E6AP and calmodulin reciprocally regulate estrogen receptor stability.
Estrogen promotes the proliferation of human breast epithelial cells by interacting with the estrogen receptor (ER). Physiological responses of cells to estrogen are regulated in part by degradation of the ER. Previous studies revealed that calmodulin binds directly to the ER, thereby enhancing its stability. Consistent with these findings, cell-permeable calmodulin antagonists dramatically reduced the number of ER in MCF-7 human breast epithelial cells. Here we investigated the molecular mechanism by which calmodulin attenuates ER degradation. MG132 and lactacystin, inhibitors of the ubiquitin-proteasome pathway, prevented the calmodulin antagonist CGS9343B from reducing the amount of ER in MCF-7 cells. In contrast, protease inhibitors afforded no protection. Moreover, CGS9343B enhanced ER ubiquitination. A point mutant ER construct that is unable to bind calmodulin, termed ERDeltaCaM, is ubiquitinated to a greater extent than wild type ER. The ubiquitin-protein isopeptide ligase E6-associated protein (E6AP) associated with and promoted the degradation of ER. The possible convergence of calmodulin and E6AP on ER degradation was examined. ERDeltaCaM bound E6AP with higher affinity than that of wild type ER. Moreover, calmodulin attenuated the in vitro interaction between ER and E6AP in a Ca(2+)-dependent manner. Collectively, our data reveal that E6AP is a component of ER degradation via the ubiquitin-proteasome pathway and that Ca(2+)/calmodulin modulates this degradation mechanism. These results have potential implications for the development of selectively targeted therapeutic agents for breast cancer.
Li
Lu
L
Department of Pathology, Brigham and Women's Hospital and Harvard Medical School, 75 Francis Street, Boston, MA 02115, USA.
Li
Zhigang
Z
Howley
Peter M
PM
Sacks
David B
DB
D000818:Animals; D001562:Benzimidazoles / Q000494:pharmacology; D015153:Blotting, Western; D001943:Breast Neoplasms / Q000378:metabolism; D019556:COS Cells; D002147:Calmodulin / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D045744:Cell Line, Tumor; D002522:Chlorocebus aethiops; D004591:Electrophoresis, Polyacrylamide Gel; D017353:Gene Deletion; D006801:Humans; D047468:Immunoprecipitation; D007976:Leupeptins / Q000494:pharmacology; D009154:Mutation; D017354:Point Mutation; D011480:Protease Inhibitors / Q000494:pharmacology; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D011960:Receptors, Estrogen / Q000378:metabolism*; D014162:Transfection; D025801:Ubiquitin / Q000737:chemistry; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism / Q000502:physiology*
D001562:Benzimidazoles; D002147:Calmodulin; D007976:Leupeptins; D011480:Protease Inhibitors; D011960:Receptors, Estrogen; D025801:Ubiquitin; C052087:CGS 9343B; C471639:UBE3A protein, human; D044767:Ubiquitin-Protein Ligases; D046988:Proteasome Endopeptidase Complex; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde