Chaim Kahana

Insulin induces transcription of target genes through the hypoxia-inducible factor HIF-1alpha/ARNT.

Hypoxic stress induces the expression of genes associated with increased energy flux, including the glucose transporters Glut1 and Glut3, several glycolytic enzymes, nitric oxide synthase, tyrosine hydroxylase, erythropoietin and vascular endothelial growth factor (VEGF). Induction of these genes is mediated by a common basic helix–loop–helix‐PAS transcription complex, the hypoxia‐inducible factor‐1α (HIF‐1α)/aryl hydrocarbon nuclear translocator (ARNT). Insulin also induces some of these genes; however, the underlying mechanism is unestablished. We report here that insulin shares with hypoxia the ability to induce the HIF‐1α/ARNT transcription complex in various cell types. This induction was demonstrated by electrophoretic mobility shift of the hypoxia response element (HRE), and abolished by specific antisera to HIF‐1α and ARNT, and by transcription activation of HRE reporter vectors. Furthermore, basal and insulin‐induced expression of Glut1, Glut3, aldolase A, phosphoglycerate kinase and VEGF was reduced in cells having a defective ARNT. Similarly, the insulin‐induced activation of HRE reporter vectors and VEGF was impaired in these cells and was rescued by re‐introduction of ARNT. Finally, insulin‐like growth factor‐I (IGF‐I) also induced the HIF‐1α/ARNT transcription complex. These observations establish a novel signal transduction pathway of insulin and IGF‐I and broaden considerably the scope of activity of HIF‐1α/ARNT.

Mdm-2 and ubiquitin-independent p53 proteasomal degradation regulated by NQO1

The tumor suppressor p53 is a labile protein whose level is known to be regulated by the Mdm-2–ubiquitin–proteasome degradation pathway. We have found another pathway for p53 proteasomal degradation regulated by NAD(P)H quinone oxidoreductase 1 (NQO1). Inhibition of NQO1 activity by dicoumarol induces p53 and p73 proteasomal degradation. A mutant p53 (p53[22,23]), which is resistant to Mdm-2-mediated degradation, was susceptible to dicoumarol-induced degradation. This finding indicates that the NQO1-regulated proteasomal p53 degradation is Mdm-2-independent. The tumor suppressor p14ARF and the viral oncogenes SV40 LT and adenovirus E1A that are known to stabilize p53 inhibited dicoumarol-induced p53 degradation. Unlike Mdm-2-mediated degradation, the NQO1-regulated p53 degradation pathway was not associated with accumulation of ubiquitin-conjugated p53. In vitro studies indicate that dicoumarol-induced p53 degradation was ubiquitin-independent and ATP-dependent. Inhibition of NQO1 activity in cells with a temperature-sensitive E1 ubiquitin-activating enzyme induced p53 degradation and inhibited apoptosis at the restrictive temperature without ubiquitination. Mdm-2 failed to induce p53 degradation under these conditions. Our results establish a Mdm-2- and ubiquitin-independent mechanism for proteasomal degradation of p53 that is regulated by NQO1. The lack of NQO1 activity that stabilizes a tumor suppressor such as p53 can explain why humans carrying a polymorphic inactive NQO1 are more susceptible to tumor development.

Poliovirus 2A Protease Induces Apoptotic Cell Death

ABSTRACT A cell line was generated that expresses the poliovirus 2A protease in an inducible manner. Tightly controlled expression was achieved by utilizing the muristerone A-regulated expression system. Upon induction, cleavage of the eukaryotic translation initiation factor 4GI (eIF4GI) and eIF4GII is observed, with the latter being cleaved in a somewhat slower kinetics. eIF4G cleavage was accompanied by a severe inhibition of protein synthesis activity. Upon induction of the poliovirus 2A protease, the cells displayed fragmented nuclei, chromatin condensation, oligonucleosome-size DNA ladder, and positive TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling) staining; hence, their death can be characterized as apoptosis. These results indicate that the expression of the 2A protease in mammalian cells is sufficient to induce apoptosis. We suggest that the poliovirus 2A protease induces apoptosis either by arresting cap-dependent translation of some cellular mRNAs that encode proteins required for cell viability, by preferential cap-independent translation of cellular mRNAs encoding apoptosis inducing proteins, or by cleaving other, yet unidentified cellular target proteins.

Structural Motifs Involved in Ubiquitin-Mediated Processing of the NF-κB Precursor p105: Roles of the Glycine-Rich Region and a Downstream Ubiquitination Domain

ABSTRACT The ubiquitin proteolytic system plays a major role in a variety of basic cellular processes. In the majority of these processes, the target proteins are completely degraded. In one exceptional case, generation of the p50 subunit of the transcriptional regulator NF-κB, the precursor protein p105 is processed in a limited manner: the N-terminal domain yields the p50 subunit, whereas the C-terminal domain is degraded. The identity of the mechanisms involved in this unique process have remained elusive. It has been shown that a Gly-rich region (GRR) at the C-terminal domain of p50 is an important processing signal. Here we show that the GRR does not interfere with conjugation of ubiquitin to p105 but probably does interfere with the processing of the ubiquitin-tagged precursor by the 26S proteasome. Structural analysis reveals that a short sequence containing a few Gly residues and a single essential Ala is sufficient to generate p50. Mechanistically, the presence of the GRR appears to stop further degradation of p50 and to stabilize the molecule. It appears that the localization of the GRR within p105 plays an important role in directing processing: transfer of the GRR within p105 or insertion of the GRR into homologous or heterologous proteins is not sufficient to promote processing in most cases, which is probably due to the requirement for an additional specific ubiquitination and/or recognition domain(s). Indeed, we have shown that amino acid residues 441 to 454 are important for processing. In particular, both Lys 441 and Lys 442 appear to serve as major ubiquitination targets, while residues 446 to 454 are independently important for processing and may serve as the ubiquitin ligase recognition motif.

Transcriptional activation of mammalian ornithine decarboxylase during stimulated growth.

Cloned ornithine decarboxylase (ODC) (EC 4.1.1.17) cDNA was used to investigate the mechanisms which mediate the mitogenic induction of mammalian ODC. Stimulation of quiescent BALB/c 3T3 mouse fibroblasts with purified fibroblast and platelet-derived growth factors and with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate results in a rapid and dramatic increase in ODC mRNA, similar to the increase caused by serum stimulation. Using nuclear runoff transcriptional analysis, we demonstrate that an increase in ODC transcription accounts for the mitogenic induction of ODC mRNA, and using cycloheximide together with the stimulating mitogen, we found that the mitogenic induction of ODC is dependent on ongoing protein synthesis in the stimulated cells.

Antizyme and antizyme inhibitor, a regulatory tango

The polyamines are small basic molecules essential for cellular proliferation and viability. An autoregulatory circuit that responds to the intracellular level of polyamines regulates their production. In the center of this circuit is a family of small proteins termed antizymes. Antizymes are themselves regulated at the translational level by the level of polyamines. Antizymes bind ornithine decarboxylase (ODC) subunits and target them to ubiquitin-independent degradation by the 26S proteasome. In addition, antizymes inhibit polyamine transport across the plasma membrane via an as yet unresolved mechanism. Antizymes may also interact with and target degradation of other growth-regulating proteins. An inactive ODC-related protein termed antizyme inhibitor regulates polyamine metabolism by negating antizyme functions. The ability of antizymes to degrade ODC, inhibit polyamine uptake and consequently suppress cellular proliferation suggests that they act as tumor suppressors, while the ability of antizyme inhibitors to negate antizyme function indicates their growth-promoting and oncogenic potential.

The role of polyamines in supporting growth of mammalian cells is mediated through their requirement for translation initiation and elongation

Polyamines are essential cell constituents whose depletion results in growth cessation. Here we have investigated potential mechanisms of action of polyamines in supporting mammalian cell proliferation. We demonstrate that polyamines regulate translation both at the initiation and at the elongation steps. l-α-Difluoromethylornithine treatment resulting in polyamine depletion reduces protein synthesis via inhibition of translation initiation. N1-Guanyl-diaminoheptane (GC7), a spermidine analogue that inhibits eukaryotic initiation factor 5A (eIF5A) hypusination, also caused inhibition of translation initiation. In contrast, depletion of eIF5A by short hairpin RNA inhibits translation elongation as was recently demonstrated in yeast and Drosophila. These results suggest that in addition to competing with spermidine in the hypusination reaction, GC7 also competes with spermidine at yet undefined sites required for translation initiation. Finally, we show that either polyamine depletion or GC7 treatment induced eIF2α phosphorylation and reduced phosphorylation of 4E-BP, thus setting the molecular basis for the observed inhibition of translation initiation.

Overexpression of antizyme-inhibitor in NIH3T3 fibroblasts provides growth advantage through neutralization of antizyme functions

Antizyme inhibitor (AzI) is a homolog of ornithine decarboxylase (ODC), a key enzyme of polyamine synthesis. Antizyme inhibitor retains no enzymatic activity, but exhibits high affinity to antizyme (Az), a negative regulator of polyamine homeostasis. As polyamines are involved in maintaining cellular proliferation, and since AzI may negate Az functions, we have investigated the role of AzI in regulating cell growth. We show here that overexpression of AzI in NIH3T3 cells increased growth rate, enabled growth in low serum, and permitted anchorage-independent growth in soft agar, while reduction of AzI levels by AzI siRNA reduced cellular proliferation. Moreover, AzI overproducing cells gave rise to tumors when injected into nude mice. AzI overexpression resulted in elevation of ODC activity and of polyamine uptake. These effects of AzI are a result of its ability to neutralize Az, as overexpression of an AzI mutant with reduced Az binding failed to alter cellular polyamine metabolism and growth properties. We also demonstrate upregulation of AzI in Ras transformed cells, suggesting its relevance to some naturally occurring transformations. Finally, increased uptake activity rendered AzI overproducing and Ras-transformed cells more sensitive to toxic polyamine analogs. Our results therefore imply that AzI has a central and meaningful role in modulation of polyamine homeostasis, and in regulating cellular proliferation and transformation properties.

Mechanisms of ubiquitin-mediated, limited processingof the NF-κB1 precursor protein p105

Abstract In most cases, target proteins of the ubiquitin system are completely degraded. In several exceptions, such as the first step in the activation of the transcriptional regulator NF-κB, the substrate, the precursor protein p105, is processed in a limited manner to yield the active subunit p50. p50 is derived from the N-terminal domain of p105, whereas the C-terminal domain is degraded. The mechanisms involved in this unique process have remained elusive. We have shown that a Gly-rich region (GRR) at the C-terminal domain of p50 is one important processing signal and that it interferes with processing of the ubiquitinated precursor by the 26S proteasome. Also, amino acid residues 441–454 are important for processing under non-stimulated conditions. Lys 441 and 442 serve as ubiquitination targets, whereas residues 446–454 may serve as a ligase recognition motif. Following IκB kinase (IKK)-mediated phosphorylation, the C-terminal domain of p105, residues 918–934, recruits the SCFβ-TrCP ubiquitin ligase, and ubiquitination by this complex leads to accelerated processing. The two sites appear to be recognized under different physiological conditions by two different ligases, targeting two distinct recognition motifs. We have shown that ubiquitin conjugation and processing of a series of precursors of p105 that lack the C-terminal IKK phosphorylation/TrCP binding domain, is progressively inhibited with increasing number of ankyrin repeats. Inhibition is due to docking of active NF-κB subunits to the ankyrin repeat domain in the C-terminal half of p105 (IκBγ). Inhibition is alleviated by phosphorylation of the C-terminal domain that leads to ubiquitin-mediated degradation of the ankyrin repeat domain and release of the anchored subunits. We propose a model that may explain the requirement for two sites: a) a basal site that may be involved in co-translational processing prior to the synthesis of the ankyrin repeat domain; and b) a signal-induced site that is involved in processing/degradation of the complete molecule following cell activation, with rapid release of stored, transcriptionally active subunits.

Regulation of cellular polyamine levels and cellular proliferation by antizyme and antizyme inhibitor

Polyamines are small aliphatic polycations present in all living cells. Polyamines are essential for cellular viability and are involved in regulating fundamental cellular processes, most notably cellular growth and proliferation. Being such central regulators of fundamental cellular functions, the intracellular polyamine concentration is tightly regulated at the levels of synthesis, uptake, excretion and catabolism. ODC (ornithine decarboxylase) is the first key enzyme in the polyamine biosynthesis pathway. ODC is characterized by an extremely rapid intracellular turnover rate, a trait that is central to the regulation of cellular polyamine homoeostasis. The degradation rate of ODC is regulated by its end-products, the polyamines, via a unique autoregulatory circuit. At the centre of this circuit is a small protein called Az (antizyme), whose synthesis is stimulated by polyamines. Az inactivates ODC and targets it to ubiquitin-independent degradation by the 26S proteasome. In addition, Az inhibits uptake of polyamines. Az itself is regulated by another ODC-related protein termed AzI (antizyme inhibitor). AzI is highly homologous with ODC, but it lacks ornithine-decarboxylating activity. Its ability to serve as a regulator is based on its high affinity to Az, which is greater than the affinity Az has to ODC. As a result, it interferes with the binding of Az to ODC, thus rescuing ODC from degradation and permitting uptake of polyamines.