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 recep tor 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 insul in receptor (tyrosines 1158, 1162, and 1163). Wild-type TrkB and TrkB mutants with Y670F, Y674F/Y675F, Y751F (the tyrosine reported to be im portant in phosphatidylinositol 3-kinase binding (Obermeier, A., Lamme rs, R., Wiesmuller, K. H., June, G., Schlessinger, J., and Ullrich, A. (1993) J. Biol. Chem. 268, 22963-22966)), and K540R (consensus ATP bi nding lysine) substitutions were transiently expressed in COS cells fo r analysis of phosphorylation sites by two-dimensional phosphopeptide mapping. TrkB phosphorylation sites were also studied in MG86 cells st ably expressing wild-type TrkB. In addition, the mutants were expresse d in Chinese hamster ovary cells for analysis of the ability of the re ceptor to mediate BDNF-stimulated transcription from a 12-O-tetradecan oylphorbol-13-acetate response element (TRE). BDNF stimulated the phos phorylation of wild type TrkB on multiple tyrosine and serine residues . This phosphorylation occurred on tyrosines 670, 674, and 675 plus tw o other tyrosines and at least two serines that were not unequivocally identified. Wild-type TrkB mediated a pronounced stimulation of TRE-d ependent transcription. A Y674F/Y675F, but not Y670F, substitution dra matically inhibited this response. Surprisingly, in COS cells, a Y751F substitution induced dramatically lower tyrosine and serine phosphory lation at all sites but mediated a normal BDNF-stimulated activation o f a TRE. Our results demonstrate a critical role for the phosphorylati on of tyrosines 674 and 675 in BDNF-dependent signaling by wild-type T rkB.