BimEL is an important determinant for induction of anoikis sensitivity by mitogen-activated protein/extracellular signal-regulated kinase kinase inhibitors.
Loss of contact with substratum triggers apoptosis in many normal cell types, a phenomenon termed anoikis. We reported previously that mitogen-activated protein/extracellular signal-regulated kinase kinase (MEK) inhibitors induced apoptosis in nonanchored MDA-MB231 and HBC4 human breast cancer cells, whereas anchored cells remained viable. Here, we report that activation of the BH3-only protein BimEL is the major mechanism for induction of anoikis sensitivity by MEK inhibitors in MDA-MB231 and HBC4 cells. On treatment with MEK inhibitors, BimEL in MDA-MB231 and HBC4 cells rapidly increased, irrespective of the state of anchorage. However, it translocated to mitochondria only in nonanchored cells, explaining why attached cells remain viable. MDA-MB231 and HBC4 cells had exceedingly low basal levels of BimEL compared with other breast cancer cells, suggesting that maintenance of low BimEL amount is important for survival of these cells. MEK inhibitors also induced the electrophoretic mobility shift of BimEL, indicative of reduced phosphorylation. In vitro, BimEL was phosphorylated by extracellular signal-regulated kinase on Ser(69), which resides in the BimEL-specific insert region. Using phosphospecific antibody against this site, we show that this residue is actually phosphorylated in cells. We also show that phosphorylation of Ser(69) promotes ubiquitination of BimEL. We conclude that MEK inhibitors sensitize MDA-MB231 and HBC4 cells to anoikis by blocking phosphorylation and hence degradation of BimEL, a mechanism that these cells depend on to escape anoikis.
Department of Bioactive Molecules, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8640, Japan. fukazawa@nih.go.jp
Hidesuke
H
Fukazawa
Kohji
K
Noguchi
Atsuko
A
Masumi
Yuko
Y
Murakami
Yoshimasa
Y
Uehara
D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry* / Q000502:physiology; D000595:Amino Acid Sequence; D023102:Anoikis; D051017:Apoptosis Regulatory Proteins; D051023:BH3 Interacting Domain Death Agonist Protein; D000072224:Bcl-2-Like Protein 11; D002352:Carrier Proteins / Q000737:chemistry* / Q000502:physiology*; D002448:Cell Adhesion; D002460:Cell Line; D045744:Cell Line, Tumor; D002470:Cell Survival; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000378:metabolism; D005091:Exons; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D047468:Immunoprecipitation; D020935:MAP Kinase Signaling System; D008565:Membrane Proteins / Q000737:chemistry* / Q000502:physiology*; D008928:Mitochondria / Q000378:metabolism; D008969:Molecular Sequence Data; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D020033:Protein Isoforms; D021381:Protein Transport; D011518:Proto-Oncogene Proteins / Q000737:chemistry* / Q000502:physiology*; D012694:Serine / Q000737:chemistry; D014162:Transfection; D025801:Ubiquitin / Q000378:metabolism
D048868:Adaptor Proteins, Signal Transducing; D051017:Apoptosis Regulatory Proteins; C000606775:BCL2L11 protein, human; D051023:BH3 Interacting Domain Death Agonist Protein; C494814:BID protein, human; D000072224:Bcl-2-Like Protein 11; D002352:Carrier Proteins; D004791:Enzyme Inhibitors; D008565:Membrane Proteins; D020033:Protein Isoforms; D011518:Proto-Oncogene Proteins; D025801:Ubiquitin; D012694:Serine; D048049:Extracellular Signal-Regulated MAP Kinases