The deubiquitylase Ataxin-3 restricts PTEN transcription in lung cancer cells.

The phosphatidylinositol-3-kinase (PI3K) pathway is commonly hyperactivated in cancer. One mechanism by which this occurs is by silencing of the phosphatase and tensin homolog (PTEN), a tumor suppressor and major antagonist of the pathway, through genetic, epigenetic or posttranscriptional mechanisms. Here, we used an unbiased siRNA screen in non-small-cell lung cancer cells to identify deubiquitylases (DUBs) that have an impact on PI3K signaling by regulating the abundance of PTEN. We found that PTEN expression was induced by depleting any of three members of the Josephin family DUBs: ataxin 3 (ATXN3), ataxin 3-like (ATXN3L) and Josephin domain containing 1 (JOSD1). However, this effect is not mediated through altered PTEN protein stability. Instead, depletion of each DUB increases expression of both the PTEN transcript and its competing endogenous RNA, PTENP1. In ATXN3-depleted cells, under conditions of transcriptional inhibition, PTEN and PTENP1 mRNAs rapidly decay, suggesting that ATXN3 acts primarily by repressing their transcription. Importantly, the PTEN induction observed in response to ATXN3 siRNA is sufficient to downregulate Akt phosphorylation and hence PI3K signaling. Histone deacetylase inhibitors (HDACi) have been suggested as potential mediators of PTEN transcriptional reactivation in non-small-cell lung cancer. Although PTEN exhibits a very limited response to the broad-spectrum HDACi Vorinostat (SAHA) in A549 cells, we find that combination with ATXN3 depletion enhances PTEN induction in an additive manner. Similarly, these interventions additively decrease cell viability. Thus, ATXN3 provides an autonomous, complementary therapeutic target in cancers with epigenetic downregulation of PTEN.

Ras isoform abundance and signalling in human cancer cell lines.

The ubiquitously expressed major Ras isoforms: H-, K- and N-Ras, are highly conserved, yet exhibit different biological outputs. We have compared the relative efficiencies with which epidermal or hepatocyte growth factor activates Ras isoforms and the requirement for specific isoforms in the activation of downstream pathways. We find that the relative coupling efficiencies to each Ras isoform are conserved between stimuli. Furthermore, in both cases, inhibition of receptor endocytosis led to reduced N- and H-Ras activation, but K-Ras was unaffected. Acute knockdown of each isoform with siRNA allows endogenous Ras isoform function and abundance to be probed. This revealed that there is significant variation in the contribution of individual isoforms to total Ras across a panel of cancer cell lines although typically K> or =N>>H. Intriguingly, cancer cell lines where a significant fraction of endogenous Ras is oncogenically mutated showed attenuated activation of canonical Ras effector pathways. We profiled the contribution of each Ras isoform to the total Ras pool allowing interpretation of the effect of isoform-specific knockdown on signalling outcomes. In contrast to previous studies indicating preferential coupling of isoforms to Raf and PtdIns-3-kinase pathways, we find that endogenous Ras isoforms show no specific coupling to these major Ras pathways.

Control of growth factor receptor dynamics by reversible ubiquitination.

Activated tyrosine kinase receptors acquire ubiquitin tags. Ubiquitination governs receptor down-regulation through interaction with components of the endosomal ESCRT (endosomal sorting complexes required for transport) machinery that shepherds receptors into luminal vesicles of multivesicular bodies en route to the lysosome. We have characterized two de-ubiquitinating enzymes that interact with components of this machinery. AMSH [associated molecule with the SH3 domain (Src homology 3 domain) of STAM (signal transducing adapter molecule)] shows specificity for Lys63- over Lys48-linked ubiquitin and may act to rescue receptors from taking the lysosomal pathway. In contrast, UBPY (ubiquitin-specific processing protease Y) does not discriminate between Lys48 and Lys63-linked chains and is required for lysosomal sorting.

Met receptor dynamics and signalling.

The receptor for hepatocyte growth factor (HGF), Met, controls a programme of invasive growth that combines proliferation with various moto- and morphogenetic processes. This process is important for development and organ regeneration, but dysregulation in transformed tissues can contribute to cancer progression and metastasis. Acute stimulation of tissue culture cells with HGF leads to Met downregulation via degradation through an endocytic mechanism that also requires proteasome activity. Perturbation of Met trafficking on the endocytic pathway, either at the level of the internalisation step or during sorting at the early endosome, leads to altered signalling outputs. Ubiquitination of Met through the E3-ligase Cbl is required for receptor downregulation, and a mutant receptor defective in Cbl binding is able to transform cells. We discuss the hypothesis that some naturally occurring Met mutants implicated in cancer may transform cells owing to defects in their trafficking along the endosomal degradation pathway.

The interface of receptor trafficking and signalling.

The intimate relationship between receptor trafficking and signalling is beginning to reveal its secrets. Receptor endocytosis provides a mechanism for attenuation of signalling by transfer of receptors to degradative compartments. However, it can also determine signalling output by providing a different combination of downstream effectors at endocytic compartments compared with the plasma membrane. Rab5, Hrs and Cbl, are three examples of proteins that can influence both tyrosine kinase receptor trafficking and signalling pathways. By operating at this intersection, they are well placed to couple these aspects of cell function. Each element of the Rab5 GTPase cycle is influenced by signal transduction events, which will correspondingly influence recruitment of effector proteins and receptor distribution. Hrs and Cbl, which both undergo tyrosine phosphorylation in response to growth factor stimulation, are believed to influence receptor sorting in the early endosome and engage in multiple interactions, which may play a direct role in signalling cascades.

Characterization of MTMR3. an inositol lipid 3-phosphatase with novel substrate specificity.

Inositol lipids play key roles in many fundamental cellular processes that include growth, cell survival, motility, and membrane trafficking. Recent studies on the PTEN and Myotubularin proteins have underscored the importance of inositol lipid 3-phosphatases in cell function. Inactivating mutations in the genes encoding PTEN and Myotubularin are key steps in the progression of some cancers and in the onset of X-linked myotubular myopathy, respectively. Myotubularin-related protein 3 (MTMR3) shows extensive homology to Myotubularin, including the catalytic domain, but additionally possesses a C-terminal extension that includes a FYVE domain. We show that MTMR3 is an inositol lipid 3-phosphatase, with a so-far-unique substrate specificity. It is able to hydrolyze PtdIns3P and PtdIns3,5P2, both in vitro and when heterologously expressed in S. cerevisiae, and to thereby provide the first clearly defined route for the cellular production of PtdIns5P. Overexpression of a catalytically dead MTMR3 (C413S) in mammalian cells induces a striking formation of vacuolar compartments that enclose membranous structures that are highly concentrated in mutant proteins.

Down-regulation of MET, the receptor for hepatocyte growth factor.

The ligand-dependent degradation of activated tyrosine kinase receptors provides a means by which mitogenic signalling can be attenuated. In many cell types the ligand-dependent degradation of the tyrosine kinase receptor Met is completely dependent on the activity of the 26S proteasome (Jeffers et al., 1997b). We now show that degradation also requires trafficking to late endosomal compartments and the activity of acid dependent proteases as determined by the effects of a dominant negative form of dynamin (K44A) and a vacuolar-ATPase inhibitor, concanamycin. We show that in the presence of the proteasome inhibitor lactacystin, Met fails to redistribute from the plasma membrane to intracellular compartments. This observation is most consistent with the interpretation that proteasome activity is required for Met internalization and only indirectly for its degradation.

Relationships between EEA1 binding partners and their role in endosome fusion.

Homotypic fusion between early endosomes requires the phosphatidylinositol 3-phosphate (PI3P)-binding protein, Early Endosomal Autoantigen 1 (EEA1). We have investigated the role of other proteins that interact with EEA1 in the fusion of early endosomes derived from Baby Hamster Kidney (BHK) cells. We confirm a requirement for syntaxin 13, but additionally show that the assay is equally sensitive to reagents specifically targeted against syntaxin 6. Binding of EEA1 to immobilised GST-syntaxin 6 and 13 was directly compared; only syntaxin 6 formed a stable complex with EEA1. Early endosome fusion requires the release of intravesicular calcium, and calmodulin plays a vital role in the fusion pathway, as judged by sensitivity to antagonists. We demonstrate that both EEA1 and syntaxin 13 interact with calmodulin. In the case of EEA1, binding to calmodulin requires an IQ domain, which is adjacent to a C-terminal FYVE domain that specifically binds to PI3P. We have assessed the influence of protein binding partners on EEA1 interaction with PI3P and find that both calmodulin and rab5-GTP are antagonistic to PI3P binding, whilst syntaxins 6 and 13 have no effect. These studies reveal a complex network of interactions between the proteins required for endosome fusion.

Membrane transport: deciphering fusion.

Membrane fusion events that occur in yeast have been reconstituted with a minimal set of SNARE protein components. This system has been exploited to establish the syntax underlying specificity of intracellular fusion events from yeast to mammals.

Endosomal localization and receptor dynamics determine tyrosine phosphorylation of hepatocyte growth factor-regulated tyrosine kinase substrate.

Hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) is a prominent substrate for activated tyrosine kinase receptors that has been proposed to play a role in endosomal membrane trafficking. The protein contains a FYVE domain, which specifically binds to the lipid phosphatidylinositol (PI) 3-phosphate (PI 3-P). We show that this interaction is required both for correct localization of the protein to endosomes that only partially coincides with early endosomal autoantigen 1 and for efficient tyrosine phosphorylation of the protein in response to epidermal growth factor stimulation. Treatment with wortmannin reveals that Hrs phosphorylation also requires PI 3-kinase activity, which is necessary to generate the PI 3-P required for localization. We have used both hypertonic media and expression of a dominant-negative form of dynamin (K44A) to inhibit endocytosis; under which conditions, receptor stimulation fails to elicit phosphorylation of Hrs. Our results provide a clear example of the coupling of a signal transduction pathway to endocytosis, from which we propose that activated receptor (or associated factor) must be delivered to the appropriate endocytic compartment in order for Hrs phosphorylation to occur.

Detection of thiol modification following generation of reactive nitrogen species: analysis of synaptic vesicle proteins.

S-nitrosylation is an important means of regulating the activity of proteins. We have developed a method which allows unbiased identification of thiol modified proteins within a complex mixture following NO generation, by taking advantage of the fact that prior nitrosylation will block subsequent modification of cysteine residues with 1-biotinamido-4-[4'-(maleimidomethyl)-cyclohexane-carboxamid o] butane (biotin-BMCC). Thiol modified proteins are reduced in intensity when revealed by blotting and overlay with avidin-horseradish peroxidase. In the case of a purified synaptic vesicle fraction we observe a high degree of enrichment of specific biotinylated proteins relative to homogenate. We find that thiol modification of proteins in the presence of NO donors is widespread, occurring in the majority of proteins that will react with biotin-BMCC. In a further development of this technique we have depleted the biotinylated proteins from solubilised synaptic vesicles using avidin-agarose and analysed the supernatants with a panel of antibodies. This has allowed us to identify SNARE proteins (soluble NSF attachment protein receptors) as potential targets for S-nitrosylation.

Localization of a class II phosphatidylinositol 3-kinase, PI3KC2alpha, to clathrin-coated vesicles.

We have analysed phosphatidylinositol 3-kinase activity associated with subcellular fractions prepared from rat brains. Phosphatidylinositol 3-kinase activity is not markedly enriched with synaptic vesicle purification; whilst the activity associated with the most pure fractions is inhibited at low concentrations of wortmannin (IC50 approximately 4-5 nM). In contrast, clathrin-coated vesicle (CCV) fractions showed increased enzyme activity compared to light membrane fractions from which they are purified. In addition to a wortmannin-sensitive activity, we also detected an activity that could only be inhibited at higher concentrations of wortmannin (IC50 approximately 400 nM), characteristic of certain class II enzymes (including phosphatidylinositol 3-kinase C2alpha) to be highly enriched in CCV fractions. Immunoblotting with an antibody raised against phosphatidylinositol 3-kinase C2alpha, confirmed that this enzyme is highly enriched in CCVs and displays an enrichment profile during the purification that mirrors enrichment of the low nanomolar wortmannin-insensitive activity. If the CCV purification protocol is adapted to favour nerve terminally derived vesicles, we find reduced levels of the C2alpha enzyme in the CCV fractions, suggesting that the enzyme may principally reside on vesicles associated with the cell body.

Regulation of endosome fusion.

Homotypic fusion between early endosomes can be reconstituted in vitro. By using wortmannin and LY294002, inhibitors of phosphatidylinositol (Pl) 3-kinase, a requirement for this activity has been established in order for fusion to proceed efficiently. It has been shown that Pl 3-kinase activity is required downstream of rab5 activation, although a large excess of activated rab5 can overcome wortmannin inhibition. A series of experiments have also been performed which indicate a role for early endosomal autoantigen 1 (EEA1) in determining fusion efficiency. EEA1 dissociates from membranes following wortmannin treatment. It is proposed that the requirement of endosome fusion for Pl 3-kinase activity is to promote the association of EEA1 with endosomes.

Membrane transport: Take your fusion partners.

Recent studies of how vesicles are targeted to fuse with specific membranes inside cells highlight a role for extended coiled-coil proteins in tethering partner membranes prior to formation of the 'SNARE complex' that mediates the fusion reaction. The tethering protein is recruited to membranes by a Rab family GTPase

Molecular aspects of the endocytic pathway.

Observation of the flow of material along the endocytic pathway has lead to the description of the basic architecture of the pathway and provided insight into the relationship between compartments. Significant advances have been made in the study of endocytic transport steps at the molecular level, of which studies of cargo selection, vesicle budding and membrane fusion events comprise the major part. Progress in this area has been driven by two approaches, yeast genetics and in vitro or cell-free assays, which reconstitute particular transport steps and allow biochemical manipulation. The complex protein machineries that control vesicle budding and fusion are significantly conserved between the secretory and endocytic pathways such that proteins that regulate particular steps are often part of a larger family of proteins which exercise a conserved function at other locations within the cell. Well characterized examples include vesicle coat proteins, rabs (small GTPases) and soluble N-ethylmaleimide-sensitive fusion protein (NSF) attachment protein (SNAP) receptors (SNAREs). Intracompartmental pH, lipid composition and cytoskeletal organization have also been identified as important determinants of the orderly flow of material within the endocytic pathway.

Involvement of the endosomal autoantigen EEA1 in homotypic fusion of early endosomes.

In mammalian cells, fusion between early endocytic vesicles has been shown to require the ubiquitous intracellular fusion factors N-ethylmaleimide-sensitive factor (NSF) and alpha-SNAP, as well as a factor specific for early endosomes, the small GTPase Rab5 [1-3]. We have previously demonstrated an additional requirement for phosphatidylinositol 3-kinase (PI 3-kinase) activity [4]. The membrane association of early endosomal antigen 1 (EEA1), a specific marker of early endosomes [5,6], has recently been shown to be similarly dependent on PI 3-kinase activity [7], and we therefore postulated that it might be involved in endosome fusion. Here, we present evidence that EEA1 has an important role in determining the efficiency of endosome fusion in vitro. Both the carboxy-terminal domain of EEA1 (residues 1098-1411) and specific antibodies against EEA1 inhibited endosome fusion when included in an in vitro assay. Furthermore, depletion of EEA1, both from the membrane fraction used in the assay by washing with salt and from the cytosol using an EEA1-specific antibody, resulted in inhibition of endosome fusion. The involvement of EEA1 in endosome fusion accounts for the sensitivity of the endosome fusion assay to inhibitors of PI 3-kinase.

Inhibition of endosome fusion by wortmannin persists in the presence of activated Rab5.

Rab5-dependent endosome fusion is sensitive to the phosphoinositide 3-kinase inhibitor, wortmannin. It has been proposed that phosphoinositide 3-kinase activity may be required for activation of rab5 by influencing its nucleotide cycle such as to promote its active GTP state. In this report we demonstrate that endosome fusion remains sensitive to wortmannin despite preloading of endosomes with stimulatory levels of a GTPase-defective mutant rab5(Q79L) or of a xanthosine triphosphate-binding mutant, rab5(D136N), in the presence of the nonhydrolysable analogue XTPgammaS. These results suggest that activation of rab5 cannot be the principal function of the wortmannin-sensitive factor on the endosome fusion pathway. This result is extrapolated to all GTPases by demonstrating that endosome fusion remains wortmannin sensitive despite prior incubation with the nonhydrolysable nucleotide analogue GTPgammaS. Consistent with these results, direct measurement of clathrin-coated vesicle-stimulated nucleotide dissociation from exogenous rab5 was insensitive to the presence of wortmannin. A large excess of rab5(Q79L), beyond levels required for maximal stimulation of the fusion assay, afforded protection against wortmannin inhibition, and partial protection was also observed with an excess of wild-type rab5 independent of GTPgammaS.

Formation of N delta-cyanoornithine from NG-hydroxy-L-arginine and hydrogen peroxide by neuronal nitric oxide synthase: implications for mechanism.

Neuronal nitric oxide synthase (nNOS) catalyzes the oxidation of NG-hydroxy-L-arginine (NHA) by hydrogen peroxide. The amino acid products were characterized by high-performance liquid chromatography/mass spectrometry of the o-phthalaldehyde/2-mercaptoethanol derivatives and identified as citrulline and N delta-cyanoornithine (CN-orn). The assignment of CN-orn was confirmed by independent chemical synthesis and comparison of the properties of the enzyme-derived product with those of synthetic CN-orn. The inorganic products detected in the enzymatic reaction were NO2- and NO3-, presumably from oxidation of NO-. The reaction of H2O2 and NHA with nNOS was at least 10-fold slower than the reaction of NADPH, O2, and NHA (Vmax,app = 49 +/- 2 nmol min-1 mg-1 for the reactions with 10 microM added H4B). The reaction exhibited saturation kinetics with respect to hydrogen peroxide [K(m,app)(H2O2) = 10 +/- 1 mM for the reactions with 10 microM added H4B]. No H2O2-dependent reaction was observed with L-arginine as the amino acid substrate. The different products for the NADPH- and H2O2-dependent transformations of NHA are of mechanistic significance in the NOS reaction.

Meta-stability of the hemifusion intermediate induced by glycosylphosphatidylinositol-anchored influenza hemagglutinin.

Fusion between influenza virus and target membranes is mediated by the viral glycoprotein hemagglutinin (HA). Replacement of the transmembrane domain of HA with a glycosylphosphatidylinositol (GPI) membrane anchor allows lipid mixing but not the establishment of cytoplasmic continuity. This observation led to the proposal that the fusion mechanism passes through an intermediate stage corresponding to hemifusion between outer monolayers. We have used confocal fluorescence microscopy to study the movement of probes for specific bilayer leaflets of erythrocytes fusing with HA-expressing cells. N-Rh-PE and NBD-PC were used for specific labeling of the outer and inner membrane leaflet, respectively. In the case of GPI-HA-induced fusion, different behaviors of lipid transfer were observed, which include 1) exclusive movement of N-Rh-PE (hemifusion), 2) preferential movement of N-Rh-PE relative to NBD-PC, and 3) equal movement of both lipid analogs. The relative population of these intermediate states was dependent on the time after application of a low pH trigger for fusion. At early time points, hemifusion was more common and full redistribution of both bilayers was rare, whereas later full redistribution of both probes was frequently observed. In contrast to wild-type HA, the latter was not accompanied by mixing of the cytoplasmic marker Lucifer Yellow. We conclude that 1) the GPI-HA-mediated hemifusion intermediate is meta-stable and 2) expansion of an aqueous fusion pore requires the transmembrane and/or cytoplasmic domain of HA.