Ceramide regulation of the tumor suppressor phosphatase PTEN in rafts isolated from neurotumor cell lines.
The neutral sphingolipid ceramide has been implicated in the apoptotic death of cells by a number of different mechanisms, including activation of protein kinase B (Akt) phosphatase. Here we present evidence that ceramide recruits the tumor suppressor PTEN (phosphatase and tensin homolog deleted from chromosome 10) into membrane microdomains (rafts), where it could act to reduce the levels of polyphosphoinositides necessary for the activation of Akt. A PTEN construct with a red-fluorescent protein (RFP) tag was overexpressed in both a human cell line derived from oligodendroglioma (HOG) and a rat pheochromocytoma cell line (PC12) by means of an inducible promoter system (Tet-Off). Induction of PTEN by removal of doxycycline enhanced both capsase-3 and cell death with staurosporine, wortmannin, or C2-ceramide, whereas antisense PTEN had the reverse effect. Overexpression of PTEN also increased acid sphingomyelinase (ASMase) activity. PTEN normally has a generalized (cytosolic/membrane) distribution, but treatment with C2-ceramide translocated a fraction of the PTEN to the plasma membrane, showing a plasma membrane distribution similar to that observed for a prenylated green-fluorescent (GFP) construct. PTEN was then shown to translocate to the detergent-resistant membrane microdomain fraction (raft) of the plasma membrane. The colocalization of sphingomyelinases, ceramide, polyphosphoinositides, and PTEN in the raft fraction further suggests that the association of these lipids is critical for regulating cell death.
Goswami
R
R
Department of Pediatrics, University of Chicago, Chicago, Illinois 60637, USA.
Singh
D
D
Phillips
G
G
Kilkus
J
J
Dawson
G
G
D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D001932:Brain Neoplasms; D053148:Caspase 3; D053181:Caspase 8; D020169:Caspases / Q000378:metabolism; D002455:Cell Division / Q000187:drug effects; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D021962:Membrane Microdomains / Q000378:metabolism*; D009837:Oligodendroglioma; D016716:PC12 Cells; D051059:PTEN Phosphohydrolase; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010744:Phosphoric Monoester Hydrolases / Q000235:genetics / Q000378:metabolism*; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D051381:Rats; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000494:pharmacology; D019311:Staurosporine / Q000494:pharmacology; D014162:Transfection; D025521:Tumor Suppressor Proteins / Q000235:genetics / Q000378:metabolism*; D000077191:Wortmannin
D000730:Androstadienes; D004791:Enzyme Inhibitors; C064769:N-acetylsphingosine; D018129:Phosphatidylinositol Phosphates; D000081082:Phosphoinositide-3 Kinase Inhibitors; D047428:Protein Kinase Inhibitors; D025521:Tumor Suppressor Proteins; D010744:Phosphoric Monoester Hydrolases; D051059:PTEN Phosphohydrolase; C494929:PTEN protein, human; C505407:CASP3 protein, human; C505530:CASP8 protein, human; C505410:Casp3 protein, rat; C505528:Casp8 protein, rat; D053148:Caspase 3; D053181:Caspase 8; D020169:Caspases; D019311:Staurosporine; D013110:Sphingosine; D000077191:Wortmannin