title: Acidic substitution of the activation loop tyrosines in TrkA supports nerve growth factor-dependent, but not nerve growth factor-independent, differentiation and cell cycle arrest in the human neuroblastoma cell line, SY5Y.
abstract: TrkA is the receptor tyrosine kinase (RTK) for nerve growth factor (NGF) and stimulates NGF-dependent cell survival and differentiation in primary neurons and also differentiation of neuroblastomas and apoptosis of medulloblastomas. We have previously shown that aspartic acid and glutamic acid substitution (AspGlu and GluAsp) of the activation loop tyrosines in TrkA (Tyr(683) and Tyr(684)) supports NGF-independent neuritogenesis and cell survival in PC12 cell-derived nnr5 cells. In this study, the AspGlu and GluAsp mutant Trks have been analysed for their ability to support NGF-independent and NGF-dependent neuritogenesis, proliferation and cell signalling in the human neuroblastoma cell line, SY5Y. We find that the AspGlu and GluAsp mutant Trks support NGF-dependent, but not NGF-independent, autophosphorylation, neuritogenic responses and/or inhibit cell cycle progression. The NGF-dependent neuritogenic responses are lower for the mutant Trks (approximately 30-60% for AspGlu and 50-60% for GluAsp), relative to wild-type TrkA. While both the AspGlu and GluAsp mutant Trks support NGF-dependent transient phosphorylation of Shc, PLCgamma-1, AKT, FRS2, SH2B as well as prolonged MAP kinase activation, the GluAsp mutant induces stronger NGF-dependent tyrosine phosphorylation of FRS2 and SH2B, as well as a stronger reduction in bromodeoxyuridine (BrdU) incorporation. Collectively, these data suggest that neither absolute levels of receptor autophosphorylation, high levels of TrkA expression nor the activation of a specific signalling pathway is dominant and absolutely essential for neuritogenesis and cell cycle arrest of SY5Y cells.
authors.affiliation: Laboratory of Neural Signalling, Cell Biology Group, The Robarts Research Institute, 100 Perth Drive, London, Ontario, Canada N6A 5K8.
authors.forename: Ela A
authors.initials: EA
authors.lastname: Gryz
authors.forename: Susan O
authors.initials: SO
authors.lastname: Meakin
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport / Q000378:metabolism; D019943:Amino Acid Substitution; D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology; D002453:Cell Cycle; D002454:Cell Differentiation; D045744:Cell Line, Tumor; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D020932:Nerve Growth Factor / Q000502:physiology*; D016501:Neurites / Q000502:physiology; D009447:Neuroblastoma / Q000378:metabolism / Q000473:pathology; D051966:Phospholipase C gamma; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt; D011817:Rabbits; D020917:Receptor, trkA; D055765:Shc Signaling Adaptor Proteins; D015398:Signal Transduction; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; D010738:Type C Phospholipases / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism*
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D002352:Carrier Proteins; C106534:FRS2 protein, human; D008565:Membrane Proteins; D010750:Phosphoproteins; D011518:Proto-Oncogene Proteins; C518910:SH2B1 protein, human; C528024:SHC1 protein, human; D055765:Shc Signaling Adaptor Proteins; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; D014443:Tyrosine; D020932:Nerve Growth Factor; D020917:Receptor, trkA; C494918:AKT1 protein, human; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D020928:Mitogen-Activated Protein Kinases; D010738:Type C Phospholipases; D051966:Phospholipase C gamma