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.
Mesh Terms:
Amino Acid Sequence, Animals, COS Cells, Carrier Proteins, Cell Nucleus, Cell-Free System, Circular Dichroism, DNA, DNA-Binding Proteins, Down-Regulation, Green Fluorescent Proteins, HSP70 Heat-Shock Proteins, Immunoblotting, Ligands, Luminescent Proteins, Microscopy, Confocal, Microscopy, Fluorescence, Molecular Sequence Data, Phosphorylation, Protein Binding, Protein Isoforms, Protein Structure, Secondary, Receptors, Glucocorticoid, Sequence Homology, Amino Acid, Surface Plasmon Resonance, Time Factors, Transcription Factors, Transcriptional Activation, Transfection
Amino Acid Sequence, Animals, COS Cells, Carrier Proteins, Cell Nucleus, Cell-Free System, Circular Dichroism, DNA, DNA-Binding Proteins, Down-Regulation, Green Fluorescent Proteins, HSP70 Heat-Shock Proteins, Immunoblotting, Ligands, Luminescent Proteins, Microscopy, Confocal, Microscopy, Fluorescence, Molecular Sequence Data, Phosphorylation, Protein Binding, Protein Isoforms, Protein Structure, Secondary, Receptors, Glucocorticoid, Sequence Homology, Amino Acid, Surface Plasmon Resonance, Time Factors, Transcription Factors, Transcriptional Activation, Transfection
EMBO J.
Date: Dec. 01, 2000
PubMed ID: 11101523
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