HER2 Carboxyl-terminal Fragments Regulate Cell Migration and Cortactin Phosphorylation

Jesús García-Castillo,Kim Pedersen,Pier-Davide Angelini,Joan Josep Bech-Serra,Núria Colomé,Matthew Paul Cunningham,Josep Lluis Parra-Palau,Francesc Canals,José Baselga,Joaquín Arribas

doi:10.1074/jbc.m109.001982

Abstract

A group of breast cancer patients with a higher probability of developing metastasis expresses a series of carboxyl-terminal fragments (CTFs) of the tyrosine kinase receptor HER2. One of these fragments, 611-CTF, is a hyperactive form of HER2 that constitutively establishes homodimers maintained by disulfide bonds, making it an excellent model to study overactivation of HER2 during tumor progression and metastasis. Here we show that expression of 611-CTF increases cell motility in a variety of assays. Since cell motility is frequently regulated by phosphorylation/dephosphorylation, we looked for phosphoproteins mediating the effect of 611-CTF using two alternative proteomic approaches, stable isotope labeling with amino acids in cell culture and difference gel electrophoresis, and found that the latter is particularly well suited to detect changes in multiphosphorylated proteins. The difference gel electrophoresis screening identified cortactin, a cytoskeleton-binding protein involved in the regulation of cell migration, as a phosphoprotein probably regulated by 611-CTF. This result was validated by characterizing cortactin in cells expressing this HER2 fragment. Finally, we showed that the knockdown of cortactin impairs 611-CTF-induced cell migration. These results suggest that cortactin is a target of 611-CTF involved in the regulation of cell migration and, thus, in the metastatic behavior of breast tumors expressing this CTF.

Highlights

The epidermal growth factor receptor is the prototype of a family that includes HER2 (ErbB2, Neu), HER3, and HER4 (ErbB3 and ErbB4)
Effect of carboxyl-terminal fragments (CTFs) on Cell Migration—To analyze the effect of individual CTFs on cell migration, we used MCF7 Tet-Off cells stably transfected with plasmids encoding HER2 or 611, 648, or 687-CTFs (Fig. 1A; see Ref. 7) and, as control, the same cells transfected with the empty vector
E, MCF7 Tet-Off/611-CTF cells treated with or without doxycycline for 48 h were analyzed in a confocal microscope by indirect immunofluorescence with the antibodies H-191 against cortactin and CB11 against HER2, as described under “Experimental Procedures.”

Summary

EXPERIMENTAL PROCEDURES

Cells—MCF7 Tet-Off (BD Biosciences) transfected with fulllength HER2, 611-CTF, 648-CTF, and 687-CTF have been recently characterized [7]. DIGE—107 MCF7 Tet-Off/611-CTF cells were seeded and cultured with or without doxycycline for 18 h in the presence of serum and an additional 6 h in serum-free media and lysed. The kinase activity was determined by incubating the immunoprecipitated material for 4 h with lysates of MCF7 Tet-Off cells stably transfected with 611-CTF treated or not with doxycycline, in 150 mM NaCl, 20 mM HEPES, pH 7.9, 0.1% Nonidet P-40, 5 mM MgCl2, 5 mM MnCl2, 1 mM dithiothreitol, 1 ␮M cold ATP, and 0.3 ␮M [␥-32P]ATP (3000 Ci/mmol). To analyze in vitro kinase activity of 611-CTF, it was purified by immunoprecipitation with CB11 antibody from MCF7 TetOff cells treated or not with doxycycline and incubated for 24 h with cortactin-FLAG purified as above in the presence of [␥-32P]ATP. For blocking and antibody binding, we used phosphate-buffered saline with 1% bovine serum albumin, 0.1% saponin, and 0.02% NaN3, and for mounting, we used Vectashield with DAPI (Vector Laboratories)

RESULTS

DISCUSSION

Confirming a role of cortactin in