Regulation of SRC-3 intercompartmental dynamics by estrogen receptor and phosphorylation.
The steroid receptor coactivator 3 gene (SRC-3) (AIB1/ACTR/pCIP/RAC3/TRAM1) is a p160 family transcription coactivator and a known oncogene. Despite its importance, the functional regulation of SRC-3 remains poorly understood within a cellular context. Using a novel combination of live-cell, high-throughput, and fluorescent microscopy, we report SRC-3 to be a nucleocytoplasmic shuttling protein whose intracellular mobility, solubility, and cellular localization are regulated by phosphorylation and estrogen receptor alpha (ERalpha) interactions. We show that both chemical inhibition and small interfering RNA reduction of the mitogen-activated protein kinase/extracellular signal-regulated kinase 1/2 (MEK1/2) pathway induce a cytoplasmic shift in SRC-3 localization, whereas stimulation by epidermal growth factor signaling enhances its nuclear localization by inducing phosphorylation at T24, S857, and S860, known participants in the phosphocode that regulates SRC-3 activity. Accordingly, the cytoplasmic localization of a nonphosphorylatable SRC-3 mutant further supported these results. In the presence of ERalpha, U0126 also dramatically reduces (i) ligand-dependent colocalization of SRC-3 and ERalpha, (ii) the formation of ER-SRC-3 complexes in cell lysates, and (iii) SRC-3 targeting to a visible, ERalpha-occupied and -regulated prolactin promoter array. Taken together, these results indicate that phosphorylation coordinates SRC-3 coactivator function by linking the probabilistic formation of transient nuclear receptor-coactivator complexes with its molecular dynamics and cellular compartmentalization. Technically and conceptually, these findings have a new and broad impact upon evaluating mechanisms of action of gene regulators at a cellular system level.
Amazit
Larbi
L
Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX 77030, USA.
Pasini
Luigi
L
Szafran
Adam T
AT
Berno
Valeria
V
Wu
Ray-Chang
RC
Mielke
Marylin
M
Jones
Elizabeth D
ED
Mancini
Maureen G
MG
Hinojos
Cruz A
CA
O'Malley
Bert W
BW
Mancini
Michael A
MA
D021581:Active Transport, Cell Nucleus / Q000502:physiology; D000818:Animals; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism; D004815:Epidermal Growth Factor / Q000378:metabolism; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D051548:Histone Acetyltransferases / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D006801:Humans; D007150:Immunohistochemistry; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure; D056921:Nuclear Receptor Coactivator 3; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D034622:RNA Interference; D011960:Receptors, Estrogen / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology; D013347:Subcellular Fractions / Q000378:metabolism* / Q000648:ultrastructure; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure
D009687:Nuclear Proteins; D011960:Receptors, Estrogen; D011993:Recombinant Fusion Proteins; D015534:Trans-Activators; D004815:Epidermal Growth Factor; D051548:Histone Acetyltransferases; C538866:NCOA3 protein, human; D056921:Nuclear Receptor Coactivator 3; D048049:Extracellular Signal-Regulated MAP Kinases