Activation loop tyrosines contribute varying roles to TrkB autophosphorylation and signal transduction

Abstract

The TrkB receptor tyrosine kinase (RTK) is a high a nity receptor for the neurotrophins brain derived neurotrophic factor (BDNF) and neurotrophin-4/5 (NT-4/5). Following exposure to BDNF or NT-4/5, TrkB is autophosphorylated on ®ve cytoplasmic tyrosines: Y484, Y670, Y674, Y675, and Y785. Based on crystallographic analyses for others RTKs, TrkB tyrosines Y670, Y674, and Y675 are expected to lie within a putative kinase activation loop. Phosphorylation of these activation loop tyrosines is postulated to be a conserved event required for complete RTK activation. Here, we have assessed the importance these activation loop tyrosines play in regulating TrkB autophosphorylation, cytoplasmic signal transduction, and cell proliferation. We show that while tyrosine 670 is dispensable for BDNF-inducible TrkB autophosphorylation and the activation of certain signal transduction events, it is required for complete TrkBmediated cellular proliferation. Combinatorial mutagenesis of tyrosines 674 and 675 only moderately a ects TrkB autophosphorylation, but signi®cantly impairs the BDNF-inducible stimulation of cytoplasmic signaling events and cellular proliferation. The combined mutation of all three activation loop tyrosines results in an inactive receptor, which is unable to autophosphorylate, stimulate signaling events, or induce mitogenesis. The data highlight the varying degrees of importance of the three activation loop tyrosines in TrkB mediated biological responses.

Mutual Regulation of Src Family Kinases and the Neurotrophin Receptor TrkB

“…By contrast, addition of zinc to HEK cells expressing Y515F mutant was still capable of inducing increased phosphorylation of Tyr-705/Tyr-706. Note that Y705F/Y706F mutant was still able to be phosphorylated at Tyr-515, probably due to overexpression-induced forced phosphorylation and consistent with previous findings (16,18). Collectively, these findings support the assertion that phosphorylation of Tyr-705/Tyr-706 is the initial event in zinc-mediated transactivation of TrkB.…”

Acidic substitution of the activation loop tyrosines in TrkA supports nerve growth factor-independent cell survival and neuronal differentiation

“…Tyr 684 in TrkA, the positional equivalent of Tyr 1163 in the IRK, is thought to stabilize an active conformation. Consistent with this model, phenylalanine mutagenesis of the catalytic core tyrosines in TrkA, TrkB, the IRK, the IGF-1 receptor, the FGF receptor and Met e ectively inactivates kinase activity (Ellis et al, 1986;Wilden et al, 1992;Li et al, 1994;Longati et al, 1994;Hernandez-Sanchez et al, 1995;Stannard et al, 1995;Cunningham et al, 1997;McCarty and Feinstein, 1998).…”

SLAM-associated Protein as a Potential Negative Regulator in Trk Signaling

“…This leads to the subsequent trans-phosphorylation of five tyrosine residues (Tyr 484 , Tyr 670 , Tyr 674 , Tyr 675 , and Tyr 785 on TrkB) on the adjacent receptor (5). Among the five phosphotyrosines, Tyr 484 and Tyr 785 are located outside the kinase domain, whereas Tyr 670 , Tyr 674 , and Tyr 675 are situated in the activation loop of the kinase domain.…”

“…Tyr 785 , on the other hand, serves as docking site for PLC␥. Association of these adaptor molecules with activated Trk receptors results in the initiation of signaling pathways, including the mitogen-activated protein kinase, phosphatidylinositol 3-kinase, and PLC␥ pathways, thereby mediating the actions of neurotrophins (5)(6)(7)(8)(9)(10)(11)(12).…”