Role of the promyelocytic leukemia body in the dynamic interaction between the androgen receptor and steroid receptor coactivator-1 in living cells.
The dynamic interaction between the androgen receptor (AR) and steroid receptor coactivator-1 (SRC-1) was explored in living cells expressing chimeric forms of the receptor and the coactivator containing two spectral variants of jellyfish fluorescent protein. Laser scanning confocal imaging of transfected cells expressing fluorescently labeled SRC-1 revealed that in an unsynchronized cell population, the coactivator is distributed in approximately 40% cells as nuclear bodies of 0.2-1.0 microm in diameter. Immunostaining of cyan fluorescent protein-labeled SRC-1 (CFP-SRC1)-expressing cells with antibody to promyelocytic leukemia (PML) protein showed significant overlap of the CFP fluorescence with the antibody stain. Cotransfection of cells with a plasmid expressing the CFP conjugate of Sp100 (another marker protein for the PML nuclear body) also showed colocalization of the yellow fluorescent protein (YFP)-SRC1 containing nuclear foci with the PML bodies in living cells. Analysis of the three-dimensional structure revealed that the PML bodies are round to elliptical in shape with multiple satellite bodies on their surface. Some of these satellite bodies contain the SRC-1. Activation and nuclear import of CFP-AR by the agonistic ligand 5alpha-dihydrotestosterone, but not by the antagonist casodex, transferred YFP-SRC1 from the PML bodies to an interlacing filamentous structure. In a single living cell, agonist-activated AR caused a time-dependent movement of YFP-SRC1 from the PML bodies to this filamentous structure. Additionally, coexpression of a constitutively active mutant of AR (AR-deltaligand binding domain) also displaced YFP-SRC1 from the PML bodies to this intranuclear filamentous structure. The fluorescence recovery after photobleaching approach was used to examine changes in the kinetics of movement of YFP-SRC1 during its mobilization from the PML bodies to the intranuclear filamentous structure by the agonist-activated AR. Results of the relative half-times (t(1/2)) of replacement of YFP-SRC1 within the photobleached region of a single PML body from its surrounding nuclear space supported the conclusion that SRC-1 is actively transported from the PML bodies to the intranuclear filamentous structure by the ligand-activated AR. This observation also suggests an interaction between AR and SRC-1 before its binding to the target gene. The PML bodies have been implicated as a cross-road for multiple regulatory pathways that control cell proliferation, cellular senescence, and apoptosis. Our present results along with other recent reports expand the role of this subnuclear structure to include the regulation of steroid hormone action.
Rivera
Omar J
OJ
Department of Cellular & Structural Biology, The University of Texas Health Science Center at San Antonio, San Antonio, Texas 78245-3207, USA.
Song
Chung S
CS
Centonze
Victoria E
VE
Lechleiter
James D
JD
Chatterjee
Bandana
B
Roy
Arun K
AK
D021581:Active Transport, Cell Nucleus / Q000502:physiology; D000818:Animals; D034961:Antigens, Nuclear / Q000378:metabolism; D001324:Autoantigens / Q000378:metabolism; D019556:COS Cells; D002467:Cell Nucleus / Q000378:metabolism*; D036681:Fluorescence Recovery After Photobleaching; D005455:Fluorescent Antibody Technique; D005786:Gene Expression Regulation / Q000502:physiology; D051548:Histone Acetyltransferases; D006801:Humans; D009363:Neoplasm Proteins / Q000502:physiology*; D009687:Nuclear Proteins / Q000378:metabolism / Q000502:physiology*; D056920:Nuclear Receptor Coactivator 1; D000071156:Promyelocytic Leukemia Protein; D021381:Protein Transport / Q000502:physiology; D011944:Receptors, Androgen / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000502:physiology; D014157:Transcription Factors / Q000502:physiology*; D014162:Transfection; D014407:Tumor Cells, Cultured; D025521:Tumor Suppressor Proteins
D034961:Antigens, Nuclear; D001324:Autoantigens; D009363:Neoplasm Proteins; D009687:Nuclear Proteins; D000071156:Promyelocytic Leukemia Protein; D011944:Receptors, Androgen; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D025521:Tumor Suppressor Proteins; C066642:SP100 protein, human; C070576:PML protein, human; D051548:Histone Acetyltransferases; C538859:NCOA1 protein, human; D056920:Nuclear Receptor Coactivator 1