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-deri ved neurotrophic factor and neurotrophin-3. In response to brain-deriv ed neurotrophic factor and neurotrophin-3 treatment, TrkB expressed ex ogenously in Rat-2 cells is rapidly phosphorylated on tyrosine residue s. At least 2 regions of TrkB contain phosphorylated tyrosines. The ma jor 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 p60(c -Src). Tyr-785, which lies just to the COOH-terminal side of the kinas e domain in a relatively short tail characteristic of the Trk family o f protein-tyrosine kinase receptors, is also phosphorylated in respons e to neurotrophin-3 treatment. The sequence around Tyr-785 fits a cons ensus sequence for binding phospholipase C-gamma 1. The simplest inter pretation of these results is that, in response to neurotrophin bindin g, at least two and perhaps all three of the tyrosines in the Tyr-670/ 674/675 region are autophosphorylated independently and Tyr-785 is aut ophosphorylated in vivo. Following activation of TrkB, phospholipase C -gamma 1 is phosphorylated on Tyr-783, Tyr-771, and Tyr-1254. Phosphol ipase C-gamma 1 also forms a complex with TrkB in response to neurotro phin-3 treatment, consistent with the possibility that one of the TrkB autophosphorylation sites provides a binding site for the phospholipa se C-gamma 1 SH2 domains, as is the case for other receptor protein-ty rosine kinases. We conclude that phospholipase C-gamma 1 is directly p hosphorylated by TrkB. Since phosphorylation of Tyr-783 and Tyr-1254 r esults in activation of phospholipase C-gamma 1, we predict that neuro trophin-3 leads to activation of phospholipase C-gamma 1 following bin ding to TrkB in Rat-2 cells.