Purification and identification of p68 RNA helicase acting as a transcriptional coactivator specific for the activation function 1 of human estrogen receptor alpha.
The estrogen receptor (ER) regulates the expression of target genes in a ligand-dependent manner. The ligand-dependent activation function AF-2 of the ER is located in the ligand binding domain (LBD), while the N-terminal A/B domain (AF-1) functions in a ligand-independent manner when isolated from the LBD. AF-1 and AF-2 exhibit cell type and promoter context specificity. Furthermore, the AF-1 activity of the human ERalpha (hERalpha) is enhanced through phosphorylation of the Ser(118) residue by mitogen-activated protein kinase (MAPK). From MCF-7 cells, we purified and cloned a 68-kDa protein (p68) which interacted with the A/B domain but not with the LBD of hERalpha. Phosphorylation of hERalpha Ser(118) potentiated the interaction with p68. We demonstrate that p68 enhanced the activity of AF-1 but not AF-2 and the estrogen-induced as well as the anti-estrogen-induced transcriptional activity of the full-length ERalpha in a cell-type-specific manner. However, it did not potentiate AF-1 or AF-2 of ERbeta, androgen receptor, retinoic acid receptor alpha, or mineralocorticoid receptor. We also show that the RNA helicase activity previously ascribed to p68 is dispensable for the ERalpha AF-1 coactivator activity and that p68 binds to CBP in vitro. Furthermore, the interaction region for p68 in the ERalpha A/B domain was essential for the full activity of hERalpha AF-1. Taken together, these findings show that p68 acts as a coactivator specific for the ERalpha AF-1 and strongly suggest that the interaction between p68 and the hERalpha A/B domain is regulated by MAPK-induced phosphorylation of Ser(118).
Molecular Medicine Laboratories, Institute for Drug Discovery Research, Yamanouchi Pharmaceutical, Tsukuba, Ibaraki 305-8585, Japan.
H
H
Endoh
K
K
Maruyama
Y
Y
Masuhiro
Y
Y
Kobayashi
M
M
Goto
H
H
Tai
J
J
Yanagisawa
D
D
Metzger
S
S
Hashimoto
S
S
Kato
D000230:Adenocarcinoma / Q000473:pathology; D000595:Amino Acid Sequence; D000818:Animals; D001943:Breast Neoplasms / Q000473:pathology; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000502:physiology; D053487:DEAD-box RNA Helicases; D047628:Estrogen Receptor alpha; D005260:Female; D005786:Gene Expression Regulation; D006801:Humans; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins / Q000302:isolation & purification / Q000502:physiology; D010766:Phosphorylation; D011485:Protein Binding; D011494:Protein Kinases; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D020365:RNA Helicases / Q000302:isolation & purification* / Q000502:physiology; D011817:Rabbits; D011960:Receptors, Estrogen / Q000737:chemistry / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000502:physiology; D017421:Sequence Analysis; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured
D047628:Estrogen Receptor alpha; D009363:Neoplasm Proteins; D011960:Receptors, Estrogen; D011993:Recombinant Fusion Proteins; D011494:Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; C506749:Ddx5 protein, human; D053487:DEAD-box RNA Helicases; D020365:RNA Helicases