Annexin A6 inhibits Ras signalling in breast cancer cells.
Overexpression of epidermal growth factor receptor (EGFR) is associated with enhanced activation of wild-type (hyperactive) Ras in breast cancer. Little is known about the regulation of Ras inactivation and GTPase-activating proteins (GAPs), such as p120GAP, in cells with hyperactive Ras. Recently, we showed that in EGFR-overexpressing A431 cells, which lack endogenous Annexin A6 (AnxA6), ectopic expression of AnxA6 stimulates membrane recruitment of p120GAP to modulate Ras signalling. We now demonstrate that, AnxA6 is downregulated in a number of EGFR-overexpressing and estrogen receptor (ER)-negative breast cancer cells. In these cells, AnxA6 overexpression promotes Ca(2+)- and EGF-inducible membrane targeting of p120GAP. In ER-negative MDA-MB-436 cells, overexpression of p120GAP, but not CAPRI or a p120GAP mutant lacking the AnxA6-binding domain inhibits Ras/MAPK activity. AnxA6 knockdown in MDA-MB-436 increases Ras activity and cell proliferation in anchorage-independent growth assays. Furthermore, AnxA6 co-immunoprecipitates with H-Ras in a Ca(2+)- and EGF-inducible manner and fluorescence resonance energy transfer (FRET) microscopy confirmed that AnxA6 is in close proximity of active (G12V), but not inactive (S17N) H-Ras. Thus, association of AnxA6 with H-Ras-containing protein complexes may contribute to regulate p120GAP/Ras assembly in EGFR-overexpressing and ER-negative breast cancer cells.
Departament de Biologia Cellular, Facultat de Medicina, Universitat de Barcelona, Barcelona, Spain.
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Vilá de Muga
P
P
Timpson
L
L
Cubells
R
R
Evans
T E
TE
Hayes
C
C
Rentero
A
A
Hegemann
M
M
Reverter
J
J
Leschner
A
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Pol
F
F
Tebar
R J
RJ
Daly
C
C
Enrich
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Grewal
D000818:Animals; D017317:Annexin A6 / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D049109:Cell Proliferation; D006224:Cricetinae; D003412:Cricetulus; D019938:Cyclin D1; D066246:ErbB Receptors / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D005455:Fluorescent Antibody Technique; D006801:Humans; D047468:Immunoprecipitation; D016283:Proto-Oncogene Proteins p21(ras) / Q000235:genetics / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000494:pharmacology; D011960:Receptors, Estrogen / Q000378:metabolism; D015398:Signal Transduction; D014407:Tumor Cells, Cultured / Q000187:drug effects / Q000378:metabolism; D020729:p120 GTPase Activating Protein / Q000235:genetics / Q000378:metabolism*
D017317:Annexin A6; C530104:CCND1 protein, human; D034741:RNA, Small Interfering; D011960:Receptors, Estrogen; D020729:p120 GTPase Activating Protein; D019938:Cyclin D1; D066246:ErbB Receptors; C501009:HRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras); D002118:Calcium