Figure 8: ERBB2 indirectly promotes TOM1L1-Ser321 phosphorylation for interaction with TOLLIP and MT1-MMP trafficking.
From: TOM1L1 drives membrane delivery of MT1-MMP to promote ERBB2-induced breast cancer cell invasion
(a) Lysates from 3T3 cells infected as indicated were immunoprecipitated with an anti-TOM1L1 antibody and immunoblotted to visualize TOM1L1 phosphorylation. (b) SILAC mass spectrometry analysis. 3T3-neu cells transfected with GFP–hTOM1L1 were cultured for 2 weeks in medium containing light (12C614N4-Arg and 12C614N2-Lys) or heavy (13C615N4-Arg and 13C615N2-Lys) arginines and lysines and treated with 1 μM Lapatinib or not (DMSO) for 3 h before lysis. GFP–hTOM1L1 was immunoprecipitated using the GFP-Nanotrap technology then digested using trypsin. The hTOM1L1 peptides phosphorylation was then analysed by mass spectrometry (see Methods for details). Upper panel: fragmentation spectra of the single peptide find phosphorylated (localization on Ser321 with a probability >0.75 as calculated by MaxQuant). Lower panel: Heavy/Light SILAC ratio (H/L) for this peptide, traducing the phosphorylation ratio changes between the tested conditions. (c) 3T3-neu and 293 T cells infected as indicated were transfected with HA–TOLLIP. Lysates were then immunoprecipitated and immunoblotted as shown. (d) 3T3-neu cells infected as indicated were seeded in Boyden chambers with matrigel for 24 h and cells present in the lower chamber were counted. The histogram shows the invasion ratio normalized to control (n=3). **P≤0.01 (Student’s t-test). (e) 3T3-neu cells infected as indicated were cultured on Oregon Green 488 gelatin for 24 h to visualize gelatin degradation areas. The quantification (mean±s.e.m.) of degradation areas per cell is shown (n=26-48). **P≤0.01; ***P≤0.001 (Student’s t-test). (f) BT-474 cells infected with TOM1L1 shRNA and transfected with indicated constructs were imaged by confocal orthogonal imaging. Arrowheads show the change of MT1-MMP apico-basal polarity. Scale bar, 10 μm. (g) Quantification of f. The fraction of mCherry–MT1-MMP in contact with the gelatin layer was evaluated as in Fig. 5b (n=4). Mean±s.e.m. *P≤0.05 (Student’s t-test). (h) 3T3-neu cells transfected with mCherry–MT1-MMP, HA–TOLLIP and GFP–TOM1L1 or the phosphomimetic mutant (S320E) were treated or not with 1 μM Lapatinib for 2 h. Localization of colocated spots was visualized by confocal imaging. Insets show higher magnification of the boxed areas. Scale bar, 10 μm.
