Reduced phosphorylation of AS160 contributes to glucocorticoid-mediated inhibition of glucose uptake in human and murine adipocytes.

Excess glucocorticoids induce insulin resistance and reduce glucose uptake although the underlying mechanisms are unclear. Here we demonstrate that Dex (1 microM for 24h) inhibits basal and insulin (1 nM) stimulated glucose uptake in human and murine adipocytes by 50% with a concomitant reduction in the levels of GLUT1/4 at the plasma membrane but no change in total GLUT1/4 levels. Expression and phosphorylation of proximal insulin signalling molecules (IRS1, PI3K, AKT) was unaffected by Dex as was phosphorylation of mTOR and FOXO1. In contrast, phosphorylation of AKT substrate 160kDa (AS160) at T642, which is essential for 14-3-3 recruitment and GLUT4 translocation, was reduced by 50% in basal and insulin-stimulated cells and this was mirrored by decreased 14-3-3 association. Co-treatment with the glucocorticoid receptor antagonist RU486 (10 microM) abrogated the Dex effect on AS160-T642 phosphorylation and restored glucose uptake by 80%. These data suggest Dex inhibits glucose uptake in adipocytes, at least in part, by reducing AS160 phosphorylation and interaction with 14-3-3.

A single nucleotide in an estrogen-related receptor alpha site can dictate mode of binding and peroxisome proliferator-activated receptor gamma coactivator 1alpha activation of target promoters.

The orphan nuclear receptor estrogen-related receptor alpha (ERRalpha, NR3B1) is a constitutively active transcription factor that controls multiple processes, most notably mitochondrial function. ERRalpha preferentially binds to a nine-nucleotide extended half-site sequence TNAAGGTCA, referred to as the ERRE, as either a monomer or a dimer, although how the mode of DNA binding is dictated remains to be determined. Here, we used variants of the extended half-site sequence and selective DNA binding domain mutants of ERRalpha to investigate the effects of ERRE sequence specificity on ERRalpha DNA binding mode, transactivation and interaction with the coactivator protein peroxisome proliferator-activated receptor gamma coactivator 1alpha (PGC-1alpha). We found that the base at the N position of the TNAAGGTCA sequence dictated ERRalpha binding preference as a monomer or dimer. In addition, we demonstrated that the threonine residue at position 124 (Thr(124)) was a determinant of ERRalpha DNA-dependent dimerization. Transfection experiments also indicated that substituting a thymidine for a cytosine at the N position in the ERRE of the native ERRalpha target promoter trefoil factor 1 (TFF1) considerably diminished the transcriptional response of the ERRalpha/PGC-1alpha complex. These results suggest that a single nucleotide in an ERRalpha binding site can determine specific configuration to the receptor and productive interaction with the coactivator PGC-1alpha.

Epidermal growth factor-induced signaling in breast cancer cells results in selective target gene activation by orphan nuclear receptor estrogen-related receptor alpha.

The orphan nuclear hormone receptor estrogen-related receptor alpha (ERRalpha, NR3B1) is a constitutive transcription factor that is structurally and functionally related to the classic estrogen receptors. ERRalpha can recognize both the estrogen response element and its own binding site (ERRE) in either dimeric or monomeric forms. ERRalpha is also a phosphoprotein whose expression in human breast tumors correlates with that of the receptor tyrosine kinase ErbB2, suggesting that its transcriptional activity could be regulated by signaling cascades. Here, we investigated growth factor regulation of ERRalpha function and found that it is phosphorylated in MCF-7 breast cancer cells in response to epidermal growth factor (EGF), an event that enhances its DNA binding. Interestingly, treatment with alkaline phosphatase shifts ERRalpha from a dimeric to a monomeric DNA-binding factor, and only the dimeric form interacts with the coactivator PGC-1alpha. In vitro, the DNA-binding domain of ERRalpha is selectively phosphorylated by protein kinase Cdelta (PKCdelta), which increases its DNA-binding activity, whereas expression of constitutively active PKCdelta enhances TFF1 promoter activity via the ERRE. However, whereas treatment of MCF-7 cells with the phorbol ester phorbol-12-myristate 13-acetate also enhances ERRalpha activation of the TFF1 promoter reporter, it does not affect ERRalpha activity on its own promoter. In agreement, chromatin immunoprecipitation analysis shows that ERRalpha and RNA polymerase II are preferentially recruited to the TFF1 promoter after EGF treatment, whereas recruitment of these factors to its own promoter is not affected. These results reveal a mechanism through which growth factor signaling can selectively activate ERRalpha target genes in breast cancer cells.

Ski-interacting protein, a bifunctional nuclear receptor coregulator that interacts with N-CoR/SMRT and p300.

Ski-interacting protein (SKIP), a vitamin D receptor (VDR) coactivator, also functions as a repressor in Notch signalling in association with the corepressor SMRT. Here we show that SKIP bifunctionally modulates (activates or represses) Retinoid-X receptor (RXR)- and VDR-dependent gene transcription in a cell line-specific manner, with activation in CV-1 and repression in P19 cells. The coactivator function of SKIP in these cells appeared to correlate with the relative level and ratio of expression of N-CoR and p300, with greater SKIP activation in higher p300-expressing and lower N-CoR-expressing cell-lines. C-terminal deletion of SKIP (delta334-536 aa) was associated with strong activation in both CV-1 and P19 cells. The corepressors N-CoR and SMRT and the coregulator p300 interacted with SKIP through the same N-terminal region (1-200 aa). Overall these results suggest that transcriptional action of SKIP may depend on distinct functional domains and cell line-specific interactions with both corepressors and coactivators.

Interactions of SKIP/NCoA-62, TFIIB, and retinoid X receptor with vitamin D receptor helix H10 residues.

The vitamin D receptor (VDR) is a ligand-dependent transcription factor that heterodimerizes with retinoid X receptor (RXR) and interacts with the basal transcription machinery and transcriptional cofactors to regulate target gene activity. The p160 coactivator GRIP1 and the distinct coregulator Ski-interacting protein (SKIP)/NCoA-62 synergistically enhance ligand-dependent VDR transcriptional activity. Both coregulators bind directly to and form a ternary complex with VDR, with GRIP1 contacting the activation function-2 (AF-2) domain and SKIP/NCoA-62 interacting through an AF-2 independent interface. It was previously reported that SKIP/NCoA-62 interaction with VDR was independent of the heterodimerization interface (specifically, helices H10/H11). In contrast, the present study defines specific residues within a conserved and surface-exposed region of VDR helix H10 that are required for interaction with SKIP/NCoA-62 and for full ligand-dependent transactivation activity. SKIP/NCoA-62, the basal transcription factor TFIIB, and RXR all interacted with VDR helix H10 mutants at reduced levels compared with wild type in the absence of ligand and exhibited different degrees of increased interaction upon ligand addition. Thus, SKIP/NCoA-62 interacts with VDR at a highly conserved region not previously associated with coregulator binding to regulate transactivation by a molecular mechanism distinct from that of p160 coactivators.