Sumoylation and proteasomal activity determine the transactivation properties of the mineralocorticoid receptor.

MR is a hormone-activated transcription factor that carries a strong synergy inhibitory function at its N-terminus. Using this region as bait in a yeast two-hybrid screening, we isolated major components of the sumoylation pathway, including the SUMO-1-conjugating enzyme Ubc9, and SUMO-1 itself. We found that MR interacts with both, Ubc9 and SUMO-1 in mammalian cells, and that the receptor is sumoylated at four acceptor sites which are clustered within its AF-1 domain. We observed that MR can be poly-ubiquitinated and that proteasome activity is essential for MR-activated transcription. Disruption of the SUMO-1 attachment sites abolished MR sumoylation but interfered with neither the poly-ubiquitination of the receptor nor its transactivation potential on MMTV. However, the hormone-activated mutant displayed enhanced synergistic potential on a compound promoter and delayed mobility in the nucleus. FRAP analysis further showed that proteasome inhibition immobilizes a subpopulation of unliganded MR receptors in the nucleus, a phenomenon that is significantly attenuated in the presence of aldosterone. Interestingly, the ability of the hormone to counteract the immobilizing effect of MG132 requires the sumoylation-competent form of MR. Moreover, increasing exogenously SUMO-1 cellular levels resulted in a selective, dose-dependent inhibition of the activity of the sumoylation-deficient MR. This effect was observed only on a synergy-competent promoter, revealing a mode for negative regulation of synergy that might involve sumoylation of factors different from MR. The data suggest that the overall transcriptional activity of MR can be modulated by its sumoylation potential as well as the sumoylation level of MR-interacting proteins, and requires the continuous function of the proteasome.

Soluble beta-amyloid1-40 induces NMDA-dependent degradation of postsynaptic density-95 at glutamatergic synapses.

Amyloid-beta (Abeta) has been implicated in memory loss and disruption of synaptic plasticity observed in early-stage Alzheimer's disease. Recently, it has been shown that soluble Abeta oligomers target synapses in cultured rat hippocampal neurons, suggesting a direct role of Abeta in the regulation of synaptic structure and function. Postsynaptic density-95 (PSD-95) is a postsynaptic scaffolding protein that plays a critical role in synaptic plasticity and the stabilization of AMPA (AMPARs) and NMDA (NMDARs) receptors at synapses. Here, we show that exposure of cultured cortical neurons to soluble oligomers of Abeta(1-40) reduces PSD-95 protein levels in a dose- and time-dependent manner and that the Abeta1(1-40)-dependent decrease in PSD-95 requires NMDAR activity. We also show that the decrease in PSD-95 requires cyclin-dependent kinase 5 activity and involves the proteasome pathway. Immunostaining analysis of cortical cultured neurons revealed that Abeta treatment induces concomitant decreases in PSD-95 at synapses and in the surface expression of the AMPAR glutamate receptor subunit 2. Together, these data suggest a novel pathway by which Abeta triggers synaptic dysfunction, namely, by altering the molecular composition of glutamatergic synapses.

The manifold actions of the protein inhibitor of activated STAT proteins on the transcriptional activity of mineralocorticoid and glucocorticoid receptors in neural cells.

Corticosteroid actions in the brain are exerted via the mineralocorticoid receptor (MR) and glucocorticoid receptor (GR). These receptors share several structural and functional similarities but their activation in the brain triggers distinct biological actions, for instance on neuronal survival or the regulation of the hypothalamo-pituitary-adrenal axis. Like other hormone-activated receptors, the transcriptional properties of the MR and GR depend on their ability to recruit a variety of co-regulators, which modulate their activity on target promoters, in a specific manner. The N-terminal regions of the MR and GR share the smallest degree of sequence conservation, whereas they display opposite effects on the transactivation properties of these receptors; thus, they may provide surfaces suitable for receptorspecific interactions with co-regulatory proteins. Here, we employed a yeast two-hybrid system to identify molecules interacting with the N-terminal part of the MR (amino acids 170-433). This approach resulted in the isolation of representative cDNAs from all members of the protein inhibitor of activated STAT (PIAS) family of proteins as potential MR-interacting partners. In neural cells, PIAS3 exhibited a strong and specific interaction with MR, but not GR, as indicated by mammalian two-hybrid assays and co-immunoprecipitation experiments in vivo. The interaction with MR was enhanced in the presence of aldosterone, an MR agonist, and was found to occur through a conserved, serine- and acidic amino acid residue-rich domain of PIAS3. To compare the modulatory properties of PIAS proteins on MR and GR transcriptional activity in a neural environment, MMTV reporter gene assays were performed in the human neuroblastoma cell line SK-N-MC. This analysis revealed that PIAS3 can inhibit MR, but not GR, transactivation in response to their corresponding ligands. Further, it showed that PIAS1 and PIASxbeta, but not PIASy, could also inhibit MR-mediated transcription despite the lack of detected physical interaction with MR. Interestingly, PIASxbeta and PIASy dose-dependently co-activated GR, whereas PIAS1 impaired GR-induced transcription. Taken together the results reveal differential modulatory roles of the PIAS proteins on the transcriptional properties of MR and GR, thus providing new insights into the bifurcating actions of these two receptors in neural cells where they are frequently co-localized.

DAXX, FLASH, and FAF-1 modulate mineralocorticoid and glucocorticoid receptor-mediated transcription in hippocampal cells--toward a basis for the opposite actions elicited by two nuclear receptors?

Mineralocorticoid (MR) and glucocorticoid (GR) receptors are two closely-related members of the steroid nuclear receptor family of transcription factors that bind common ligands in the brain (corticosterone and cortisol) and supposedly have identical hormone response elements. This raises the important question of how they can elicit differential biological actions in neurons in which they are often colocalized. One plausible explanation is that they differentially recruit proteins (coregulators or other receptor-interacting factors) through cell-specific interactions with regions that diverge between MR and GR to modulate target gene transcription in a receptor-specific manner. We therefore performed a yeast-two-hybrid screening of a human brain cDNA library with an AF1-containing region of the human MR as bait. This screening revealed several potential MR-interacting partners; among them were several clones bearing homology to DAXX, FLASH, and FAF-1, all previously implicated in apoptosis. Coexpression of candidate clones in a mouse hippocampal cell line confirmed these interactions in a mammalian neural cell environment as well. In transient transactivation assays, DAXX and FLASH influenced MR- and GR-driven transcription of the MMTV-Luc reporter similarly; in contrast, although FAF-1 did not transactivate GR, it did selectively stimulate MR-mediated transcription. Thus, the present findings, that 1) DAXX, FLASH, and FAF-1 modulate the transcriptional activities of MR and GR and that 2) FAF-1 selectively coactivates only MR, provide possible clues for how these closely related receptors might differentially influence neuronal function.