Figure 4.

Analysis of the requirement of individual tyrosine residues in the YRASYY activation loop motif for full length ALK activation. (A) Detection of phosphorylation of ALK (Y1604) and downstream signalling activity (pERK1/2) of various ALK activation loop mutants by immunoblotting using the indicated antibodies. PanERK was employed as a loading control. Blots are representative of three independent experiments; (B) Neurite outgrowth assay of PC12 cells transfected with various ALK mutants, as indicated in the figure, in the presence or absence of mAb46 stimulation. Black bars represent unstimulated cells and grey bars denote cells that are stimulated with mAb46. Bars represent mean percentage ± STD of neurite-carrying cells among GFP-positive cells from three independent experiments; (C) In vitro kinase assay of various ALK TKDs. Substrate phosphorylation activity of the ALK TKDs mutated in various combinations of Y1278, Y1282 and Y1283 to phenylalanine was assayed employing a peptide mimic of the ALK activation loop. Increasing amounts of ALK TKD proteins were assayed at 30 °C for 30 min.