Inhibition of CIN85-Mediated Invasion by a Novel SH3 Domain Binding Motif in the Lysyl Oxidase Propeptide
Figure 2
LOX-PP reduces CIN85 mono-ubiquitination and ability to interact with c-Cbl.
(A) GST or LOX-PP-GST (PP-GST) was expressed in ZR-75 (left panel) or Hs578T (right panel) cells. GST and associated proteins were precipitated as described in Fig. 1B and subjected to WB for CIN85 (Upstate Biotechnology and Calbiochem antibodies for ZR-75 and Hs578T cells, respectively), CD2AP (H-290), c-Cbl, AMAP1, EGFR, p130Cas and GST. Input, 4%. (B–C) HEK293T cells were transfected with AMAP1-FLAG, CIN85, HA-c-Cbl, HA-ubiquitin and LOX-PP-GST (PP-GST) as indicated and subjected to a ubiquitination assay. FLAG-tagged AMAP1 was immunoprecipitated and total whole cell extracts were subjected to WB with an HA antibody (upper panel), or the indicated antibodies (lower panel). (B). Data were quantified and relative mono-ubiquitination of AMAP1 with and without LOX-PP was determined by averaging the results of three independent experiments (C). P value was calculated using Student's t-test. *, P<0.03. (D) FLAG-CIN85 was expressed in Hs578T (left panel) or MCF-7 (right panel) cells. After lysis, the indicated amount of recombinant LOX-PP-myc-His was added and the mixture incubated at 4°C for 2 h. Proteins were then immunoprecipitated with a FLAG antibody and subjected to WB with FLAG and c-Cbl antibodies. (L Exp, longer exposure).