Suppression of mitochondrial respiration through recruitment of p160 myb binding protein to PGC-1α : modulation by p38 MAPK

The transcriptional coactivator PPAR gamma coactivator 1 α (PGC-1α) is a key regulator of metabolic processes such as mitochondrial biogenesis and respiration in muscle and gluconeogenesis in liver. Reduced levels of PGC-1α in humans have been associated with type II diabetes. PGC-1α contains a negative regulatory domain that attenuates its transcriptional activity. This negative regulation is removed by phosphorylation of PGC-1α by p38 MAPK, an important kinase downstream of cytokine signaling in muscle and β-adrenergic signaling in brown fat. We describe here the identification of p160 myb binding protein (p160MBP) as a repressor of PGC-1α. The binding and repression of PGC-1α by p160MBP is disrupted by p38 MAPK phosphorylation of PGC-1α. Adenoviral expression of p160MBP in myoblasts strongly reduces PGC-1α's ability to stimulate mitochondrial respiration and the expression of the genes of the electron transport system. This repression does not require removal of PGC-1α from chromatin, suggesting that p160MBP is or recruits a direct transcriptional suppressor. Overall, these data indicate that p160MBP is a powerful negative regulator of PGC-1α function and provide a molecular mechanism for the activation of PGC-1α by p38 MAPK. The discovery of p160MBP as a PGC-1α regulator has important implications for the understanding of energy balance and diabetes

Ubiquitination regulates PTEN nuclear import and tumor suppression

The PTEN tumor suppressor is frequently affected in cancer cells, and inherited PTEN mutation causes cancer-susceptibility conditions such as Cowden syndrome. PTEN acts as a plasma-membrane lipid-phosphatase antagonizing the phosphoinositide 3-kinase/AKT cell survival pathway. However, PTEN is also found in cell nuclei, but mechanism, function, and relevance of nuclear localization remain unclear. We show that nuclear PTEN is essential for tumor suppression and that PTEN nuclear import is mediated by its monoubiquitination. A lysine mutant of PTEN, K289E associated with Cowden syndrome, retains catalytic activity but fails to accumulate in nuclei of patient tissue due to an import defect. We identify this and another lysine residue as major monoubiquitination sites essential for PTEN import. While nuclear PTEN is stable, polyubiquitination leads to its degradation in the cytoplasm. Thus, we identify cancer-associated mutations of PTEN that target its posttranslational modification and demonstrate how a discrete molecular mechanism dictates tumor progression by differentiating between degradation and protection of PTEN

MBD2 is a transcriptional repressor belonging to the MeCP1 histone deacetylase complex

Mammalian DNA is methylated at many CpG dinucleotides. The biological consequences of methylation are mediated by a family of methyl-CpG binding proteins (1–4). The best characterized family member is MeCP2, a transcriptional repressor that recruits histone deacetylases (5–7). Our report concerns MBD2, which can bind methylated DNA in vivo and in vitro4 and has been reported to actively demethylate DNA (ref. 8). As DNA methylation causes gene silencing, the MBD2 demethylase is a candidate transcriptional activator. Using specific antibodies, however, we find here that MBD2 in HeLa cells is associated with histone deacetylase (HDAC) in the MeCP1 repressor complex (1,9). An affinity-purified HDAC1 corepressor complex (10,11) also contains MBD2, suggesting that MeCP1 corresponds to a fraction of this complex. Exogenous MBD2 represses transcription in a transient assay, and repression can be relieved by the deacetylase inhibitor trichostatin A (TSA; ref. 12). In our hands, MBD2 does not demethylate DNA. Our data suggest that HeLa cells, which lack the known methylationdependent repressor MeCP2, use an alternative pathway involving MBD2 to silence methylated genes

Single active-site histidine in D-xylose isomerase from Streptomyces violaceoruber. Identification by chemical derivatization and peptide mapping.

Group-specific chemical modifications of D-xylose isomerase from Streptomyces violaceruber indicated that complete loss of activity is fully correlated with the acylation of a single histidine. Active-site protection, by the ligand combination of xylitol plus Mg2+, completely blocked diethyl pyrocarbonate derivatization of this particular residue [Vangrysperre, Callens, Kersters-Hilderson & De Bruyne (1988) Biochem. J. 250, 153-160]. Differential peptide mapping between D-xylose isomerase, which has previously been treated with diethyl pyrocarbonate in the presence or absence of xylitol plus Mg2+, allowed specific isolation and sequencing of a peptide containing this active-site histidine. For this purpose we used two essentially new techniques: first, a highly reproducible peptide cleavage protocol for protease-resistant, carbethoxylated proteins with guanidinium hydrochloride as denaturing agent and subtilisin for proteolysis; and second, reverse-phase liquid chromatography with dual-wavelength detection at 214 and 238 nm, and calculation of absorbance ratios. It allowed us to locate the single active-site histidine at position 54 in the primary structure of Streptomyces violaceoruber D-xylose isomerase. The sequence around this residue is conserved in D-xylose isomerases from a diversity of micro-organisms, suggesting that this is a structurally and/or functionally essential part of the molecule