Specific amino acid residues in the basic helix-loop-helix domain of SRC-3 are essential for its nuclear localization and proteasome-dependent turnover.
SRC-3/AIB1/ACTR/pCIP/RAC3/TRAM-1 is a primary transcriptional coactivator for the estrogen receptor. Here we report that deletion of the SRC-3 basic helix-loop-helix (bHLH) domain blocks its proteasome-dependent turnover. We further identified two residues (K17 and R18) in the SRC-3 bHLH domain that are essential for its stability. Moreover, we found that the bHLH domain contains a bipartite nuclear localization signal (NLS). SRC-3 NLS mutants block its translocation into the nucleus, and this correlates with its insensitivity to proteasome-dependent turnover. SRC-3 shows a time-dependent decay in the presence of cycloheximide which is not apparent for the cytoplasmic mutant. Fusion of a simian virus 40 T antigen NLS to the cytoplasmic localized SRC-3 mutant drives it back into the nucleus and restores its proteasomal sensitivity. In addition, the cytoplasmic mutants are inactive for transcriptional coactivation and cancer cell growth. Taken together, our data indicate that proteasome-dependent turnover of SRC-3 occurs in the nucleus and that two amino acid residues in the bHLH domain provide a signal for its nuclear localization and proteasome-dependent degradation as well as for regulation of SRC-3 transcriptional coactivator capacity.
Li
Chao
C
Department of Molecular and Cellular Biology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA.
Wu
Ray-Chang
RC
Amazit
Larbi
L
Tsai
Sophia Y
SY
Tsai
Ming-Jer
MJ
O'Malley
Bert W
BW
D021581:Active Transport, Cell Nucleus; D000595:Amino Acid Sequence; D000952:Antigens, Polyomavirus Transforming / Q000378:metabolism; D001120:Arginine / Q000378:metabolism*; D048708:Cell Growth Processes; D002467:Cell Nucleus / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism; D017930:Genes, Reporter; D006367:HeLa Cells; D018257:Helix-Loop-Helix Motifs; D051548:Histone Acetyltransferases / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D008239:Lysine / Q000378:metabolism*; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000378:metabolism; D019913:Nuclear Localization Signals / Q000737:chemistry; D056921:Nuclear Receptor Coactivator 3; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D013329:Structure-Activity Relationship; D013816:Thermodynamics; D015534:Trans-Activators / Q000737:chemistry* / Q000378:metabolism*; D014158:Transcription, Genetic
D000952:Antigens, Polyomavirus Transforming; D050505:Mutant Proteins; D019913:Nuclear Localization Signals; D015534:Trans-Activators; D001120:Arginine; D051548:Histone Acetyltransferases; C538866:NCOA3 protein, human; D056921:Nuclear Receptor Coactivator 3; D046988:Proteasome Endopeptidase Complex; D008239:Lysine