Developmental control of apoptosis by the immunophilin aryl hydrocarbon receptor-interacting protein (AIP) involves mitochondrial import of the survivin protein

Survivin is a multifunctional protein with essential roles in cell division and inhibition of apoptosis, but the molecular underpinnings of its cytoprotective properties are poorly understood. Here we show that homozygous deletion of the aryl hydrocarbon receptor-interacting protein (AIP), a survivin-associated immunophilin, causes embryonic lethality in mice by embryonic day 13.5-14, increased apoptosis of Ter119(-)/CD71(-) early erythropoietic progenitors, and loss of survivin expression in its cytosolic and mitochondrial compartments in vivo. In import assays using recombinant proteins, AIP directly mediated the import of survivin to mitochondria, thus enabling its anti-apoptotic function, whereas a survivin 1-141 mutant that does not bind AIP was not imported to mitochondria and failed to inhibit apoptosis. AIP-directed mitochondrial import of survivin did not affect cell division, was independent of the organelle transmembrane potential, did not require the chaperone Heat Shock Protein 90 (Hsp90), and was inhibited by cytosolic factor(s) present in normal cells. shRNA knockdown of the mitochondrial import receptor Tom20 abolished mitochondrial import of survivin and sensitized tumor cells to apoptosis, whereas silencing of Tom70 had no effect. Therefore, an AIP-Tom20 recognition contributes to cell survival in development and cancer by mediating the mitochondrial import of survivin.     

Regulation of constitutive gene expression through interactions of Sp1 protein with the nuclear aryl hydrocarbon receptor complex.

The region of residues -145 to -119 (CD/L) of the cathepsin D gene promoter contains a GC-rich motif that binds Sp1 protein and an adjacent pentanucleotide (CACGC) that corresponds to the core sequence of a dioxin responsive element (DRE) and binds the aryl hydrocarbon receptor (AhR)-AhR nuclear translocator (Arnt) complex. This Sp1(N)(4)DRE(core) motif has been identified in promoters of several genes in which Sp1 plays an important role in basal gene expression. In transient transfection assays with MCF-7 human breast cancer cells using wild-type pCD/L and constructs mutated in the core DRE (pCD/L(m1)) and Sp1 (pCD/L(m2)) sites, it was shown that both motifs were required for maximal basal activity. The requirements for AhR-Arnt interactions with Sp1 protein for maximal activity of pCD/L were confirmed in wild-type MCF-7 and Hepa 1c1c7 cells and Arnt-deficient Hepa 1c1c7 cells using antisense Arnt and Arnt expression plasmids. The functional interactions of Sp1 with AhR-Arnt were paralleled by physical interactions showing that AhR-Arnt and Sp1 proteins were co-immunoprecipitated and AhR-Arnt enhanced Sp1-[(32)P]CD/L binding in electrophoretic mobility shift assays. The physical and functional interactions of Sp1 with AhR-Arnt proteins bound to the Sp1(N)(4)DRE(core) motif were also dependent on the proximity of these sites, and both the activity and the extent of Sp1-DNA binding decreased as the number of intervening nucleotides increased from 4 to 20. These studies show that regulation of basal expression of some genes by Sp1 may also require interactions with AhR-Arnt.     

The aryl hydrocarbon receptor, more than a xenobiotic-interacting protein

The aryl hydrocarbon (dioxin) receptor (AhR) has been studied for several decades largely because of its critical role in xenobiotic-induced toxicity and carcinogenesis. Albeit this is a major issue in basic and clinical research, an increasing number of investigators are turning their efforts to try to understand the physiology of the AhR under normal cellular conditions. This is an exciting area that covers cell proliferation and differentiation, endogenous mechanisms of activation, gene regulation, tumor development and cell motility and migration, among others. In this review, we will attempt to summarize the studies supporting the implication of the AhR in those endogenous cellular processes.     

A novel Arnt-interacting protein Ainp2 enhances the aryl hydrocarbon receptor signalling

In an effort to better understand the Ah receptor nuclear translocator (Arnt)-dependent signaling mechanisms, we employed a phage display system to identify Arnt-interacting peptides. Human liver cDNA library was utilized to screen for Arnt-interacting peptides using an Arnt construct fused to thioredoxin (TH-ArntCDelta418). Two clones, namely Ainp1 and Ainp2 (Arnt-interacting peptide), were identified and subsequently Ainp2 was further characterized. Ainp2 interacts with TH-ArntCDelta418 in the GST pull-down and mammalian two-hybrid assays. Northern blot results revealed that Ainp2 is predominantly expressed in human liver. The putative full-length Ainp2 cDNA sequence was subsequently cloned using RACE PCR. Endogenous expression of Ainp2 was found in Jurkat cells at the mRNA and protein levels. Results from the transient transfection studies using a DRE-driven reporter plasmid and the real-time QPCR experiments examining the endogenous CYP1A1 expression showed that Ainp2 enhances the 3-methylchloranthrene-induced activity in HepG2 cells, suggesting that Ainp2 plays a role in the Arnt-dependent function     

Regulation of protein stability by GSK3 mediated phosphorylation

Glycogen synthase kinase-3 (GSK3) plays important roles in numerous signaling pathways that regulate a variety of cellular processes including cell proliferation, differentiation, apoptosis and embryonic development. In the canonical Wnt signaling pathway, GSK3 phosphorylation mediates proteasomal targeting and degradation of β-catenin via the destruction complex. We recently reported a biochemical screen that discovered multiple additional protein substrates whose stability is regulated by Wnt signaling and/or GSK3 and these have important implications for Wnt/GSK3 regulation of different cellular processes.1 In this article, we also present a bio-informatics based screen for proteins whose stability may be controlled by GSK3 and β-Trcp, the SCF E3 ubiquitin ligase that is responsible for β-catenin degradation in the Wnt signaling pathway. Furthermore, we review various GSK3 regulated proteolysis substrates described in the literature. We propose that GSK3 phosphorylation dependent proteolysis is a widespread mechanism that the cell employs to regulate a variety of cell processes in response to signals.     

Regulation of Cidea protein stability by the ubiquitin-mediated proteasomal degradation pathway

Cidea, one of three members of the CIDE (cell-death-inducing DNA-fragmentation-factor-45-like effector) family of proteins, is highly enriched in brown adipose tissue, in which it plays a critical role in adaptive thermogenesis and fat accumulation. Cidea-null mice have increased energy expenditure with resistance to high-fat-diet-induced obesity and diabetes. However, little is known as to how the Cidea protein is regulated. In the present study we show that Cidea is a short-lived protein as measured by cycloheximide-based protein chase experiments in different cell lines or in differentiated brown adipocytes. Proteasome inhibitors specifically increased the stability of both transfected and endogenous Cidea protein. Furthermore, Cidea protein was found to be polyubiquitinated when overexpressed in different culture cells as well as in differentiated mature brown adipocytes. Extensive mutational analysis of individual lysine residues revealed that ubiquitinated lysine residues are located in the N-terminal region of Cidea, as alteration of these lysine residues to alanine (N-5KA mutant) renders Cidea much more stable when compared with wild-type or C-terminal lysine-less mutant (C-5KA). Furthermore, K23 (Lys23) within the N-terminus of the Cidea was identified as the major contributor to its polyubiquitination signal and the protein instability. Taken together, the results of our study demonstrated that the ubiquitin-proteasome system confers an important post-translational modification that controls the protein stability of Cidea.     

Characterization of an inducible aryl hydrocarbon receptor-like protein in rat liver.

The individual pretreatment of Sprague-Dawley rats with either 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or 2,2',4,4',5,5'-hexachlorobiphenyl (HCB) has been previously shown to result in the "induction" of [3H]TCDD specific binding activity in hepatic tissue. In the present work, the coadministration of TCDD and HCB increased the concentration of hepatic proteins capable of binding [3H]TCDD specifically by at least 2-3-fold. This increase was shown not to be the result of activation, by HCB, of a form of the receptor having low affinity toward [3H]TCDD into a form with high affinity. Kinetic analysis of the time course of binding of [3H]TCDD to induced cytosol was consistent with the presence of an "inducible" binding protein in addition to the "constitutive" aryl hydrocarbon (Ah) receptor present in cytosol from untreated animals. The liganded ([3H]TCDD) form of the inducible binding component lost its ligand much faster than the liganded form of the constitutive Ah receptor at 37 degrees C; apparent first order rate constants for loss of [3H]TCDD were 0.55 min-1 and less than 0.0024 min-1, respectively. Conversely, the unliganded form of the induced binding component was slightly more stable (approximately 2-fold) toward thermal inactivation than the unbound constitutive Ah receptor. The [3H]TCDD-bound protein(s) in uninduced and induced cytosols behaved identically in a sucrose gradient; 8.7-8.9 S in the absence of salt, shifted to 5.5 S by 0.4 M KCl. They were also indistinguishable by gel permeation chromatography, and by photoaffinity labeling their TCDD-binding subunits, approximate molecular weights 105,000. These results show the hepatic TCDD-binding protein(s) induced upon pretreatment of Sprague-Dawley rats with TCDD/HCB to be kinetically distinct from the Ah receptor, but structurally very similar.     

Glutamate receptor-interacting protein 1 protein binds to the microtubule-associated protein

To identify the interaction proteins for the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptor subunit glutamate receptor-interacting protein 1 (GRIP1), GRIP1 interactions with microtubule-associated protein (MAP)-1B light chain (LC) were investigated. GRIP1 interacts with MAP-1A and MAP-1B in the yeast two-hybrid assay, as is indicated also by glutathione S-transferase (GST) pull-down and coimmunoprecipitation with MAP-1B LC antibody in brain fractions. These results suggest a novel mechanism for localizing AMPA receptors to synaptic sites.