Naturally occurring tyrosine kinase inserts block high affinity binding of phospholipase C gamma and Shc to TrkC and neurotrophin-3 signaling.
Neurotrophin-3 binds to the receptor tyrosine kinase, TrkC. Several naturally occurring splice variants of TrkC exist including those with 14- and 39-amino acid inserts within the tyrosine kinase homology region. When expressed in fibroblasts, full-length TrkC, but not the kinase insert variants, mediated neurotrophin-3-stimulated cell proliferation. We investigated the molecular basis of this signaling defect. The kinase inserts blocked the ability of TrkC to mediate neurotrophin-3 stimulated c-myc and c-fos transcription and activation of the AP-1 transcriptional complex. In cells expressing full-length TrkC, neurotrophin-3 promoted a sustained activation of mitogen-activated protein kinase; TrkC containing kinase inserts only mediated transient activation of mitogen-activated protein kinase. The kinase inserts specifically blocked neurotrophin-3-stimulated autophosphorylation of the phospholipase C gamma binding site on TrkC (tyrosine 789) resulting in a severe reduction in phospholipase C gamma association with TrkC and its tyrosine phosphorylation. Neurotrophin-3-stimulated phosphorylation of the Shc binding site (tyrosine 485) on TrkC, and tyrosine phosphorylation of Shc itself, was unaffected by the kinase inserts; however, the kinase inserts blocked high affinity Shc association with TrkC. It is proposed that the lack of high affinity binding of Shc and/or phospholipase C gamma to the TrkC kinase insert variants may be responsible for the inability of these variants to bring about a full biological response in fibroblasts.
Guiton
M
M
Department of Biochemistry, School of Medical Sciences, University of Bristol, United Kingdom.
Gunn-Moore
F J
FJ
Glass
D J
DJ
Geis
D R
DR
Yancopoulos
G D
GD
Tavaré
J M
JM
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D002455:Cell Division / Q000187:drug effects; D017931:DNA Primers; D004789:Enzyme Activation / Q000187:drug effects; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D007527:Isoenzymes / Q000378:metabolism; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D009414:Nerve Growth Factors / Q000494:pharmacology*; D020933:Neurotrophin 3; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D020794:Receptor Protein-Tyrosine Kinases / Q000378:metabolism*; D020801:Receptor, Ciliary Neurotrophic Factor; D020812:Receptor, trkC; D017475:Receptors, Nerve Growth Factor / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D015398:Signal Transduction; D013379:Substrate Specificity; D014162:Transfection; D010738:Type C Phospholipases / Q000378:metabolism*
D017931:DNA Primers; D007527:Isoenzymes; D009414:Nerve Growth Factors; D020933:Neurotrophin 3; D020801:Receptor, Ciliary Neurotrophic Factor; D017475:Receptors, Nerve Growth Factor; D011994:Recombinant Proteins; D011505:Protein-Tyrosine Kinases; D020794:Receptor Protein-Tyrosine Kinases; D020812:Receptor, trkC; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D010738:Type C Phospholipases