Fig. 1: Abundance-dependent activation of TrkB kinase and changes in actin morphology strongly depend on phosphorylation in the YxxxYY-motif.

A Model depicting TrkB kinase signaling. Neurotrophin (BDNF/NT-4)) binding induces conformational changes and supports receptor dimerization. The kinase is released from cis-autoinhibition, ATP binds to the intracellular kinase domain and allows trans-autophosphorylation. Three tyrosine residues in the consensus motif YxxxYY, Y⁷⁰¹, Y⁷⁰⁵, and Y⁷⁰⁶ are phosphorylated in the activation loop of the receptor. ATP-binding and phosphorylation of the YxxxYY motif occur upstream of autophosphorylation and downstream signaling. In the intracellular domain (ICD), phosphorylation of Y⁵¹⁵ recruits Shc and activates Ras/ERK and Pi3K-Akt pathways. Y⁸¹⁶ forms an adapter site for PLCγ. S⁴⁷⁸ signals to the TIAM-Rac1 pathway. B TrkB phosphorylation in the absence of BDNF is unaffected by serum depletion. Western blotting of whole-cell lysates generated from HEK293 cells expressing TrkB. Control cultures were maintained in serum before total lysates were produced. The cell cultures were treated as indicated. Serum depletion was performed for 3 h. To inhibit TrkB kinase activity, the cultures were preincubated with 150 nM K252a, a Trk kinase inhibitor, for 30 min. DMSO was used as the solvent control. When indicated, cells were also treated with 20 ng/ml BDNF for 15 min and compared with BDNF-stimulated cells under K252a treatment. C Quantification of Western blots for pTrkB-kin normalized to the total Trk-kinase levels by densitometry. The relative integrated densities are also presented. K252a treatment for 30 min caused a reduction in TrkB phosphorylation levels under control, serum-depleted, and BDNF stimulation conditions. Bar graph: Mean ± SEM, overlaid with single data points; n = 3. D In the absence of neurotrophins, TrkB overexpression induces TrkB phosphorylation and changes cell morphology. Immunofluorescence of the TrkB receptor (green) and pTrk-PLCγ (red). F-actin was labeled with Acti-stain-670 phalloidin (blue). HEK293 cells were transfected with either TrkB- or TrkB-YFY kinase mutant. The cells were immunostained for 30 h. Yellow arrows indicate round pTrk-positive cells. Cyan arrows indicate filamentous pTrk-negative cells. Confocal images; scale bar:25 µm. E Filopodia phenotype of TrkB-expressing cells (high-resolution confocal stack image). F Round-shaped cells expressing kinase-active TrkB mutants. Immunostaining of HEK293 cells expressing either TrkB-wt or TrkB mutants (for details see Fig. S1). Cells expressing TrkB-wt or the constitutively active mutant TrkB-YDY were also treated with K252a. Typically, filamentous cells express kinase-dead ATP mutants of TrkB or YxxxYY mutants (YFY and YFF). Treatment with 150 nM K252a for 30 min reversed the round shape of the cells expressing TrkB-wt or TrkB-YDY. Confocal images; scale bar: 25 µm. G Quantification of the percentage of cells showing either a round shape or typical filopodia. Round cells were further subdivided into those that were either positive (+) or negative (−) for pTrk. Data were acquired from ten fields of view in three biological replicates (n = 3). Scale bar: 25 µm. H TrkB phosphorylation is correlated with TrkB expression. Linear positive correlation of the integrated density of anti-TrkB and anti-pTrkB-kin immunoreactivity in TrkB-expressing HEK293 cells. Immunolabels per cell were measured as the integrated density per cell from the maximum intensity projection images of confocal z-stacks. Single-cell data, n = 280 cells; data collected from 20 confocal image fields and 4 cell cultures (n = 4).