Abstract
Nuclear receptors and their coactivators are key regulators of numerous physiological functions. GRIP1 (glucocorticoid receptor-interacting protein) is a member of the steroid receptor coactivator family. Here, we show that GRIP1 is regulated by cAMP-dependent protein kinase (PKA) that induces its degradation through the ubiquitin-proteasome pathway. GRIP1 was down-regulated in transiently transfected COS-1 cells after treatment with 8-para-chlorophenylthio-cAMP or forskolin and 3-isobutyl-1-methylxanthine and in adrenocortical Y1 cells after incubation with adrenocorticotropic hormone. Pulse-chase experiments with transiently transfected COS-1 cells demonstrated that the half-life of GRIP1 was markedly reduced in cells overexpressing the PKA catalytic subunit, suggesting that activation of PKA increases the turnover of GRIP1 protein. The proteasome inhibitors MG132 and lactacystin abolished the PKA-mediated degradation of GRIP1. Using ts20 cells, a temperature-sensitive cell line that contains a thermolabile ubiquitin-activating E1 enzyme, it was confirmed that PKA-mediated degradation of GRIP1 is dependent upon the ubiquitin-proteasome pathway. Coimmunoprecipitation studies of COS-1 cells transfected with expression vectors encoding GRIP1 and ubiquitin using anti-GRIP1 and anti-ubiquitin antibodies showed that the ubiquitination of GRIP1 was increased by overexpression of PKA. Finally, we show that PKA regulates the intracellular distribution pattern of green fluorescent protein-GRIP1 and stimulates recruitment of GRIP1 to subnuclear foci that are colocalized with the proteasome. Taken together, these data demonstrate that GRIP1 is ubiquitinated and degraded through activation of the PKA pathway. This may represent a novel regulatory mechanism whereby hormones down-regulate a nuclear receptor coactivator.
Highlights
Nuclear receptors and their coactivators are key regulators of numerous physiological functions
Using COS-1 cells transiently transfected with GAL4-GRIP1 expression plasmid and a GAL4-responsive reporter plasmid, we observed that cotransfection with an expression plasmid encoding the catalytic subunit of protein kinase (PKA) (PKA-C␣) resulted in a marked decrease in GRIP1 activation function (Fig. 1A)
It is demonstrated that stimulation of the cAMP/PKA pathway, which mediates the intracellular effects of a number of peptide hormones, leads to ubiquitination and proteasomal degradation of the nuclear receptor coactivator GRIP1
Summary
Plasmid Constructs—The expression plasmid pCMV5-SF-1, and the luciferase reporter plasmid pT81– 4CRS2-luc (4CRS2-luc) that contains four copies of the SF-1 binding site from the proximal promoter region of the bovine cyp gene, have been previously described [37]. At each desired time point, the cells were washed twice with PBS and lysed in an immunoprecipitation buffer containing 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 2 mM EDTA, 0.5% (w/v) SDS, 1.0% Triton X-100, 1 g/ml aprotinin, 5 mM N-ethylmaleimide, 0.2 mM PMSF, and Complete Mini EDTA-free protease inhibitors (Roche Applied Science). Cell lysates in the immunoprecipitation buffer were supplemented with four volumes of a buffer containing 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 2 mM EDTA, 1 g/ml aprotinin, 5 mM N-ethylmaleimide, 0.2 mM PMSF, and Complete Mini protease inhibitors and incubated with 30 l of protein G-Sepharose (Amersham Biosciences) for 1 h at 4 °C on a rotating wheel. Confocal Microscopy, Immunofluorescence, and Image Analysis— COS-1 cells were plated onto microscopic coverglasses in 35-mm Petri dishes and transfected with expression plasmids encoding GFP-GRIP1 (400 ng), SF-1 (100 ng), and PKA-C␣ (100 ng) as described above. The background intensity was subtracted from the fluorescence intensity of the nucleus
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
