Identification of TrkB autophosphorylation sites and evidence that phospholipase C-gamma 1 is a substrate of the TrkB receptor.
The TrkB receptor protein-tyrosine kinase is a receptor for brain-derived neurotrophic factor and neurotrophin-3. In response to brain-derived neurotrophic factor and neurotrophin-3 treatment, TrkB expressed exogenously in Rat-2 cells is rapidly phosphorylated on tyrosine residues. At least 2 regions of TrkB contain phosphorylated tyrosines. The major sites of autophosphorylation are in the region containing Tyr-670, Tyr-674, and Tyr-675, which lies in the kinase domain and corresponds by sequence homology to the Tyr-416 autophosphorylation site in p60c-Src. Tyr-785, which lies just to the COOH-terminal side of the kinase domain in a relatively short tail characteristic of the Trk family of protein-tyrosine kinase receptors, is also phosphorylated in response to neurotrophin-3 treatment. The sequence around Tyr-785 fits a consensus sequence for binding phospholipase C-gamma 1. The simplest interpretation of these results is that, in response to neurotrophin binding, at least two and perhaps all three of the tyrosines in the Tyr-670/674/675 region are autophosphorylated independently, and Tyr-785 is autophosphorylated in vivo. Following activation of TrkB, phospholipase C-gamma 1 is phosphorylated on Tyr-783, Tyr-771, and Tyr-1254. Phospholipase C-gamma 1 also forms a complex with TrkB in response to neurotrophin-3 treatment, consistent with the possibility that one of the TrkB autophosphorylation sites provides a binding site for the phospholipase C-gamma 1 SH2 domains, as is the case for other receptor protein-tyrosine kinases. We conclude that phospholipase C-gamma 1 is directly phosphorylated by TrkB. Since phosphorylation of Tyr-783 and Tyr-1254 results in activation of phospholipase C-gamma 1, we predict that neurotrophin-3 leads to activation of phospholipase C-gamma 1 following binding to TrkB in Rat-2 cells.
Middlemas
D S
DS
Molecular Biology and Virology Laboratory, Salk Institute, San Diego, California 92186.
Meisenhelder
J
J
Hunter
T
T
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D015870:Gene Expression / Q000187:drug effects; D007527:Isoenzymes / Q000378:metabolism*; D008969:Molecular Sequence Data; D009414:Nerve Growth Factors / Q000494:pharmacology; D009419:Nerve Tissue Proteins / Q000494:pharmacology; D020933:Neurotrophin 3; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D010449:Peptide Mapping; D010748:Phosphopeptides / Q000737:chemistry / Q000302:isolation & purification; D010766:Phosphorylation; D051381:Rats; D020794:Receptor Protein-Tyrosine Kinases / Q000096:biosynthesis / Q000378:metabolism*; D020813:Receptor, trkB; D017475:Receptors, Nerve Growth Factor / Q000096:biosynthesis / Q000378:metabolism*; D013379:Substrate Specificity; D014162:Transfection; D014357:Trypsin; D010738:Type C Phospholipases / Q000378:metabolism*
D007527:Isoenzymes; D009414:Nerve Growth Factors; D009419:Nerve Tissue Proteins; D020933:Neurotrophin 3; D010446:Peptide Fragments; D010748:Phosphopeptides; D017475:Receptors, Nerve Growth Factor; D020794:Receptor Protein-Tyrosine Kinases; D020813:Receptor, trkB; D010738:Type C Phospholipases; D014357:Trypsin