cAMP-dependent protein kinase regulates ubiquitin-proteasome-mediated degradation and subcellular localisation of the nuclear receptor coactivator GRIP1

Abstract

Nuclear receptors and their coactivators are key regulators of numerous physiological functions. GEIP1 (glucocorticoid receptor-interacting grotein) is a member of the steroid receptor coactivator family. Here, we show that GEIP1 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-cAMDP 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 GEIP1 was markedly reduced in cells over-expressing the PKA catalytic subunit, suggesting that activation of PKA increases the turnover of GRIP1 protein. The proteasome inhibitors HG132 and lactacystin abolished the PKA-mediated degradation of GEIP1. 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-proteaseme 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.