Hsp70-RAP46 interaction in downregulation of DNA binding by glucocorticoid receptor.
Receptor-associating protein 46 (RAP46) is a cochaperone that regulates the transactivation function of several steroid receptors. It is transported into the nucleus by a liganded glucocorticoid receptor where it downregulates DNA binding and transactivation by this receptor. The N- and C-termini of RAP46 are both implicated in its negative regulatory function. In metabolic labelling experiments, we have shown that the N-terminus of RAP46 is modified by phosphorylation, but this does not contribute to the downregulation of glucocorticoid receptor activity. However, deletion of a sequence that binds 70 kDa heat shock protein (Hsp70) and the constitutive isoform of Hsp70 (Hsc70) at the C-terminus of RAP46 abrogated its negative regulatory action. Surface plasmon resonance studies showed that RAP46 binds the glucocorticoid receptor only when it has interacted with Hsp70/Hsc70, and confocal immunofluorescence analyses revealed a nuclear transport of Hsp70/Hsc70 by the liganded receptor. Together these findings demonstrate an important contribution of Hsp70/Hsc70 in the binding of RAP46 to the glucocorticoid receptor and suggest a role for this molecular chaperone in the RAP46-mediated downregulation of glucocorticoid receptor activity.
Schneikert
J
J
Forschungszentrum Karlsruhe, Institute of Toxicology and Genetics, PO Box 3640, D-76021 Karlsruhe, Schering AG, Enabling Technologies, D-13342 Berlin, Germany.
Hübner
S
S
Langer
G
G
Petri
T
T
Jäättelä
M
M
Reed
J
J
Cato
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AC
D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002467:Cell Nucleus / Q000378:metabolism; D002474:Cell-Free System; D002942:Circular Dichroism; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins; D015536:Down-Regulation; D049452:Green Fluorescent Proteins; D018840:HSP70 Heat-Shock Proteins / Q000737:chemistry / Q000378:metabolism*; D015151:Immunoblotting; D008024:Ligands; D008164:Luminescent Proteins / Q000378:metabolism; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011485:Protein Binding; D020033:Protein Isoforms; D017433:Protein Structure, Secondary; D011965:Receptors, Glucocorticoid / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D020349:Surface Plasmon Resonance; D013997:Time Factors; D014157:Transcription Factors; D015533:Transcriptional Activation; D014162:Transfection
C091732:BCL2-associated athanogene 1 protein; D002352:Carrier Proteins; D004268:DNA-Binding Proteins; D018840:HSP70 Heat-Shock Proteins; D008024:Ligands; D008164:Luminescent Proteins; D020033:Protein Isoforms; D011965:Receptors, Glucocorticoid; D014157:Transcription Factors; D049452:Green Fluorescent Proteins; D004247:DNA