Search

Glycogen synthase kinase 3 (GSK3) is a highly conserved protein kinase regulating key cellular functions. Its two isoforms, GSK3α and GSK3β, are encoded by distinct genes. In most tissues the two isoforms are functionally interchangeable, except in the developing embryo where GSK3β is essential. One functional allele of either of the two isoforms is sufficient to maintain normal tissue functions. Both GSK3 isoforms, present in sperm from several species including human, are suggested to play a role in epididymal initiation of sperm motility. Using genetic approaches, we have tested requirement for each of the two GSK3 isoforms in testis and sperm. Both GSK3 isoforms are expressed at high levels during the onset of spermatogenesis. Conditional knockout of GSK3α, but not GSK3β, in developing testicular germ cells in mice results in male infertility. Mice lacking one allele each of GSK3α and GSK3β are fertile. Despite overlapping expression and localization in differentiating spermatids, GSK3β does not substitute for GSK3α. Loss of GSK3α impairs sperm hexokinase activity resulting in low ATP levels. Net adenine nucleotide levels in caudal sperm lacking GSK3α resemble immature caput epididymal sperm. Changes in the association of the protein phosphatase PP1γ2 with its protein interactors occurring during epididymal sperm maturation is impaired in sperm lacking GSK3α. The isoform-specific requirement for GSK3α is likely due to its specific binding partners in the sperm principal piece. Testis and sperm are unique in their specific requirement of GSK3α for normal function and male fertility.

Over 80% of diffuse intrinsic pontine gliomas (DIPGs) harbor a point mutation in histone H3.3 where lysine 27 is substituted with methionine (H3.3K27M); however, how the mutation affects kinetics and function of PcG proteins remains elusive. We demonstrate that H3.3K27M prolongs the residence time and search time of Ezh2, but has no effect on its fraction bound to chromatin. In contrast, H3.3K27M has no effect on the residence time of Cbx7, but prolongs its search time and decreases its fraction bound to chromatin. We show that increasing expression of Cbx7 inhibits the proliferation of DIPG cells and prolongs its residence time. Our results highlight that the residence time of PcG proteins directly correlates with their functions and the search time of PcG proteins is critical for regulating their genomic occupancy. Together, our data provide mechanisms in which the cancer-causing histone mutation alters the binding and search dynamics of epigenetic complexes.

Members of the B'/B56/PR61 family regulatory subunits of PP2A determine the subcellular localization, substrate specificity, and catalytic activity of PP2A in a wide range of biological processes. Here, we summarize the structure and intracellular localization of B56-containing PP2As and review functions of B56-containing PP2As in several major developmental/cancer signaling pathways., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Eye induction and eye field separation are the earliest events during vertebrate eye development. Both of these processes occur much earlier than the formation of optic vesicles. The insulin-like growth factor (IGF) pathway appears to be essential for eye induction, yet it remains unclear how IGF downstream pathways are involved in eye induction. As a consequence of eye induction, a single eye anlage is specified in the anterior neural plate. Subsequently, this single eye anlage is divided into two symmetric eye fields in response to Sonic Hedgehog (Shh) secreted from the prechordal mesoderm. Here, we report that B56epsilon regulatory subunit of protein phosphatase 2A (PP2A) is involved in Xenopus eye induction and subsequent eye field separation. We provide evidence that B56epsilon is required for the IGF/PI3K/Akt pathway and that interfering with the PI3K/Akt pathway inhibits eye induction. In addition, we show that B56epsilon regulates the Hedgehog (Hh) pathway during eye field separation. Thus, B56epsilon is involved in multiple signaling pathways and plays critical roles during early development.

Alzheimer's disease is characterized by the extracellular deposition of the amyloid beta-peptide that derives from its precursor betaAPP by sequential actions of beta- and gamma- secretases, respectively. Recent studies aimed at identifying these enzymes have been reported as it is thougth that their inhibition should hopefully lead to reduce Abeta load in the AD brains. beta-secretase seems to be due to BACE1, a novel membrane-bound aspartyl protease. gamma-secretase identification is still a matter of controversy. Invalidation of presenilin genes was reported to impair both gamma-secretase-mediated Abeta production and Notch cleavage leading to NICD production. This observation together with another biochemical and pharmacological evidences led to suggest that presenilins could be the genuine long-searched gamma-secretase that would be responsible for both APP and Notch cleavages. We have designed novel non peptidic potential inhibitors of gamma-secretase (referred to as JLK inhibitors) and examined their ability to prevent Abeta40 and Abeta42 secretions as well as NICD production. Three out of a series of these agents drastically lower the recoveries of both Abeta40 and Abeta42 produced by betaAPP-expressing cell lines and concomitantly protect intracellular C99 and C83 recoveries. These inhibitors also prevent Abeta40/42 productions by C99-expressing cells. Interestingly, these inhibitors were totally unable to affect the DeltaENotch cleavage leading to NICD generation. Here, we also further characterize the pharmacological properties and specificity of these JLK inhibitors.

gamma-Secretase activity is involved in the generation of Abeta and therefore likely contributes to the pathology of Alzheimer's disease. Blocking this activity was seen as a major therapeutic target to slow down or arrest Abeta-related AD progression. This strategy seemed more doubtful when it was established that gamma-secretase also targets other substrates including Notch, a particularly important transmembrane protein involved in vital functions, at both embryonic and adulthood stages. We have described previously new non-peptidic inhibitors able to selectively inhibit Abeta cellular production in vitro without altering Notch pathway. We show here that in vivo, these inhibitors do not alter the Notch pathway responsible for somitogenesis in the zebrafish embryo. In addition, we document further the selectivity of JLK inhibitors by showing that, unlike other described gamma-secretase inhibitors, these agents do not affect E-cadherin processing. Finally, we establish that JLKs do not inhibit beta-site APP cleaving enzymes (BACE) 1 and BACE2, alpha-secretase, the proteasome, and GSK3beta kinase. Altogether, JLK inhibitors are the sole agents to date that are able to prevent Abeta production without triggering unwanted cleavages of other proteins., (Copyright 2003 Wiley-Liss, Inc.)

Gastrointestinal (GI) smooth muscle diseases represent a major health concern affecting in excess of 2 million people each year. Little is currently known regarding the molecular mechanisms controlling either normal or pathogenic GI smooth muscle development. In an effort to identify the specific gene products responsible for modulating GI smooth muscle cell (SMC) differentiation, we performed differential display on distinct intestinal SMC (ISMC) phenotypes. This analysis identified over 40 unique transcripts that appeared to be differentially expressed in distinct SMC phenotypes. One such transcript that appeared to be preferentially expressed in immature smooth muscle myocytes was identified as vesicle-associated membrane protein, associated protein A (VAP-A). Northern blot analysis confirmed that VAP-A was expressed threefold higher in immature smooth muscle myocytes when compared with both smooth muscle myoblasts and mature smooth muscle myocytes. VAP-A mRNA was differentially expressed during normal rat development and showed peak levels of expression in the intestine during late embryogenesis and early neonatal development. These observations provide the first evidence that VAP-A-mediated membrane trafficking may play an important role in modulating ISMC differentiation., (Copyright 2003 Wiley-Liss, Inc.)

Valproic acid is widely used to treat epilepsy and bipolar disorder and is also a potent teratogen, but its mechanisms of action in any of these settings are unknown. We report that valproic acid activates Wntdependent gene expression, similar to lithium, the mainstay of therapy for bipolar disorder. Valproic acid, however, acts through a distinct pathway that involves direct inhibition of histone deacetylase (IC(50) for HDAC1 = 0.4 mm). At therapeutic levels, valproic acid mimics the histone deacetylase inhibitor trichostatin A, causing hyperacetylation of histones in cultured cells. Valproic acid, like trichostatin A, also activates transcription from diverse exogenous and endogenous promoters. Furthermore, valproic acid and trichostatin A have remarkably similar teratogenic effects in vertebrate embryos, while non-teratogenic analogues of valproic acid do not inhibit histone deacetylase and do not activate transcription. Based on these observations, we propose that inhibition of histone deacetylase provides a mechanism for valproic acid-induced birth defects and could also explain the efficacy of valproic acid in the treatment of bipolar disorder.

The malignant potential of smooth muscle tumors correlates strongly with the disappearance of gamma-smooth muscle isoactin, a lineage-specific marker of smooth muscle development. In this paper, we identify a 36-base pair regulatory motif containing an AT-rich domain, CArG box, and a non-canonical NK-2 homeodomain-binding site that has the capacity to regulate smooth muscle-specific gene expression in cultured intestinal smooth muscle cells. Serum-response factor associates with an NK-2 transcription factor via protein-protein interactions and binds to the core CArG box element. Our studies suggest that the NK-2 transcription factor that associates with serum-response factor during smooth muscle differentiation is Nkx2-3. Myocyte-specific enhancer factor 2 binding to this regulatory complex was also observed but limited to uterine smooth muscle tissues. Smooth muscle neoplasms displayed altered transcription factor binding when compared with normal myometrium. Differential nuclear accessibility of serum-response factor protein during smooth muscle differentiation and neoplastic transformation was also observed. Thus, we have identified a unique regulatory complex whose differential binding properties and nuclear accessibility are associated with modulating gamma-smooth muscle isoactin-specific gene expression in both normal and neoplastic tissues.

Lithium is highly effective in the treatment of bipolar disorder and also has multiple effects on embryonic development, glycogen synthesis, hematopoiesis, and other processes. However, the mechanism of lithium action is still unclear. A number of enzymes have been proposed as potential targets of lithium action, including inositol monophosphatase, a family of structurally related phosphomonoesterases, and the protein kinase glycogen synthase kinase-3. These potential targets are widely expressed, require metal ions for catalysis, and are generally inhibited by lithium in an uncompetitive manner, most likely by displacing a divalent cation. Thus, the challenge is to determine which target, if any, is responsible for a given response to lithium in cells. Comparison of lithium effects with genetic disruption of putative target molecules has helped to validate these targets, and the use of alternative inhibitors of a given target can also lend strong support for or against a proposed mechanism of lithium action. In this review, lithium sensitive enzymes are discussed, and a number of criteria are proposed to evaluate which of these enzymes are involved in the response to lithium in a given setting.