Abstract
The TrkB receptor tyrosine kinase (RTK) is a high affinity receptor for the neurotrophins brain derived neurotrophic factor (BDNF) and neurotrophin-4/5 (NT-4/5). Following exposure to BDNF or NT-4/5, TrkB is autophosphorylated on five cytoplasmic tyrosines: Y484, Y670, Y674, Y675, and Y785. Based on crystallographic analyses for others RTKs, TrkB tyrosines Y670, Y674, and Y675 are expected to lie within a putative kinase activation loop. Phosphorylation of these activation loop tyrosines is postulated to be a conserved event required for complete RTK activation. Here, we have assessed the importance these activation loop tyrosines play in regulating TrkB autophosphorylation, cytoplasmic signal transduction, and cell proliferation. We show that while tyrosine 670 is dispensable for BDNF-inducible TrkB autophosphorylation and the activation of certain signal transduction events, it is required for complete TrkB-mediated cellular proliferation. Combinatorial mutagenesis of tyrosines 674 and 675 only moderately affects TrkB autophosphorylation, but significantly impairs the BDNF-inducible stimulation of cytoplasmic signaling events and cellular proliferation. The combined mutation of all three activation loop tyrosines results in an inactive receptor, which is unable to autophosphorylate, stimulate signaling events, or induce mitogenesis. The data highlight the varying degrees of importance of the three activation loop tyrosines in TrkB mediated biological responses.
Publication types
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Research Support, Non-U.S. Gov't
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Research Support, U.S. Gov't, Non-P.H.S.
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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3T3 Cells
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Adaptor Proteins, Signal Transducing*
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Adaptor Proteins, Vesicular Transport*
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Animals
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Brain-Derived Neurotrophic Factor / metabolism
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Brain-Derived Neurotrophic Factor / pharmacology
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Calcium-Calmodulin-Dependent Protein Kinases / metabolism
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Cell Division
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Cell Survival
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Enzyme Activation
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Gene Expression
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Humans
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Isoenzymes / metabolism
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Mice
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Mitogen-Activated Protein Kinase 1
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Mitogen-Activated Protein Kinase 3
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Mitogen-Activated Protein Kinases*
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Mutagenesis, Site-Directed
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Phospholipase C gamma
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Phosphorylation
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Proteins / metabolism
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Proto-Oncogene Proteins c-fos / biosynthesis
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Receptor Protein-Tyrosine Kinases / genetics
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Receptor Protein-Tyrosine Kinases / physiology*
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Receptor, Ciliary Neurotrophic Factor
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Receptors, Nerve Growth Factor / genetics
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Receptors, Nerve Growth Factor / physiology*
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Shc Signaling Adaptor Proteins
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Signal Transduction*
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Src Homology 2 Domain-Containing, Transforming Protein 1
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Type C Phospholipases / metabolism
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Tyrosine / metabolism*
Substances
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Adaptor Proteins, Signal Transducing
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Adaptor Proteins, Vesicular Transport
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Brain-Derived Neurotrophic Factor
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Isoenzymes
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Proteins
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Proto-Oncogene Proteins c-fos
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Receptor, Ciliary Neurotrophic Factor
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Receptors, Nerve Growth Factor
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SHC1 protein, human
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Shc Signaling Adaptor Proteins
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Shc1 protein, mouse
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Src Homology 2 Domain-Containing, Transforming Protein 1
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Tyrosine
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Receptor Protein-Tyrosine Kinases
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Calcium-Calmodulin-Dependent Protein Kinases
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Mitogen-Activated Protein Kinase 1
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Mitogen-Activated Protein Kinase 3
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Mitogen-Activated Protein Kinases
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Type C Phospholipases
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Phospholipase C gamma