Biomedical subjects

Ben E Black

Publications and source records attributed to Ben E Black.

7 recordsLinked to original sources

PubMed2025

Maternal CENP-C restores centromere symmetry in mammalian zygotes to ensure proper chromosome segregation.

Across metazoan species, the centromere-specific histone variant CENP-A is essential for accurate chromosome segregation, yet its regulation during the mammalian parental-to-zygote transition is poorly understood. To address this, we generated a CENP-A-mScarlet mouse model that revealed sex-specific dynamics: mature sperm retain 10% of the CENP-A levels present in MII oocytes. However, this difference is resolved in zygotes prior to the first mitosis, using maternally inherited cytoplasmic CENP-A. Notably, the increase in CENP-A at paternal centromeres is independent of sensing CENP-A asymmetry or the presence of maternal chromosomes. Instead, CENP-A equalization relies on the asymmetric recruitment of maternal CENP-C to paternal centromeres. Depletion of maternal CENP-A decreases total CENP-A in both pronuclei without disrupting equalization. In contrast, reducing maternal CENP-C or disruption of its dimerization function impairs CENP-A equalization and chromosome segregation. Therefore, maternal CENP-C acts as a key epigenetic regulator that resets centromeric symmetry at fertilization to preserve genome integrity.

Animals↗

PubMed2005

Stress kinase signaling regulates androgen receptor phosphorylation, transcription, and localization.

Activation of signal transduction kinase cascades is known to alter androgen receptor (AR) activity, but the molecular mechanisms are still poorly defined. Here we show that stress kinase signaling regulates Ser 650 phosphorylation and AR nuclear export. In LNCaP prostate cancer cells, activation of either MAPK kinase (MKK) 4:c-Jun N-terminal kinase (JNK) or MKK6:p38 signaling pathways increased Ser 650 phosphorylation, whereas pharmacologic inhibition of JNK or p38 signaling led to a reduction of AR Ser 650 phosphorylation. Both p38alpha and JNK1 phosphorylated Ser 650 in vitro. Small interfering RNA-mediated knockdown of either MKK4 or MKK6 increased endogenous prostate-specific antigen (PSA) transcript levels, and this increase was blocked by either bicalutamide or AR small interfering RNA. Stress kinase inhibition of PSA transcription is, therefore, dependent on the AR. Similar experiments involving either activation or inhibition of MAPK/ERK kinase:ERK signaling had little effect on Ser 650 phosphorylation or PSA mRNA levels. Ser 650 is proximal to the DNA binding domain that contains a nuclear export signal. Mutation of Ser 650 to alanine reduced nuclear export of the AR, whereas mutation of Ser 650 to the phosphomimetic amino acid aspartate restored AR nuclear export. Pharmacologic inhibition of stress kinase signaling reduced wild-type AR nuclear export equivalent to the S650A mutant without affecting nuclear export of the S650D mutant. Our data suggest that stress kinase signaling and nuclear export regulate AR transcriptional activity.

Active Transport, Cell Nucleus↗

PubMed2005

Retrotranslocation of the chaperone calreticulin from the endoplasmic reticulum lumen to the cytosol.

Polypeptide folding and quality control in the endoplasmic reticulum (ER) are mediated by protein chaperones, including calreticulin (CRT). ER localization of CRT is specified by two types of targeting signals, an N-terminal hydrophobic signal sequence that directs insertion into the ER and a C-terminal KDEL sequence that is responsible for retention in the ER. CRT has been implicated in a number of cytoplasmic and nuclear processes, suggesting that there may be a pathway for generating cytosolic CRT. Here we show that CRT is fully inserted into the ER, undergoes processing by signal peptidase, and subsequently undergoes retrotranslocation to the cytoplasm. A transcription-based reporter assay revealed an important role for the C-terminal Ca(2+) binding domain in CRT retrotranslocation. Neither ubiquitylation nor proteasome activity was necessary for retrotranslocation, which indicates that the pathway is different from that used by unfolded proteins targeted for destruction. Forced expression of cytosolic CRT is sufficient to rescue a cell adhesion defect observed in mouse embryo fibroblasts from crt(-/-) mice. The ability of CRT to retrotranslocate from the ER lumen to the cytosol explains how CRT can change compartments and modulate cell adhesion, transcription, and translation.

Amino Acid Sequence↗

PubMed2004

Structural determinants for generating centromeric chromatin.

Mammalian centromeres are not defined by a consensus DNA sequence. In all eukaryotes a hallmark of functional centromeres--both normal ones and those formed aberrantly at atypical loci--is the accumulation of centromere protein A (CENP-A), a histone variant that replaces H3 in centromeric nucleosomes. Here we show using deuterium exchange/mass spectrometry coupled with hydrodynamic measures that CENP-A and histone H4 form sub-nucleosomal tetramers that are more compact and conformationally more rigid than the corresponding tetramers of histones H3 and H4. Substitution into histone H3 of the domain of CENP-A responsible for compaction is sufficient to direct it to centromeres. Thus, the centromere-targeting domain of CENP-A confers a unique structural rigidity to the nucleosomes into which it assembles, and is likely to have a role in maintaining centromere identity.

Amino Acid Sequence↗

PubMed2004

Intranuclear organization and function of the androgen receptor.

We have entered an exciting era for androgen-receptor (AR) research that should provide a detailed description of how the AR functions as a ligand-regulated transcription factor. That AR activity is regulated by subcellular compartmentalization was first established a decade ago with the finding that binding of androgen to the AR induces its translocation from the cytoplasm to the nucleus. The contribution of compartmentalization to AR activity is, however, likely to extend beyond simple delivery to the nucleus. Defects in AR and coregulator compartmentalization in the nucleus have been demonstrated in prostate cancer, androgen-insensitivity syndrome, and spinal and bulbar muscular atrophy. A complete understanding of AR function and dysfunction in disease requires integrating transcription with the spatial and temporal regulation imposed by subnuclear organization and nuclear transport.

Active Transport, Cell Nucleus↗

PubMed2003

Transient, ligand-dependent arrest of the androgen receptor in subnuclear foci alters phosphorylation and coactivator interactions.

Here we report that mutations within the DNA-binding domain of AR, shown previously to inhibit nuclear export to the cytoplasm, cause an androgen-dependent defect in intranuclear trafficking of AR. Mutation of two conserved phenylalanines within the DNA recognition helix (F582, 583A) results in androgen-dependent arrest of AR in multiple subnuclear foci. A point mutation in one of the conserved phenylalanines (DeltaF582, F582Y) is known to cause androgen insensitivity syndrome (AIS). Both AIS mutants (DeltaF582, F582Y) and the export mutant (F582, 583A) displayed androgen-dependent arrest in foci, and all three mutants promoted androgen-dependent accumulation of the histone acetyl transferase CREB binding protein (CBP) in the foci. The foci correspond to a subnuclear compartment that is highly enriched for the steroid receptor coactivator glucocorticoid receptor-interacting protein (GRIP)-1. Agonist-bound wild-type AR induces the redistribution of GRIP-1 from foci to the nucleoplasm. This likely reflects a direct interaction between these proteins because mutation of a conserved residue within the major coactivator binding site on AR (K720A) inhibits AR-dependent dissociation of GRIP-1 from foci. GRIP-1 also remains foci-associated in the presence of agonist-bound F582, 583A, DeltaF582, or F582Y forms of AR. Two-dimensional phospho-peptide mapping and analysis with a phospho-specific antibody revealed that mutant forms of AR that arrest in the subnuclear foci are hypophosphorylated at Ser81, a site that normally undergoes androgen-dependent phosphorylation. Our working model is that the subnuclear foci are sites where AR undergoes ligand-dependent engagement with GRIP-1 and CBP, a recruitment step that occurs before Ser81 phosphorylation and association with promoters of target genes.

Androgen-Insensitivity Syndrome↗

PubMed2002

Ca2+-dependent nuclear export mediated by calreticulin.

We have characterized a pathway for nuclear export of the glucocorticoid receptor (GR) in mammalian cells. This pathway involves the Ca2+ -binding protein calreticulin (CRT), which directly contacts the DNA binding domain (DBD) of GR and facilitates its delivery from the nucleus to the cytoplasm. In the present study, we investigated the role of Ca2+ in CRT-dependent export of GR. We found that removal of Ca2+ from CRT inhibits its capacity to stimulate the nuclear export of GR in digitonin-permeabilized cells and that the inhibition is due to the failure of Ca2+-free CRT to bind the DBD. These effects are reversible, since DBD binding and nuclear export can be restored by Ca2+ addition. Depletion of intracellular Ca2+ inhibits GR export in intact cells under conditions that do not inhibit other nuclear transport pathways, suggesting that there is a Ca2+ requirement for GR export in vivo. We also found that the Ran GTPase is not required for GR export. These data show that the nuclear export pathway used by steroid hormone receptors such as GR is distinct from the Crm1 pathway. We suggest that signaling events that increase Ca2+ could positively regulate CRT and inhibit GR function through nuclear export.

Active Transport, Cell Nucleus↗