Nerve growth factor activation of the TrkA receptor induces cell death, by macropinocytosis, in medulloblastoma Daoy cells.
Ectopic expression of the TrkA receptor tyrosine kinase in tumors of the nervous system can mediate nerve growth factor (NGF)-dependent cell death by apoptosis and /or autophagy. Herein, we demonstrate that TrkA can also induce cell death in medulloblastoma Daoy cells by a caspase-independent mechanism that involves the hyperstimulation of macropinocytosis. Specifically, NGF-stimulates the uptake of AlexaFluor546-dextran into lysosome-associated membrane protein-1 positive vacuoles which fuse with microtubule associated protein light chain 3 (LC3) positive autophagosomes, to form large intracellular vacuoles (> 1 mum), which then fuse with lysotracker positive lysosomes. While LC3 cleavage and the appearance of LC3 positive vacuoles suggest the induction of autophagy, siRNA reduced expression of four proteins essential to autophagy (beclin-1, Atg5, LC3 and Atg9) neither blocks NGF-induced vacuole formation nor cell death. TrkA activated cell death does not require p38, JNK or Erk1/2 kinases but does require activation of class III PI-3 kinase and is blocked by the casein kinase 1 (CK1) inhibitor, D4476. This inhibitor does not interfere with TrkA activation but does block NGF-dependent AlexaFluor546-dextran uptake and CK1 dependent phosphorylation of beta-catenin. Collectively, these data demonstrate that TrkA stimulates cell death by a novel mechanism involving CK1-dependent hyperstimulation of macropinocytosis.
Li
Chunhui
C
Laboratory of Neural Signalling, The Robarts Research Institute, London, Ontario, Canada.
Macdonald
James I S
JI
Hryciw
Todd
T
Meakin
Susan O
SO
D000704:Analysis of Variance; D051017:Apoptosis Regulatory Proteins / Q000235:genetics / Q000378:metabolism; D001343:Autophagy / Q000187:drug effects*; D000071187:Autophagy-Related Protein 5; D000071186:Beclin-1; D045744:Cell Line, Tumor; D045304:Cytochromes c / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D047468:Immunoprecipitation / Q000379:methods; D051907:Lysosomal Membrane Proteins / Q000235:genetics / Q000378:metabolism; D008247:Lysosomes / Q000187:drug effects / Q000648:ultrastructure; D008527:Medulloblastoma / Q000473:pathology / Q000648:ultrastructure; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D046529:Microscopy, Electron, Transmission / Q000379:methods; D008869:Microtubule-Associated Proteins / Q000235:genetics / Q000378:metabolism; D020932:Nerve Growth Factor / Q000494:pharmacology*; D010873:Pinocytosis / Q000187:drug effects* / Q000502:physiology*; D034741:RNA, Small Interfering / Q000494:pharmacology; D020917:Receptor, trkA / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects; D013997:Time Factors; D014162:Transfection / Q000379:methods; D052118:Lysosomal-Associated Membrane Protein 1
D051017:Apoptosis Regulatory Proteins; D000071187:Autophagy-Related Protein 5; D000071186:Beclin-1; D045304:Cytochromes c; D004791:Enzyme Inhibitors; D049452:Green Fluorescent Proteins; D051907:Lysosomal Membrane Proteins; D008565:Membrane Proteins; D008869:Microtubule-Associated Proteins; D020932:Nerve Growth Factor; D034741:RNA, Small Interfering; D020917:Receptor, trkA; C502778:ATG5 protein, human; C491997:BECN1 protein, human; C489116:LAMP1 protein, human; D052118:Lysosomal-Associated Membrane Protein 1; C477012:MAP1LC3A protein, human; C485184:enhanced green fluorescent protein