Essential role of tuberous sclerosis genes TSC1 and TSC2 in NF-kappaB activation and cell survival.
The TSC1-TSC2 complex has recently been implicated in cell survival responses. We observed that NF-kappaB signaling is attenuated in TSC1- and TSC2-deficient MEFs concomitant with reduced survival following DNA damage or TNFalpha stimulation. Reconstitution of TSC2 expression in TSC2(-/-) MEFs rescued survival in an NF-kappaB activity-dependent manner. Furthermore, in TSC2(-/-) MEFs, the rapamycin-mediated inhibition of deregulated mTOR activity restored NF-kappaB activation and survival. This rapamycin-mediated effect was reversed by inhibition of NF-kappaB transcriptional activation or by inhibition of ERK1/2 MAP kinase or PI-3K pathways, which lie on signaling cascades that lead to NF-kappaB activation. These results provide evidence for a crosstalk between the TSC/Rheb/mTOR pathway and the NF-kappaB induction pathways and indicate that NF-kappaB functions as an important survival factor that regulates TSC2-dependent cell survival.

title: Dynamics of ankyrin-containing complexes in chicken embryonic erythroid cells: role of phosphorylation.
abstract: Chicken erythroid ankyrin undergoes a fairly rapid cycle of cytoskeletal association, dissociation, and turnover. In addition, the cytoskeletal association of ankyrin is regulated by phosphorylation. Treatment of erythroid cells with serine and threonine phosphatase inhibitors stimulated the hyperphosphorylation of the 225- and 205-kDa ankyrin isoforms, and dissociated the bulk of these isoforms from cytoskeletal spectrin. In vitro binding studies have shown that this dissociation of ankyrin from spectrin in vivo can be attributed to a reduced ability of hyperphosphorylated ankyrin to bind spectrin. Interestingly, a significant fraction of detergent insoluble ankyrin accumulates in a spectrin-independent pool. At least some of this spectrin-independent pool of ankyrin is complexed with the AE1 anion exchanger, and the solubility properties of this pool are also regulated by phosphorylation. Treatment of cells with serine and threonine phosphatase inhibitors had no effect on ankyrin/AE1 complex formation. However, these inhibitors were sufficient to shift ankyrin/AE1 complexes from the detergent insoluble to the soluble pool. These analyses, which are the first to document the in vivo consequences of ankyrin phosphorylation, indicate that erythroid ankyrin-containing complexes can undergo dynamic rearrangements in response to changes in phosphorylation.

title: CK2 constitutively associates with and phosphorylates chicken erythroid ankyrin and regulates its ability to bind to spectrin
abstract: Previous analyses have shown that the phosphorylation state of chicken erythroid ankyrin regulates its association with the spectrin cytoskeleton in vivo. Treatment of erythroid cells with serine and threonine phosphatase inhibitors stimulates the hyperphosphorylation of ankyrin and its dissociation from spectrin. In this study, we demonstrate that a kinase that directs the phosphorylation of ankyrin in vivo coprecipitates with ankyrin-containing complexes and has properties identical to CK2. Studies using CK2-specific inhibitors have indicated that all of the phosphorylation events associated with erythroid ankyrin in vivo are CK2 dependent. Furthermore, inhibitor studies combined with in vitro binding analyses have indicated that the phosphorylation of erythroid ankyrin by CK2 regulates its ability to associate with spectrin. Additional analyses revealed that CK2 coprecipitates with ankyrin-3-containing complexes isolated from Madin Darby canine kidney epithelial cells and phosphorylates this epithelial ankyrin isoform in vivo. These results are the first demonstration of a kinase constitutively associating with the ankyrin-spectrin cytoskeleton in erythroid and kidney epithelial cells. This association provides a mechanism for rapidly reorganizing the membrane cytoskeleton in these cell types through the phosphorylation of ankyrin.

title: Chicken erythroid AE1 anion exchangers associate with the cytoskeleton during recycling to the Golgi.
abstract: Chicken erythroid AE1 anion exchangers receive endoglycosidase F (endo F)-sensitive sugar modifications in their initial transit through the secretory pathway. After delivery to the plasma membrane, anion exchangers are internalized and recycled to the Golgi where they acquire additional N-linked modifications that are resistant to endo F. During recycling, some of the anion exchangers become detergent insoluble. The acquisition of detergent insolubility correlates with the association of the anion exchanger with cytoskeletal ankyrin. Reagents that inhibit different steps in the endocytic pathway, including 0.4 M sucrose, ammonium chloride, and brefeldin A, block the acquisition of endo F-resistant sugars and the acquisition of detergent insolubility by newly synthesized anion exchangers. The inhibitory effects of ammonium chloride on anion exchanger processing are rapidly reversible. Furthermore, AE1 anion exchangers become detergent insoluble more rapidly than they acquire endo F-resistant modifications in cells recovering from an ammonium chloride block. This suggests that the cytoskeletal association of the recycling anion exchangers occurs after release from the compartment where they accumulate due to ammonium chloride treatment, and prior to their transit through the Golgi. The recycling pool of newly synthesized anion exchangers is reflected in the steady-state distribution of the polypeptide. In addition to plasma membrane staining, anion exchanger antibodies stain a perinuclear compartment in erythroid cells. This perinuclear AE1-containing compartment is also stained by ankyrin antibodies and partially overlaps the membrane compartment stained by NBD C6-ceramide, a Golgi marker. Detergent extraction of erythroid cells in situ has suggested that a substantial fraction of the perinuclear pool of AE1 is cytoskeletal associated. The demonstration that erythroid anion exchangers interact with elements of the cytoskeleton during recycling to the Golgi suggests the cytoskeleton may be involved in the post-Golgi trafficking of this membrane transporter.

title: Regulation of inducible gene expression by the transcription factor NF-kappaB.
abstract: The transcription factor NF-kappaB plays a critical role in regulating inducible gene expression in immune responses. The activation of NF-kappaB is regulated at multiple levels, probably reflecting a need to maintain a tight control of its activity. We have recently discovered that direct phosphorylation of NF-kappaB itself is essential for its transcriptional activity and that phosphorylation acts as a switch to determine association of nuclear NF-kappaB with histone acetylases vs deacetylases.

title: Cell density-dependent transcriptional activation of endocrine-related genes in human adipose tissue-derived stem cells.
abstract: Adipose tissue is recognized as an endocrine organ that plays an important role in human diseases such as type II diabetes and cancer. Human adipose tissue-derived stem cells (ASCs), a distinct cell population in adipose tissue, are capable of differentiating into multiple lineages including adipogenesis. When cultured in vitro under a confluent condition, ASCs reach a commitment stage for adipogenesis, which can be further induced into terminally differentiated adipocytes by a cocktail of adipogenic factors. Here we report that the confluent state of ASCs triggers transcriptional activation cascades for genes that are responsible for the endocrine function of adipose tissue. These include insulin-like growth factor 1 (IGF-1) and aromatase (Cyp19), a key enzyme in estrogen biosynthesis. Despite similar adipogenic potentials, ASCs from different individuals display huge variations in activation of these endocrine-related genes. Bioinformatics and experimental data suggest that transcription factor Foxo1 controls a large number of "early" confluency-response genes, which subsequently induce "late" response genes. Furthermore, siRNA-mediated knockdown of Foxo1 substantially compromises the ability of committed ASCs to stimulate tumor cell migration in vitro. Thus, our work suggests that cell density is an important determinant of the endocrine potential of ASCs.

title: Mobilization of cytoplasmic CE droplets by overexpression of human macrophage cholesteryl ester hydrolase Published, JLR Papers in Press, July 1, 2003. DOI 10.1194/jlr.M300162-JLR200
abstract: The obligatory first step in the removal of cholesterol from foam cells is the hydrolysis of stored cholesteryl esters (CEs) to release free cholesterol (FC). Neutral cholesteryl ester hydrolase (CEH) catalyzes this hydrolysis, and limiting levels of CEH could play a role in determining the susceptibility to atherosclerosis. We have recently reported the first identification and cloning of cDNA for human macrophage CEH. In the present study, we tested the hypothesis that systematically varied levels of overexpression of human macrophage CEH results in a proportional degree of reduction in cellular CE content in a cell system with known and reproducible amounts of CE accumulation. CEH expression was confirmed by demonstrating the presence of CEH mRNA and protein with an increase in CEH activity. A significant reduction in intracellular lipid droplets was observed in CEH-expressing cells, together with a decrease in cellular CE mass and a 2-fold increase in FC efflux. These results demonstrate that when human macrophage CEH is expressed in lipid-laden cells, hydrolysis and mobilization of CE (stored as lipid droplets) occur. These data establish the possibility that increased CE hydrolysis, mediated by CEH up-regulation, could represent an important mechanism to reduce the cholesterol burden of foam cells.

title: C-terminal phenylalanine of bacteriophage T7 single-stranded DNA-binding protein is essential for strand displacement synthesis by T7 DNA polymerase at a nick in DNA.
abstract: Single-stranded DNA-binding protein (gp2.5), encoded by gene 2.5 of bacteriophage T7, plays an essential role in DNA replication. Not only does it remove impediments of secondary structure in the DNA, it also modulates the activities of the other replication proteins. The acidic C-terminal tail of gp2.5, bearing a C-terminal phenylalanine, physically and functionally interacts with the helicase and DNA polymerase. Deletion of the phenylalanine or substitution with a nonaromatic amino acid gives rise to a dominant lethal phenotype, and the altered gp2.5 has reduced affinity for T7 DNA polymerase. Suppressors of the dominant lethal phenotype have led to the identification of mutations in gene 5 that encodes the T7 DNA polymerase. The altered residues in the polymerase are solvent-exposed and lie in regions that are adjacent to the bound DNA. gp2.5 lacking the C-terminal phenylalanine has a lower affinity for gp5-thioredoxin relative to the wild-type gp2.5, and this affinity is partially restored by the suppressor mutations in DNA polymerase. gp2.5 enables T7 DNA polymerase to catalyze strand displacement DNA synthesis at a nick in DNA. The resulting 5'-single-stranded DNA tail provides a loading site for T7 DNA helicase. gp2.5 lacking the C-terminal phenylalanine does not support this event with wild-type DNA polymerase but does to a limited extent with T7 DNA polymerase harboring the suppressor mutations.

title: Activation in vitro of NF-kappa B by phosphorylation of its inhibitor I kappa B.
abstract: Nuclear factor kappa B (NF-kappa B), which was first detected by its binding to the kappa B site in the immunoglobulin kappa-gene enhancer, is important for the regulated expression of the kappa-gene and is partly responsible for the induction in appropriate cells of interleukin-2 (IL-2), IL-2 alpha receptor, beta-interferon and serum amyloid A protein. NF-kappa B is present as a nuclear DNA-binding protein in B lymphocytes and mature macrophages, but is found in the cytoplasm of many cells in a form unable to bind to DNA. The cytoplasmic form is bound to an inhibitor protein, I kappa B, from which it can be released in vitro by deoxycholate and other agents. Activation of cells by various agents, notably the phorbol esters that stimulate protein kinase C (PKC), leads to dissociation in vivo of the NF-kappa B/I kappa B complex and migration of NF-kappa B to the nucleus. Therefore, it acts as a second messenger system, transducing activation signals from the cytoplasm to the nucleus. To elucidate the mechanism of signal transfer, we have used an in vitro system in which addition of purified protein kinases to a partially purified NF-kappa B/I kappa B complex leads to the activation of the DNA-binding activity of NF-kappa B. Using gel retardation assays we found that PKC, cyclic AMP-dependent protein kinase (PKA) and a haem-regulated eIF-2 kinase (HRI) could activate NF-kappa B in vitro, whereas casein kinase II was ineffective. To determine the target for the protein kinases we purified and characterized both NF-kappa B and I kappa B and found that I kappa B is phosphorylated and inactivated in the presence of PKC and HRI but not PKA.