Acidic substitution of the activation loop tyrosines in TrkA supports nerve growth factor-independent cell survival and neuronal differentiation.
TrkA is the receptor tyrosine kinase (RTK) for nerve growth factor (NGF) and stimulates NGF-dependent cell survival and differentiation in primary neurons. TrkA expression in neuronal tumors also supports NGF-dependent differentiation of neuroblastomas and apoptosis of medulloblastomas. Phosphorylation of the activation loop tyrosines in RTK's are essential to activation as well as allosteric changes that facilitate substrate interaction and phosphorylation. Acidic amino acid substitution of the activation loop tyrosines in TrkA, Tyr683Tyr684, was performed to mimic the negative charges normally induced by ligand activation and receptor phosphorylation. A total of eight independent mutants containing single or double substitutions were generated for comparison. Herein, we demonstrate that acidic substitution of the activation loop tyrosines is sufficient to induce allosteric changes required for constitutive TrkA kinase activity as well as phosphorylation of TrkA signaling proteins such as Shc, PLCgamma-1, FRS-2 and erk1/2. The strongest constitutively active TrkA mutants, GluAsp and AspGlu, support NGF-independent neuritogenesis and cell survival to levels approximately 65 and 80-100%, respectively, of NGF-activated wild type TrkA. Thus, constitutively active TrkA may provide a useful strategy in future therapeutic approaches to limit the development and progression of neuronal tumors.
Gryz
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Neurodegeneration Group, The John P. Robarts Research Institute, London, Ontario, Canada.
Meakin
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D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry / Q000502:physiology*; D002454:Cell Differentiation; D002460:Cell Line; D002470:Cell Survival; D008565:Membrane Proteins / Q000378:metabolism; D020929:Mitogen-Activated Protein Kinase Kinases / Q000378:metabolism; D009097:Multienzyme Complexes; D020932:Nerve Growth Factor / Q000494:pharmacology*; D009474:Neurons / Q000502:physiology*; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D011506:Proteins / Q000378:metabolism; D011817:Rabbits; D055765:Shc Signaling Adaptor Proteins; D015398:Signal Transduction; D010738:Type C Phospholipases / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism
D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D001426:Bacterial Proteins; C106534:FRS2 protein, human; D008565:Membrane Proteins; D009097:Multienzyme Complexes; D010750:Phosphoproteins; D011506:Proteins; D055765:Shc Signaling Adaptor Proteins; C076398:TyrA protein, Bacteria; D014443:Tyrosine; D020932:Nerve Growth Factor; D020929:Mitogen-Activated Protein Kinase Kinases; D010738:Type C Phospholipases