Identification of in vivo brain-derived neurotrophic factor-stimulated autophosphorylation sites on the TrkB receptor tyrosine kinase by site-directed mutagenesis.

Brain-derived neurotrophic factor (BDNF) interacts with the TrkB receptor tyrosine kinase, the tyrosine kinase domain of which has homology with the insulin receptor subfamily of protein kinases. This includes the conservation of three regulatory tyrosines (residues 670, 674, and 675) known to play a crucial role in signal transmission by the insulin receptor (tyrosines 1158, 1162, and 1163). Wild-type TrkB and TrkB mutants with Y670F, Y674F/Y675F, Y751F (the tyrosine reported to be important in phosphatidylinositol 3-kinase binding (Obermeier, A., Lammers, R., Wiesmuller, K. H., June, G., Schlessinger, J., and Ullrich, A. (1993) J. Biol. Chem. 268, 22963-22966)), and K540R (consensus ATP binding lysine) substitutions were transiently expressed in COS cells for analysis of phosphorylation sites by two-dimensional phosphopeptide mapping. TrkB phosphorylation sites were also studied in MG86 cells stably expressing wild-type TrkB. In addition, the mutants were expressed in Chinese hamster ovary cells for analysis of the ability of the receptor to mediate BDNF-stimulated transcription from a 12-O-tetradecanoylphorbol-13-acetate response element (TRE). BDNF stimulated the phosphorylation of wild-type TrkB on multiple tyrosine and serine residues. This phosphorylation occurred on tyrosines 670, 674, and 675 plus two other tyrosines and at least two serines that were not unequivocally identified. Wild-type TrkB mediated a pronounced stimulation of TRE-dependent transcription. A Y674F/Y675F, but not Y670F, substitution dramatically inhibited this response. Surprisingly, in COS cells, a Y751F substitution induced dramatically lower tyrosine and serine phosphorylation at all sites but mediated a normal BDNF-stimulated activation of a TRE. Our results demonstrate a critical role for the phosphorylation of tyrosines 674 and 675 in BDNF-dependent signaling by wild-type TrkB.