways. We reported previously that several 14-3-3 isotypes bind to protein kinase C (PKC)-zeta and facilitate coupling of PKC-zeta to Raf-1 [van der Hoeven, van der Wal, Ruurs, van Dijk and van Blitterswijk (2000) Biochem. J. 345, 297-306], an event that boosts the mitogen-activated protein kinase (ERK) pathway in Rat-1 fibroblasts. The present work investigated whether bound 14-3-3 would affect PKC-zeta activity. Using recombinant 14-3-3 proteins and purified PKC-zeta in a convenient, newly developed in vitro kinase assay, we found that 14-3-3 proteins stimulated PKC-zeta activity in a dose-dependent fashion up to approx. 2.5-fold. Activation of PKC-zeta by 14-3-3 isotypes was unrelated to their mutual affinity, estimated by co-immunoprecipitation from COS cell lysates. Accordingly, PKC-zeta with a defective (point-mutated) 14-3-3-binding site, showed the same 14-3-3-stimulated activity as wild-type PKC-zeta. As 14-13-3 proteins are acidic, we tested several other acidic proteins, which turned out to stimulate PKC-zeta activity in a similar fashion, whereas neutral or basic proteins did not. These effects were not restricted to the atypical PKC-zeta, but were also found for classical PKC. Together, the results suggest that the stimulation of PKC activity by 14-3-3 proteins is non-specific and solely due to the acidic nature of these proteins, quite similar to that known for acidic lipids.
Assuntos
Proteína Quinase C/metabolismo , Proteínas/química , Proteínas/farmacologia , Tirosina 3-Mono-Oxigenase , Proteínas 14-3-3 , Sequência de Aminoácidos , Animais , Sítios de Ligação , Biotinilação , Células COS , Relação Dose-Resposta a Droga , Ativação Enzimática/efeitos dos fármacos , Concentração de Íons de Hidrogênio , Ponto Isoelétrico , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/isolamento & purificação , Isoenzimas/metabolismo , Dados de Sequência Molecular , Mutação/genética , Peptídeos/química , Peptídeos/metabolismo , Fosfatidilserinas/metabolismo , Fosfatidilserinas/farmacologia , Testes de Precipitina , Ligação Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/farmacologia , Proteína Quinase C/química , Proteína Quinase C/genética , Proteína Quinase C/isolamento & purificação , Proteínas/genética , Proteínas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Especificidade por Substrato , TransfecçãoRESUMO
14-3-3 Proteins may function as adapters or scaffold in signal-transduction pathways. We found previously that protein kinase C-zeta (PKC-zeta) can phosphorylate and activate Raf-1 in a signalling complex [van Dijk, Hilkmann and van Blitterswijk (1997) Biochem. J. 325, 303-307]. We report now that PKC-zeta-Raf-1 interaction is mediated by 14-3-3 proteins in vitro and in vivo. Co-immunoprecipitation experiments in COS cells revealed that complex formation between PKC-zeta and Raf-1 is mediated strongly by the 14-3-3beta and -theta; isotypes, but not by 14-3-3zeta. Far-Western blotting revealed that 14-3-3 binds PKC-zeta directly at its regulatory domain, where a S186A mutation in a putative 14-3-3-binding domain strongly reduced the binding and the complex formation with 14-3-3beta and Raf-1. Treatment of PKC-zeta with lambda protein phosphatase also reduced its binding to 14-3-3beta in vitro. Preincubation of an immobilized Raf-1 construct with 14-3-3beta facilitated PKC-zeta binding. Together, the results suggest that 14-3-3 binds both PKC-zeta (at phospho-Ser-186) and Raf-1 in a ternary complex. Complex formation was much stronger with a kinase-inactive PKC-zeta mutant than with wild-type PKC-zeta, supporting the idea that kinase activity leads to complex dissociation. 14-3-3beta and -θ were substrates for PKC-zeta, whereas 14-3-3zeta was not. Phosphorylation of 14-3-3beta by PKC-zeta negatively regulated their physical association. 14-3-3beta with its putative PKC-zeta phosphorylation sites mutated enhanced co-precipitation between PKC-zeta and Raf-1, suggesting that phosphorylation of 14-3-3 by PKC-zeta weakens the complex in vivo. We conclude that 14-3-3 facilitates coupling of PKC-zeta to Raf-1 in an isotype-specific and phosphorylation-dependent manner. We suggest that 14-3-3 is a transient mediator of Raf-1 phosphorylation and activation by PKC-zeta.