Middlemas D S, Meisenhelder J, Hunter T
Molecular Biology and Virology Laboratory, Salk Institute, San Diego, California 92186.
J Biol Chem. 1994 Feb 18;269(7):5458-66.
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.
TrkB受体蛋白酪氨酸激酶是脑源性神经营养因子和神经营养素-3的受体。在接受脑源性神经营养因子和神经营养素-3处理后，在大鼠-2细胞中外源表达的TrkB在酪氨酸残基上迅速发生磷酸化。TrkB至少有2个区域含有磷酸化酪氨酸。自身磷酸化的主要位点位于包含Tyr-670、Tyr-674和Tyr-675的区域，该区域位于激酶结构域内，通过序列同源性与p60c-Src中的Tyr-416自身磷酸化位点相对应。Tyr-785位于激酶结构域COOH末端一侧，处于Trk家族蛋白酪氨酸激酶受体特有的相对较短的尾部，在接受神经营养素-3处理后也会发生磷酸化。Tyr-785周围的序列符合磷脂酶C-γ1结合的共有序列。对这些结果最简单的解释是，在神经营养素结合后，Tyr-670/674/675区域中的至少两个酪氨酸，也许是所有三个酪氨酸独立地发生自身磷酸化，并且Tyr-785在体内发生自身磷酸化。TrkB激活后，磷脂酶C-γ1在Tyr-783、Tyr-771和Tyr-1254上发生磷酸化。在接受神经营养素-3处理后，磷脂酶C-γ1也与TrkB形成复合物，这与TrkB自身磷酸化位点之一为磷脂酶C-γ1的SH2结构域提供结合位点的可能性一致，其他受体蛋白酪氨酸激酶也是如此。我们得出结论，磷脂酶C-γ1被TrkB直接磷酸化。由于Tyr-783和Tyr-1254的磷酸化导致磷脂酶C-γ1激活，我们预测在大鼠-2细胞中，神经营养素-3与TrkB结合后会导致磷脂酶C-γ1激活。
