TY - JOUR
T1 - Filamin A is a novel caveolin-1-dependent target in IGF-I-stimulated cancer cell migration
AU - Ravid, Dana
AU - Chuderland, Dana
AU - Landsman, Limor
AU - Lavie, Yaakov
AU - Reich, Reuven
AU - Liscovitch, Mordechai
N1 - Funding Information:
We gratefully acknowledge the excellent technical assistance of Yona Ely and we thank all members of our group for their help and support. We are grateful to Prof. Markus Keiser (University of Hannover), Prof. Richard Pagano (Mayo Clinic) and Prof. Christopher McCulloch (University of Toronto) for providing vectors. We thank Prof. Arie Admon (Technion) for mass spectrometry analyses and Dr. Maya Freund (Israel National Center of Forensic Medicine) for cell lines genotyping and authentication. We have greatly benefited from discussions with Prof. Alexander Bershadsky, Prof. Hagit Eldar-Finkelman, Dr. Merav Yoeli-Lerner, Prof. Rony Seger, Prof. Sandrine Humbert and Prof. Haim Werner. This research was supported in part by Philip Morris USA Inc. and Philip Morris International, La Fondation Raphael et Regina Levy and the Women's Health Research Center. D.R. was a recipient of the Rita Gehl Predoctoral Scholarship in Cancer Research. M.L. is the incumbent of the Harold L. Korda Professorial Chair in Biology.
PY - 2008/9/10
Y1 - 2008/9/10
N2 - Caveolin-1 is an essential structural constituent of caveolae which is involved in regulation of mitogenic signaling and oncogenesis. Caveolin-1 has been implicated in cell migration but its exact role and mechanism of action in this process remained obscure. We have previously reported that expression of caveolin-1 in stably transfected MCF-7 human breast cancer (MCF-7/Cav1) cells up-regulates phosphorylation of a putative Akt substrate protein, designated pp340 [D. Ravid, S. Maor, H. Werner, M. Liscovitch, Caveolin-1 inhibits cell detachment-induced p53 activation and anoikis by upregulation of insulin-like growth factor-I receptors and signaling, Oncogene 24 (2005) 1338-1347.]. We now show, using differential detergent extraction, SDS-PAGE and mass spectrometry, that the major protein in the pp340 band is the actin filament cross-linking protein filamin A. The identity of pp340 as filamin A was confirmed by immunoprecipitation of pp340 with specific filamin A antibodies. RT-PCR, flow cytometry and Western blot analyses show that filamin A mRNA and protein levels are respectively 3.5- and 2.5-fold higher in MCF-7/Cav1 cells than in MCF-7 cells. Basal filamin A phosphorylation on Ser-2152, normalized to total filamin A levels, is 7.8-fold higher in MCF-7/Cav1 than in MCF-7 cells. Insulin-like growth factor-I (IGF-I) stimulates phosphorylation of filamin A on Ser-2152 in MCF-7 cells and further enhances Ser-2152 phosphorylation over its already high basal level in MCF-7/Cav1 cells. The effect of IGF-I is inhibited by the PI3K inhibitor wortmannin, indicating that IGF-I-stimulated phosphorylation of filamin A occurs via the PI3K/Akt pathway. Co-immunoprecipitation experiments have confirmed a previous report showing that filamin A and caveolin-1 co-exist in a complex and have revealed the presence of active phospho-Akt in this complex. Ser-2152 phosphorylation of filamin A has been implicated in cancer cell migration. Accordingly, caveolin-1 expression dramatically enhances IGF-I-dependent MCF-7 cell migration. These data indicate that caveolin-1 specifies filamin A as a novel target for Akt-mediated filamin A Ser-2152 phosphorylation thus mediating the effects of caveolin-1 on IGF-I-induced cancer cell migration.
AB - Caveolin-1 is an essential structural constituent of caveolae which is involved in regulation of mitogenic signaling and oncogenesis. Caveolin-1 has been implicated in cell migration but its exact role and mechanism of action in this process remained obscure. We have previously reported that expression of caveolin-1 in stably transfected MCF-7 human breast cancer (MCF-7/Cav1) cells up-regulates phosphorylation of a putative Akt substrate protein, designated pp340 [D. Ravid, S. Maor, H. Werner, M. Liscovitch, Caveolin-1 inhibits cell detachment-induced p53 activation and anoikis by upregulation of insulin-like growth factor-I receptors and signaling, Oncogene 24 (2005) 1338-1347.]. We now show, using differential detergent extraction, SDS-PAGE and mass spectrometry, that the major protein in the pp340 band is the actin filament cross-linking protein filamin A. The identity of pp340 as filamin A was confirmed by immunoprecipitation of pp340 with specific filamin A antibodies. RT-PCR, flow cytometry and Western blot analyses show that filamin A mRNA and protein levels are respectively 3.5- and 2.5-fold higher in MCF-7/Cav1 cells than in MCF-7 cells. Basal filamin A phosphorylation on Ser-2152, normalized to total filamin A levels, is 7.8-fold higher in MCF-7/Cav1 than in MCF-7 cells. Insulin-like growth factor-I (IGF-I) stimulates phosphorylation of filamin A on Ser-2152 in MCF-7 cells and further enhances Ser-2152 phosphorylation over its already high basal level in MCF-7/Cav1 cells. The effect of IGF-I is inhibited by the PI3K inhibitor wortmannin, indicating that IGF-I-stimulated phosphorylation of filamin A occurs via the PI3K/Akt pathway. Co-immunoprecipitation experiments have confirmed a previous report showing that filamin A and caveolin-1 co-exist in a complex and have revealed the presence of active phospho-Akt in this complex. Ser-2152 phosphorylation of filamin A has been implicated in cancer cell migration. Accordingly, caveolin-1 expression dramatically enhances IGF-I-dependent MCF-7 cell migration. These data indicate that caveolin-1 specifies filamin A as a novel target for Akt-mediated filamin A Ser-2152 phosphorylation thus mediating the effects of caveolin-1 on IGF-I-induced cancer cell migration.
KW - Akt
KW - Cancer
KW - Caveolin
KW - Filamin
KW - Migration
UR - http://www.scopus.com/inward/record.url?scp=50049106405&partnerID=8YFLogxK
U2 - 10.1016/j.yexcr.2008.06.004
DO - 10.1016/j.yexcr.2008.06.004
M3 - ???researchoutput.researchoutputtypes.contributiontojournal.article???
AN - SCOPUS:50049106405
SN - 0014-4827
VL - 314
SP - 2762
EP - 2773
JO - Experimental Cell Research
JF - Experimental Cell Research
IS - 15
ER -
