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NORMAL GENOMIC

• Haynie DT, Xue B
• Superdomains in the protein structure hierarchy: The case of PTP-C2.
• Protein Sci. 2015; 24: 874-82
• Display abstract

• Superdomain is uniquely defined in this work as a conserved combination of different globular domains in different proteins. The amino acid sequences of 25 structurally and functionally diverse proteins from fungi, plants, and animals have been analyzed in a test of the superdomain hypothesis. Each of the proteins contains a protein tyrosine phosphatase (PTP) domain followed by a C2 domain. Four novel conserved sequence motifs have been identified, one in the PTP domain and three in the C2 domain. All contribute to the PTP-C2 domain interface in PTEN, a tumor suppressor, and all are more conserved than the PTP signature motif, HCX3 (K/R)XR, in the 25 sequences. We show that PTP-C2 was formed prior to the fungi, plant, and animal kingdom divergence. A superdomain as defined here does not fit the usual protein structure classification system. The demonstrated existence of one superdomain suggests the existence of others.

• Ku PI, Bendjennat M, Ballew J, Landesman MB, Saffarian S
• ALIX is recruited temporarily into HIV-1 budding sites at the end of gag assembly.
• PLoS One. 2014; 9: 96950-96950
• Display abstract

• Polymerization of Gag on the inner leaflet of the plasma membrane drives the assembly of Human Immunodeficiency Virus 1 (HIV-1). Gag recruits components of the endosomal sorting complexes required for transport (ESCRT) to facilitate membrane fission and virion release. ESCRT assembly is initiated by recruitment of ALIX and TSG101/ESCRT-I, which bind directly to the viral Gag protein and then recruit the downstream ESCRT-III and VPS4 factors to complete the budding process. In contrast to previous models, we show that ALIX is recruited transiently at the end of Gag assembly, and that most ALIX molecules are recycled into the cytosol as the virus buds, although a subset remains within the virion. Our experiments imply that ALIX is recruited to the neck of the assembling virion and is mostly recycled after virion release.

• Sciskala B, Kolling R
• Interaction maps of the Saccharomyces cerevisiae ESCRT-III protein Snf7.
• Eukaryot Cell. 2013; 12: 1538-46
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• The Saccharomyces cerevisiae ESCRT-III protein Snf7 is part of an intricate interaction network at the endosomal membrane. Interaction maps of Snf7 were established by measuring the degree of binding of individual binding partners to putative binding motifs along the Snf7 sequence by glutathione S-transferase (GST) pulldown. For each interaction partner, distinct binding profiles were obtained. The following observations were made. The ESCRT-III subunits Vps20 and Vps24 showed a complementary binding pattern, suggesting a model for the series of events in the ESCRT-III functional cycle. Vps4 bound to individual Snf7 motifs but not to full-length Snf7. This suggests that Vps4 does not bind to the closed conformation of Snf7. We also demonstrate for the first time that the ALIX/Bro1 homologue Rim20 binds to the alpha6 helix of Snf7. Analysis of a Snf7 alpha6 deletion mutant showed that the alpha6 helix is crucial for binding of Bro1 and Rim20 in vivo and is indispensable for the multivesicular body (MVB)-sorting and Rim-signaling functions of Snf7. The Snf7Deltaalpha6 protein still appeared to be incorporated into ESCRT-III complexes at the endosomal membrane, but disassembly of the complex seemed to be defective. In summary, our study argues against the view that the ESCRT cycle is governed by single one-to-one interactions between individual components and emphasizes the network character of the ESCRT interactions.

• Pashkova N et al.
• The yeast Alix homolog Bro1 functions as a ubiquitin receptor for protein sorting into multivesicular endosomes.
• Dev Cell. 2013; 25: 520-33
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• Sorting of ubiquitinated membrane proteins into lumenal vesicles of multivesicular bodies is mediated by the Endosomal Sorting Complex Required for Transport (ESCRT) apparatus and accessory proteins such as Bro1, which recruits the deubiquitinating enzyme Doa4 to remove ubiquitin from cargo. Here we propose that Bro1 works as a receptor for the selective sorting of ubiquitinated cargoes. We found synthetic genetic interactions between BRO1 and ESCRT-0, suggesting that Bro1 functions similarly to ESCRT-0. Multiple structural approaches demonstrated that Bro1 binds ubiquitin via the N-terminal trihelical arm of its middle V domain. Mutants of Bro1 that lack the ability to bind Ub were dramatically impaired in their ability to sort Ub-cargo membrane proteins, but only when combined with hypomorphic alleles of ESCRT-0. These data suggest that Bro1 and other Bro1 family members function in parallel with ESCRT-0 to recognize and sort Ub-cargoes.

• Dores MR et al.
• ALIX binds a YPX(3)L motif of the GPCR PAR1 and mediates ubiquitin-independent ESCRT-III/MVB sorting.
• J Cell Biol. 2012; 197: 407-19
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• The sorting of signaling receptors to lysosomes is an essential regulatory process in mammalian cells. During degradation, receptors are modified with ubiquitin and sorted by endosomal sorting complex required for transport (ESCRT)-0, -I, -II, and -III complexes into intraluminal vesicles (ILVs) of multivesicular bodies (MVBs). However, it remains unclear whether a single universal mechanism mediates MVB sorting of all receptors. We previously showed that protease-activated receptor 1 (PAR1), a G protein-coupled receptor (GPCR) for thrombin, is internalized after activation and sorted to lysosomes independent of ubiquitination and the ubiquitin-binding ESCRT components hepatocyte growth factor-regulated tyrosine kinase substrate and Tsg101. In this paper, we report that PAR1 sorted to ILVs of MVBs through an ESCRT-III-dependent pathway independent of ubiquitination. We further demonstrate that ALIX, a charged MVB protein 4-ESCRT-III interacting protein, bound to a YPX(3)L motif of PAR1 via its central V domain to mediate lysosomal degradation. This study reveals a novel MVB/lysosomal sorting pathway for signaling receptors that bypasses the requirement for ubiquitination and ubiquitin-binding ESCRTs and may be applicable to a subset of GPCRs containing YPX(n)L motifs.

• Falke JJ
• Lipid targeting domain with dual-membrane specificity that expands the diversity of intracellular targeting reactions.
• Proc Natl Acad Sci U S A. 2012; 109: 1816-7

• Banuelos C et al.
• EhADH112 is a Bro1 domain-containing protein involved in the Entamoeba histolytica multivesicular bodies pathway.
• J Biomed Biotechnol. 2012; 2012: 657942-657942
• Display abstract

• EhADH112 is an Entamoeba histolytica Bro1 domain-containing protein, structurally related to mammalian ALIX and yeast BRO1, both involved in the Endosomal Sorting Complexes Required for Transport (ESCRT)-mediated multivesicular bodies (MVB) biogenesis. Here, we investigated an alternative role for EhADH112 in the MVB protein trafficking pathway by overexpressing 166 amino acids of its N-terminal Bro1 domain in trophozoites. Trophozoites displayed diminished phagocytosis rates and accumulated exogenous Bro1 at cytoplasmic vesicles which aggregated into aberrant complexes at late stages of phagocytosis, probably preventing EhADH112 function. Additionally, the existence of a putative E. histolytica ESCRT-III subunit (EhVps32) presumably interacting with EhADH112, led us to perform pull-down experiments with GST-EhVps32 and [(35)S]-labeled EhADH112 or EhADH112 derivatives, confirming EhVps32 binding to EhADH112 through its Bro1 domain. Our overall results define EhADH112 as a novel member of ESCRT-accessory proteins transiently present at cellular surface and endosomal compartments, probably contributing to MVB formation during phagocytosis.

• Pasquo A et al.
• Structural stability of human protein tyrosine phosphatase rho catalytic domain: effect of point mutations.
• PLoS One. 2012; 7: 32555-32555
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• Protein tyrosine phosphatase rho (PTPrho) belongs to the classical receptor type IIB family of protein tyrosine phosphatase, the most frequently mutated tyrosine phosphatase in human cancer. There are evidences to suggest that PTPrho may act as a tumor suppressor gene and dysregulation of Tyr phosphorylation can be observed in diverse diseases, such as diabetes, immune deficiencies and cancer. PTPrho variants in the catalytic domain have been identified in cancer tissues. These natural variants are nonsynonymous single nucleotide polymorphisms, variations of a single nucleotide occurring in the coding region and leading to amino acid substitutions. In this study we investigated the effect of amino acid substitution on the structural stability and on the activity of the membrane-proximal catalytic domain of PTPrho. We expressed and purified as soluble recombinant proteins some of the mutants of the membrane-proximal catalytic domain of PTPrho identified in colorectal cancer and in the single nucleotide polymorphisms database. The mutants show a decreased thermal and thermodynamic stability and decreased activation energy relative to phosphatase activity, when compared to wild- type. All the variants show three-state equilibrium unfolding transitions similar to that of the wild- type, with the accumulation of a folding intermediate populated at ~4.0 M urea.

• Bardens A, Doring T, Stieler J, Prange R
• Alix regulates egress of hepatitis B virus naked capsid particles in an ESCRT-independent manner.
• Cell Microbiol. 2011; 13: 602-19
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• Hepatitis B virus (HBV) is an enveloped DNA virus that exploits the endosomal sorting complexes required for transport (ESCRT) pathway for budding. In addition to infectious particles, HBV-replicating cells release non-enveloped (nucleo)capsids, but their functional implication and pathways of release are unclear. Here, we focused on the molecular mechanisms and found that the sole expression of the HBV core protein is sufficient for capsid release. Unexpectedly, released capsids are devoid of a detectable membrane bilayer, implicating a non-vesicular exocytosis process. Unlike virions, naked capsid budding does not require the ESCRT machinery. Rather, we identified Alix, a multifunctional protein with key roles in membrane biology, as a regulator of capsid budding. Ectopic overexpression of Alix enhanced capsid egress, while its depletion inhibited capsid release. Notably, the loss of Alix did not impair HBV production, furthermore indicating that virions and capsids use diverse export routes. By mapping of Alix domains responsible for its capsid release-mediating activity, its Bro1 domain was found to be required and sufficient. Alix binds to core via its Bro1 domain and retained its activity even if its ESCRT-III binding site is disrupted. Together, the boomerang-shaped Bro1 domain of Alix appears to escort capsids without ESCRT.

• Wemmer M, Azmi I, West M, Davies B, Katzmann D, Odorizzi G
• Bro1 binding to Snf7 regulates ESCRT-III membrane scission activity in yeast.
• J Cell Biol. 2011; 192: 295-306
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• Endosomal sorting complexes required for transport (ESCRTs) promote the invagination of vesicles into the lumen of endosomes, the budding of enveloped viruses, and the separation of cells during cytokinesis. These processes share a topologically similar membrane scission event facilitated by ESCRT-III assembly at the cytosolic surface of the membrane. The Snf7 subunit of ESCRT-III in yeast binds directly to an auxiliary protein, Bro1. Like ESCRT-III, Bro1 is required for the formation of intralumenal vesicles at endosomes, but its role in membrane scission is unknown. We show that overexpression of Bro1 or its N-terminal Bro1 domain that binds Snf7 enhances the stability of ESCRT-III by inhibiting Vps4-mediated disassembly in vivo and in vitro. This stabilization effect correlates with a reduced frequency in the detachment of intralumenal vesicles as observed by electron tomography, implicating Bro1 as a regulator of ESCRT-III disassembly and membrane scission activity.

• Shi Y, Stefan CJ, Rue SM, Teis D, Emr SD
• Two novel WD40 domain-containing proteins, Ere1 and Ere2, function in the retromer-mediated endosomal recycling pathway.
• Mol Biol Cell. 2011; 22: 4093-107
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• Regulated secretion, nutrient uptake, and responses to extracellular signals depend on cell-surface proteins that are internalized and recycled back to the plasma membrane. However, the underlying mechanisms that govern membrane protein recycling to the cell surface are not fully known. Using a chemical-genetic screen in yeast, we show that the arginine transporter Can1 is recycled back to the cell surface via two independent pathways mediated by the sorting nexins Snx4/41/42 and the retromer complex, respectively. In addition, we identify two novel WD40-domain endosomal recycling proteins, Ere1 and Ere2, that function in the retromer pathway. Ere1 is required for Can1 recycling via the retromer-mediated pathway, but it is not required for the transport of other retromer cargoes, such as Vps10 and Ftr1. Biochemical studies reveal that Ere1 physically interacts with internalized Can1. Ere2 is present in a complex containing Ere1 on endosomes and functions as a regulator of Ere1. Taken together, our results suggest that Snx4/41/42 and the retromer comprise two independent pathways for the recycling of internalized cell-surface proteins. Moreover, a complex containing the two novel proteins Ere1 and Ere2 mediates cargo-specific recognition by the retromer pathway.

• Lopez-Reyes I et al.
• Detection of the Endosomal Sorting Complex Required for Transport in Entamoeba histolytica and Characterization of the EhVps4 Protein.
• J Biomed Biotechnol. 2010; 2010: 890674-890674
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• Eukaryotic endocytosis involves multivesicular bodies formation, which is driven by endosomal sorting complexes required for transport (ESCRT). Here, we showed the presence and expression of homologous ESCRT genes in Entamoeba histolytica. We cloned and expressed the Ehvps4 gene, an ESCRT member, to obtain the recombinant EhVps4 and generate specific antibodies, which immunodetected EhVps4 in cytoplasm of trophozoites. Bioinformatics and biochemical studies evidenced that rEhVps4 is an ATPase, whose activity depends on the conserved E211 residue. Next, we generated trophozoites overexpressing EhVps4 and mutant EhVps4-E211Q FLAG-tagged proteins. The EhVps4-FLAG was located in cytosol and at plasma membrane, whereas the EhVps4-E211Q-FLAG was detected as abundant cytoplasmic dots in trophozoites. Erythrophagocytosis, cytopathic activity, and hepatic damage in hamsters were not improved in trophozoites overexpressing EhVps4-FLAG. In contrast, EhVps4-E211Q-FLAG protein overexpression impaired these properties. The localization of EhVps4-FLAG around ingested erythrocytes, together with our previous results, strengthens the role for EhVps4 in E. histolytica phagocytosis and virulence.

• Dilley KA, Gregory D, Johnson MC, Vogt VM
• An LYPSL late domain in the gag protein contributes to the efficient release and replication of Rous sarcoma virus.
• J Virol. 2010; 84: 6276-87
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• The efficient release of newly assembled retrovirus particles from the plasma membrane requires the recruitment of a network of cellular proteins (ESCRT machinery) normally involved in the biogenesis of multivesicular bodies and in cytokinesis. Retroviruses and other enveloped viruses recruit the ESCRT machinery through three classes of short amino acid consensus sequences termed late domains: PT/SAP, PPXY, and LYPX(n)L. The major late domain of Rous sarcoma virus (RSV) has been mapped to a PPPY motif in Gag that binds members of the Nedd4 family of ubiquitin ligases. RSV Gag also contains a second putative late domain motif, LYPSL, positioned 5 amino acids downstream of PPPY. LYPX(n)L motifs have been shown to support budding in other retroviruses by binding the ESCRT adaptor protein Alix. To investigate a possible role of the LYPSL motif in RSV budding, we constructed PPPY and LYPSL mutants in the context of an infectious virus and then analyzed the budding rates, spreading profiles, and budding morphology. The data imply that the LYPSL motif acts as a secondary late domain and that its role in budding is amplified in the absence of a fully functional PPPY motif. The LYPXL motif proved to be a stronger late domain when an aspartic acid was substituted for the native serine, recapitulating the properties of the LYPDL late domain of equine infectious anemia virus. The overexpression of human Alix in the absence of a fully functional PPPY late domain partially rescued both the viral budding rate and viral replication, supporting a model in which the RSV LYPSL motif mediates budding through an interaction with the ESCRT adaptor protein Alix.

• Wolf JM, Davis DA
• Mutational analysis of Candida albicans SNF7 reveals genetically separable Rim101 and ESCRT functions and demonstrates divergence in bro1-domain protein interactions.
• Genetics. 2010; 184: 673-94
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• The opportunistic pathogen Candida albicans can grow over a wide pH range, which is associated with its ability to colonize and infect distinct host niches. C. albicans growth in neutral-alkaline environments requires proteolytic activation of the transcription factor Rim101. Rim101 activation requires Snf7, a member of the endosomal sorting complex required for transport (ESCRT) pathway. We hypothesized that Snf7 has distinct functions in the Rim101 and ESCRT pathways, which we tested by alanine-scanning mutagenesis. While some snf7 alleles conferred no defects, we identified alleles with solely ESCRT-dependent, solely Rim101-dependent, or both Rim101- and ESCRT-dependent defects. Thus, Snf7 function in these two pathways is at least partially separable. Both Rim101- and ESCRT-dependent functions require Snf7 recruitment to the endosomal membrane and alleles that disrupted both pathways were found to localize normally, suggesting a downstream defect. Most alleles that conferred solely Rim101-dependent defects were still able to process Rim101 normally under steady-state conditions. However, these same strains did display a kinetic defect in Rim101 processing. Several alleles with solely Rim101-dependent defects mapped to the C-terminal end of Snf7. Further analyses suggested that these mutations disrupted interactions with bro-domain proteins, Rim20 and Bro1, in overlapping but slightly divergent Snf7 domains.

• Pan S et al.
• Extracellular Alix regulates integrin-mediated cell adhesions and extracellular matrix assembly.
• EMBO J. 2008; 27: 2077-90
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• Alix (ALG-2-interacting protein X), a cytoplasmic adaptor protein involved in endosomal sorting and actin cytoskeleton assembly, is required for the maintenance of fibroblast morphology. As Alix has sequence similarity to adhesin in Entamoeba histolytica, and we observed that Alix is secreted, we determined whether extracellular Alix affects fibroblast morphology. Here, we demonstrate that secreted Alix is deposited on the substratum of non-immortalized WI38 fibroblasts. Antibody binding to extracellular Alix retards WI38 cell adhesion and spreading on fibronectin and vitronectin. Alix knockdown in WI38 cells reduces spreading and fibronectin assembly, and the effect is partially complemented by coating recombinant Alix on the cell substratum. Immortalized NIH/3T3 fibroblasts deposit less Alix on the substratum and have defects in alpha5beta1-integrin functions. Coating recombinant Alix on the culture substratum for NIH/3T3 cells promotes alpha5beta1-integrin-mediated cell adhesions and fibronectin assembly, and these effects require the aa 605-709 region of Alix. These findings demonstrate that a sub-population of Alix localizes extracellularly and regulates integrin-mediated cell adhesions and fibronectin matrix assembly.

• Collins BM et al.
• Structure of Vps26B and mapping of its interaction with the retromer protein complex.
• Traffic. 2008; 9: 366-79
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• Retromer is a heteromeric protein complex with important roles in endosomal membrane trafficking, most notably in the retrograde transport of lysosomal hydrolase receptors from endosomes to the Golgi. The core of retromer is composed of three subunits vacuolar protein sorting (Vps)35, Vps26 and Vps29, and in mammals, there are two paralogues of the medium subunit Vps26A and Vps26B. We find that both Vps26A and Vps26B bind to Vps35/Vps29 with nanomolar affinity and compete for a single-binding site to define distinct retromer complexes in vitro and in vivo. We have determined the crystal structure of mouse Vps26B and compare this structure with that of Vps26A. Vps26 proteins have a striking similarity to the arrestin family of proteins that regulate the signalling and endocytosis of G-protein-coupled receptors, although we observe that surface residues involved in arrestin function are not conserved in Vps26. Using structure-based mutagenesis, we show that both Vps26A and Vps26B are incorporated into retromer complexes through binding of Vps35 to a highly conserved surface patch within the C-terminal subdomain and that this interaction is required for endosomal recruitment of the proteins.

• Lakkaraju A, Rodriguez-Boulan E
• Itinerant exosomes: emerging roles in cell and tissue polarity.
• Trends Cell Biol. 2008; 18: 199-209
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• Cells use secreted signals (e.g. chemokines and growth factors) and sophisticated vehicles such as argosomes, cytonemes, tunneling nanotubes and exosomes to relay important information to other cells, often over large distances. Exosomes, 30-100-nm intraluminal vesicles of multivesicular bodies (MVB) released upon exocytic fusion of the MVB with the plasma membrane, are increasingly recognized as a novel mode of cell-independent communication. Exosomes have been shown to function in antigen presentation and tumor metastasis, and in transmitting infectious agents. However, little is known about the biogenesis and function of exosomes in polarized cells. In this review, we discuss new evidence suggesting that exosomes participate in the transport of morphogens and RNA, and thus influence cell polarity and developmental patterning of tissues.

• Rue SM, Mattei S, Saksena S, Emr SD
• Novel Ist1-Did2 complex functions at a late step in multivesicular body sorting.
• Mol Biol Cell. 2008; 19: 475-84
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• In Saccharomyces cerevisiae, integral plasma membrane proteins destined for degradation and certain vacuolar membrane proteins are sorted into the lumen of the vacuole via the multivesicular body (MVB) sorting pathway, which depends on the sequential action of three endosomal sorting complexes required for transport. Here, we report the characterization of a new positive modulator of MVB sorting, Ist1. We show that endosomal recruitment of Ist1 depends on ESCRT-III. Deletion of IST1 alone does not cause cargo-sorting defects. However, synthetic genetic analysis of double mutants of IST1 and positive modulators of MVB sorting showed that ist1Delta is synthetic with vta1Delta and vps60Delta, indicating that Ist1 is also a positive component of the MVB-sorting pathway. Moreover, this approach revealed that Ist1-Did2 and Vta1-Vps60 compose two functional units. Ist1-Did2 and Vta1-Vps60 form specific physical complexes, and, like Did2 and Vta1, Ist1 binds to the AAA-ATPase Vps4. We provide evidence that the ist1Delta mutation exhibits a synthetic interaction with mutations in VPS2 (DID4) that compromise the Vps2-Vps4 interaction. We propose a model in which the Ist1-Did2 and Vta1-Vps60 complexes independently modulate late steps in the MVB-sorting pathway.

• Yokota T et al.
• Crystal structure of human dual specificity phosphatase, JNK stimulatory phosphatase-1, at 1.5 A resolution.
• Proteins. 2007; 66: 272-8
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• Human JNK stimulatory phosphatase-1 (JSP-1) is a novel member of dual specificity phosphatases. A C-terminus truncated JSP-1 was expressed in Escherichia coli and was crystallized using the sitting-drop vapor diffusion method. Thin-plate crystals obtained at 278 K belong to a monoclinic space group, C2, with unit-cell parameters a = 84.0 A, b = 49.3 A, c = 47.3 A, and beta = 119.5 degrees , and diffract up to 1.5 A resolution at 100 K. The structure of JSP-1 has a single compact (alpha/beta) domain, which consists of six alpha-helices and five beta-strands, and shows a conserved structural scaffold in regard to both DSPs and PTPs. A cleft formed by a PTP-loop at the active site is very shallow, and is occupied by one sulfonate compound, MES, at the bottom. In the binary complex structure of JSP-1 with MES, the conformations of three important segments in regard to the catalytic mechanism are not similar to those in PTP1B. JSP-1 has no loop corresponding to the Lys120-loop of PTP1B, and tryptophan residue corresponding to the substrate-stacking in PTP1B is substituted by alanine residue in JSP-1.

• Shi A, Pant S, Balklava Z, Chen CC, Figueroa V, Grant BD
• A novel requirement for C. elegans Alix/ALX-1 in RME-1-mediated membrane transport.
• Curr Biol. 2007; 17: 1913-24
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• BACKGROUND: Alix/Bro1p family proteins have recently been identified as important components of multivesicular endosomes (MVEs) and are involved in the sorting of endocytosed integral membrane proteins, interacting with components of the ESCRT complex, the unconventional phospholipid LBPA, and other known endocytosis regulators. During infection, Alix can be co-opted by enveloped retroviruses, including HIV, providing an important function during virus budding from the plasma membrane. In addition, Alix is associated with the actin cytoskeleton and might regulate cytoskeletal dynamics. RESULTS: Here we demonstrate a novel physical interaction between the only apparent Alix/Bro1p family protein in C. elegans, ALX-1, and a key regulator of receptor recycling from endosomes to the plasma membrane, called RME-1. The analysis of alx-1 mutants indicates that ALX-1 is required for the endocytic recycling of specific basolateral cargo in the C. elegans intestine, a pathway previously defined by the analysis of rme-1 mutants. The expression of truncated human Alix in HeLa cells disrupts the recycling of major histocompatibility complex class I, a known Ehd1/RME-1-dependent transport step, suggesting the phylogenetic conservation of this function. We show that the interaction of ALX-1 with RME-1 in C. elegans, mediated by RME-1/YPSL and ALX-1/NPF motifs, is required for this recycling process. In the C. elegans intestine, ALX-1 localizes to both recycling endosomes and MVEs, but the ALX-1/RME-1 interaction appears to be dispensable for ALX-1 function in MVEs and/or late endosomes. CONCLUSIONS: This work provides the first demonstration of a requirement for an Alix/Bro1p family member in the endocytic recycling pathway in association with the recycling regulator RME-1.

• Dejournett RE et al.
• Phosphorylation of the proline-rich domain of Xp95 modulates Xp95 interaction with partner proteins.
• Biochem J. 2007; 401: 521-31
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• The mammalian adaptor protein Alix [ALG-2 (apoptosis-linked-gene-2 product)-interacting protein X] belongs to a conserved family of proteins that have in common an N-terminal Bro1 domain and a C-terminal PRD (proline-rich domain), both of which mediate partner protein interactions. Following our previous finding that Xp95, the Xenopus orthologue of Alix, undergoes a phosphorylation-dependent gel mobility shift during progesteroneinduced oocyte meiotic maturation, we explored potential regulation of Xp95/Alix by protein phosphorylation in hormone-induced cell cycle re-entry or M-phase induction. By MALDI-TOF (matrix-assisted laser-desorption ionization-time-of-flight) MS analyses and gel mobility-shift assays, Xp95 is phosphorylated at multiple sites within the N-terminal half of the PRD during Xenopus oocyte maturation, and a similar region in Alix is phosphorylated in mitotically arrested but not serum-stimulated mammalian cells. By tandem MS, Thr745 within this region, which localizes in a conserved binding site to the adaptor protein SETA [SH3 (Src homology 3) domain-containing, expressed in tumorigenic astrocytes] CIN85 (a-cyano-4-hydroxycinnamate)/SH3KBP1 (SH3-domain kinase-binding protein 1), is one of the phosphorylation sites in Xp95. Results from GST (glutathione S-transferase)-pull down and peptide binding/competition assays further demonstrate that the Thr745 phosphorylation inhibits Xp95 interaction with the second SH3 domain of SETA. However, immunoprecipitates of Xp95 from extracts of M-phase-arrested mature oocytes contained additional partner proteins as compared with immunoprecipitates from extracts of G2-arrested immature oocytes. The deubiquitinase AMSH (associated molecule with the SH3 domain of signal transducing adaptor molecule) specifically interacts with phosphorylated Xp95 in M-phase cell lysates. These findings establish that Xp95/Alix is phosphorylated within the PRD during M-phase induction, and indicate that the phosphorylation may both positively and negatively modulate their interaction with partner proteins.

• Liu Y, Maine EM
• The Bro1-domain protein, EGO-2, promotes Notch signaling in Caenorhabditis elegans.
• Genetics. 2007; 176: 2265-77
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• In Caenorhabditis elegans, as in other animals, Notch-type signaling mediates numerous inductive events during development. The mechanism of Notch-type signaling involves proteolytic cleavage of the receptor and subsequent transport of the receptor intracellular domain to the nucleus, where it acts as a transcriptional regulator. Notch-type signaling activity is modulated by post-translational modifications and endocytosis of ligand and receptor. We previously identified the ego-2 (enhancer of glp-1) gene as a positive regulator of germline proliferation that interacts genetically with the GLP-1/Notch signaling pathway in the germline. Here, we show that ego-2 positively regulates signaling in various tissues via both GLP-1 and the second C. elegans Notch-type receptor, LIN-12. ego-2 activity also promotes aspects of development not known to require GLP-1 or LIN-12. The EGO-2 protein contains a Bro1 domain, which is known in other systems to localize to certain endosomal compartments. EGO-2 activity in the soma promotes GLP-1 signaling in the germline, consistent with a role for EGO-2 in production of active ligand. Another C. elegans Bro1-domain protein, ALX-1, is known to interact physically with LIN-12/Notch. We document a complex phenotypic interaction between ego-2 and alx-1, consistent with their relationship being antagonistic with respect to some developmental processes and agonistic with respect to others.

• Usami Y, Popov S, Gottlinger HG
• Potent rescue of human immunodeficiency virus type 1 late domain mutants by ALIX/AIP1 depends on its CHMP4 binding site.
• J Virol. 2007; 81: 6614-22
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• The release of human immunodeficiency virus type 1 (HIV-1) and of other retroviruses from certain cells requires the presence of distinct regions in Gag that have been termed late assembly (L) domains. HIV-1 harbors a PTAP-type L domain in the p6 region of Gag that engages an endosomal budding machinery through Tsg101. In addition, an auxiliary L domain near the C terminus of p6 binds to ALIX/AIP1, which functions in the same endosomal sorting pathway as Tsg101. In the present study, we show that the profound release defect of HIV-1 L domain mutants can be completely rescued by increasing the cellular expression levels of ALIX and that this rescue depends on an intact ALIX binding site in p6. Furthermore, the ability of ALIX to rescue viral budding in this system depended on two putative surface-exposed hydrophobic patches on its N-terminal Bro1 domain. One of these patches mediates the interaction between ALIX and the ESCRT-III component CHMP4B, and mutations which disrupt the interaction also abolish the activity of ALIX in viral budding. The ability of ALIX to rescue a PTAP mutant also depends on its C-terminal proline-rich domain (PRD), but not on the binding sites for Tsg101, endophilin, CIN85, or for the newly identified binding partner, CMS, within the PRD. Our data establish that ALIX can have a dramatic effect on HIV-1 release and suggest that the ability to use ALIX may allow HIV-1 to replicate in cells that express only low levels of Tsg101.

• Gill DJ et al.
• Structural insight into the ESCRT-I/-II link and its role in MVB trafficking.
• EMBO J. 2007; 26: 600-12
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• ESCRT (endosomal sorting complex required for transport) complexes orchestrate efficient sorting of ubiquitinated transmembrane receptors to lysosomes via multivesicular bodies (MVBs). Yeast ESCRT-I and ESCRT-II interact directly in vitro; however, this association is not detected in yeast cytosol. To gain understanding of the molecular mechanisms of this link, we have characterised the ESCRT-I/-II supercomplex and determined the crystal structure of its interface. The link is formed by the vacuolar protein sorting (Vps)28 C-terminus (ESCRT-I) binding with nanomolar affinity to the Vps36-NZF-N zinc-finger domain (ESCRT-II). A hydrophobic patch on the Vps28-CT four-helix bundle contacts the hydrophobic knuckles of Vps36-NZF-N. Mutation of the ESCRT-I/-II link results in a cargo-sorting defect in yeast. Interestingly, the two Vps36 NZF domains, NZF-N and NZF-C, despite having the same core fold, use distinct surfaces to bind ESCRT-I or ubiquitinated cargo. We also show that a new component of ESCRT-I, Mvb12 (YGR206W), engages ESCRT-I directly with nanomolar affinity to form a 1:1:1:1 heterotetramer. Mvb12 does not affect the affinity of ESCRT-I for ESCRT-II in vitro. Our data suggest a complex regulatory mechanism for the ESCRT-I/-II link in yeast.

• Nikko E, Andre B
• Split-ubiquitin two-hybrid assay to analyze protein-protein interactions at the endosome: application to Saccharomyces cerevisiae Bro1 interacting with ESCRT complexes, the Doa4 ubiquitin hydrolase, and the Rsp5 ubiquitin ligase.
• Eukaryot Cell. 2007; 6: 1266-77
• Display abstract

• Targeting of membrane proteins into the lysosomal/vacuolar lumen for degradation requires their prior sorting into multivesicular bodies (MVB). The MVB sorting pathway depends on ESCRT-0, -I, -II, and -III protein complexes functioning on the endosomal membrane and on additional factors, such as Bro1/Alix and the ubiquitin ligase Rsp5/Nedd4. We used the split-ubiquitin two-hybrid assay to analyze the interaction partners of yeast Bro1 at its natural cellular location. We show that Bro1 interacts with ESCRT-I and -III components, including Vps23, the Saccharomyces cerevisiae homologue of human Tsg101. These interactions do not require the C-terminal proline-rich domain (PRD) of Bro1. Rather, this PRD interacts with the Doa4 deubiquitinating enzyme to recruit it to the endosome. This interaction is disrupted by a single amino acid substitution in the conserved ELC box motif in Doa4. The PRD of Bro1 also mediates an association with Rsp5, and this interaction appears to be conserved, as Alix, the human homologue of Bro1, coimmunoprecipitates with Nedd4 in yeast lysates. We further show that the Bro1 PRD domain is essential to MVB sorting of only cargo proteins whose sorting to the vacuolar lumen is dependent on their own ubiquitination and Doa4. The Bro1 region preceding the PRD, however, is required for MVB sorting of proteins irrespective of whether their targeting to the vacuole is dependent on their ubiquitination and Doa4. Our data indicate that Bro1 interacts with several ESCRT components and contributes via its PRD to associating ubiquitinating and deubiquitinating enzymes with the MVB sorting machinery.

• Horii M et al.
• CHMP7, a novel ESCRT-III-related protein, associates with CHMP4b and functions in the endosomal sorting pathway.
• Biochem J. 2006; 400: 23-32
• Display abstract

• All CHMPs (charged multivesicular body proteins) reported to date have common features: they all contain approx. 200 amino acid residues, have coiled-coil regions and have a biased distribution of charged residues (basic N-terminal and acidic C-terminal halves). Yeast orthologues of CHMPs, including an ESCRT-III component Snf7, are required for the sorting of cargo proteins to intraluminal vesicles of multivesicular bodies. We have characterized a novel human ESCRT-III-related protein, designated CHMP7, which consists of 453 amino acid residues. CHMP7 contains an SNF7 domain and a distantly SNF7-related domain in its C-terminal half and N-terminal half respectively. Among the ten CHMP proteins classified previously in six subfamilies (CHMP1-CHMP6), the C-terminal SNF7 domain of CHMP7 is most similar to the SNF7 domain of CHMP6, which associates with CHMP4 proteins and EAP20, a component of ESCRT-II. Pull-down assays using lysates of HEK-293T (human embryonic kidney) cells that overexpressed Strep-tagged CHMP7 and GFP (green fluorescent protein)-fused CHMP4b (also named Shax1) revealed a positive interaction between the C-terminal half of CHMP7 and CHMP4b. However, interaction was not observed between CHMP7 and EAP20. Confocal fluorescence microscopic analyses revealed that FLAG-CHMP7 is distributed in HeLa cells diffusely throughout the cytoplasm, but with some accumulation, especially in the perinuclear area. The distribution of FLAG-CHMP7 was altered to a cytoplasmic punctate pattern by overexpression of either CHMP4b-GFP or GFP-Vps4B(E235Q), a dominant-negative mutant of the AAA (ATPase associated with various cellular activities) Vps4B, and partially co-localized with them. Ubiquitinated proteins and endocytosed EGF accumulated in GFP-CHMP7-expressing cells. A dominant-negative effect of overexpressed GFP-CHMP7 was also observed in the release of virus-like particles from HEK-293T cells that transiently expressed the MLV (murine leukaemia virus) Gag protein. These results suggest that CHMP7, a novel CHMP4-associated ESCRT-III-related protein, functions in the endosomal sorting pathway.

• Amerik A, Sindhi N, Hochstrasser M
• A conserved late endosome-targeting signal required for Doa4 deubiquitylating enzyme function.
• J Cell Biol. 2006; 175: 825-35
• Display abstract

• Enzyme specificity in vivo is often controlled by subcellular localization. Yeast Doa4, a deubiquitylating enzyme (DUB), removes ubiquitin from membrane proteins destined for vacuolar degradation. Doa4 is recruited to the late endosome after ESCRT-III (endosomal sorting complex required for transport III) has assembled there. We show that an N-terminal segment of Doa4 is sufficient for endosome association. This domain bears four conserved elements (boxes A-D). Deletion of the most conserved of these, A or B, prevents Doa4 endosomal localization. These mutants cannot sustain ubiquitin-dependent proteolysis even though neither motif is essential for deubiquitylating activity. Ubiquitin-specific processing protease 5 (Ubp5), the closest paralogue of Doa4, has no functional overlap. Ubp5 concentrates at the bud neck; its N-terminal domain is critical for this. Importantly, substitution of the Ubp5 N-terminal domain with that of Doa4 relocalizes the Ubp5 enzyme to endosomes and provides Doa4 function. This is the first demonstration of a physiologically important DUB subcellular localization signal and provides a striking example of the functional diversification of DUB paralogues by the evolution of alternative spatial signals.

• Teo H et al.
• ESCRT-I core and ESCRT-II GLUE domain structures reveal role for GLUE in linking to ESCRT-I and membranes.
• Cell. 2006; 125: 99-111
• Display abstract

• ESCRT complexes form the main machinery driving protein sorting from endosomes to lysosomes. Currently, the picture regarding assembly of ESCRTs on endosomes is incomplete. The structure of the conserved heterotrimeric ESCRT-I core presented here shows a fan-like arrangement of three helical hairpins, each corresponding to a different subunit. Vps23/Tsg101 is the central hairpin sandwiched between the other subunits, explaining the critical role of its "steadiness box" in the stability of ESCRT-I. We show that yeast ESCRT-I links directly to ESCRT-II, through a tight interaction of Vps28 (ESCRT-I) with the yeast-specific zinc-finger insertion within the GLUE domain of Vps36 (ESCRT-II). The crystal structure of the GLUE domain missing this insertion reveals it is a split PH domain, with a noncanonical lipid binding pocket that binds PtdIns3P. The simultaneous and reinforcing interactions of ESCRT-II GLUE domain with membranes, ESCRT-I, and ubiquitin are critical for ubiquitinated cargo progression from early to late endosomes.

• Motiwala T, Jacob ST
• Role of protein tyrosine phosphatases in cancer.
• Prog Nucleic Acid Res Mol Biol. 2006; 81: 297-329

• Mahul-Mellier AL, Hemming FJ, Blot B, Fraboulet S, Sadoul R
• Alix, making a link between apoptosis-linked gene-2, the endosomal sorting complexes required for transport, and neuronal death in vivo.
• J Neurosci. 2006; 26: 542-9
• Display abstract

• Alix/apoptosis-linked gene-2 (ALG-2)-interacting protein X is an adaptor protein involved in the regulation of the endolysosomal system through binding to endophilins and to endosomal sorting complexes required for transport (ESCRT) proteins, TSG101 and CHMP4b. It was first characterized as an interactor of ALG-2, a calcium-binding protein necessary for cell death, and several observations suggest a role for Alix in controlling cell death. We used electroporation in the chick embryo to test whether overexpressed wild-type or mutated Alix proteins influence cell death in vivo. We show that Alix overexpression is sufficient to induce cell death of neuroepithelial cells. This effect is strictly dependent on its capacity to bind to ALG-2. On the other hand, expression of Alix mutants lacking the ALG-2 or the CHMP4b binding sites prevents early programmed cell death in cervical motoneurons at day 4.5 of chick embryo development. This protection afforded by Alix mutants was abolished after deletion of the TSG101, but not of the endophilin, binding sites. Our results suggest that the interaction of the ALG-2/Alix complex with ESCRT proteins is necessary for naturally occurring death of motoneurons. Therefore, Alix represents a molecular link between the endolysosomal system and the cell death machinery.

• Blanchin-Roland S, Da Costa G, Gaillardin C
• ESCRT-I components of the endocytic machinery are required for Rim101-dependent ambient pH regulation in the yeast Yarrowia lipolytica.
• Microbiology. 2005; 151: 3627-37
• Display abstract

• Ambient pH signalling involves a cascade of conserved Rim or Pal products in ascomycetous yeasts or filamentous fungi, respectively. Insertional mutagenesis in the yeast Yarrowia lipolytica identified two components of the endosome-associated ESCRT-I complex involved in multivesicular body (MVB) vesicle formation, YlVps28p and YlVps23p. They were shown to be required at alkaline pH, like Rim factors, for transcriptional activation of alkaline-induced genes and repression of acid-induced genes. The constitutively active YlRIM101-1119 allele, which suppresses the pH-signalling defects of Ylrim mutations, also suppresses Ylvps defects in pH response, but not in endocytosis. The contribution of the ESCRT-III component Snf7p could not be assessed due to the essential nature of this component in Y. lipolytica. Unlike Rim factors, YlVps4p, a component of the MVB pathway acting downstream from ESCRT complexes, seems not to be required for the alkaline response. In Y. lipolytica, all vps mutations including those affecting YlVPS4, affected growth at acidic pH, a feature not exhibited by Ylrim mutations. These results suggest that Rim and Vps pathways cooperate in ambient pH signalling and that this relation is conserved across the full range of hemiascomycetous yeasts.

• Slagsvold T et al.
• Eap45 in mammalian ESCRT-II binds ubiquitin via a phosphoinositide-interacting GLUE domain.
• J Biol Chem. 2005; 280: 19600-6
• Display abstract

• Ubiquitination serves as a key sorting signal in the lysosomal degradation of endocytosed receptors through the ability of ubiquitinated membrane proteins to be recognized and sorted by ubiquitin-binding proteins along the endocytic route. The ESCRT-II complex in yeast contains one such protein, Vps36, which harbors a ubiquitin-binding NZF domain and is required for vacuolar sorting of ubiquitinated membrane proteins. Surprisingly, the presumptive mammalian ortholog Eap45 lacks the ubiquitin-binding module of Vps36, and it is thus not clear whether mammalian ESCRT-II functions to bind ubiquitinated cargo. In this paper, we provide evidence that Eap45 contains a novel ubiquitin-binding domain, GLUE (GRAM-like ubiquitin-binding in Eap45), which binds ubiquitin with similar affinity and specificity as other ubiquitin-binding domains. The GLUE domain shares similarities in its primary and predicted secondary structures to phosphoinositide-binding GRAM and PH domains. Accordingly, we find that Eap45 binds to a subset of 3-phosphoinositides, suggesting that ubiquitin recognition could be coordinated with phosphoinositide binding. Furthermore, we show that Eap45 colocalizes with ubiquitinated proteins on late endosomes. These results are consistent with a role for Eap45 in endosomal sorting of ubiquitinated cargo.

• Gruenberg J, Stenmark H
• The biogenesis of multivesicular endosomes.
• Nat Rev Mol Cell Biol. 2004; 5: 317-23

• Johnstone CN et al.
• ARHGAP8 is a novel member of the RHOGAP family related to ARHGAP1/CDC42GAP/p50RHOGAP: mutation and expression analyses in colorectal and breast cancers.
• Gene. 2004; 336: 59-71
• Display abstract

• The RHO family of small GTPases has multiple functions, including regulation of cytoskeletal organization, cell cycle progression and cell migration, among others. The key members of this family are RHO, RAC and CDC42. Active GTP-bound RHO proteins are down-regulated by RHO GTPase-activating proteins (RHOGAPs). Herein, we describe the identification, characterization and mutational analysis of a novel RHOGAP designated as ARHGAP8, which is located within a critical region of loss-of-heterozygosity on chromosome 22q13.31 in breast and colorectal carcinomas. ARHGAP8 shares an identical genomic organization with ARHGAP1/CDC42GAP/p50RHOGAP and the corresponding proteins share the same functional domains that distinguish them from other ARHGAP members. These domains include the C-terminal RHOGAP domain, a central SH3-binding motif, and an N-terminal BNIP-2/CDC42GAP homology (BCH)/Sec14p-like domain. Three alternatively spliced ARHGAP8 transcripts were expressed in normal mammary gland and colon, which differed in the size of the BCH/Sec14p-like domain only. PCR-SSCP analyses revealed a total of six germline missense variants in individuals with colorectal or breast cancer; however, somatic mutations were not identified. Surprisingly, ARHGAP8 expression was up-regulated in the majority of primary colorectal tumors analyzed. Taken together, ARHGAP8 encodes a novel RHOGAP with unique functional domains that is highly homologous to ARHGAP1/CDC42GAP/p50RHOGAP.

• Senchenko VN et al.
• Discovery of frequent homozygous deletions in chromosome 3p21.3 LUCA and AP20 regions in renal, lung and breast carcinomas.
• Oncogene. 2004; 23: 5719-28
• Display abstract

• We searched for chromosome 3p homo- and hemizygous losses in 23 lung cancer cell lines, 53 renal cell and 22 breast carcinoma biopsies using 31 microsatellite markers located in frequently deleted 3p regions. In addition, two sequence-tagged site markers (NLJ-003 and NL3-001) located in the Alu-PCR clone 20 region (AP20) and lung cancer (LUCA) regions, respectively, were used for quantitative real-time PCR (QPCR). We found frequent (10-18%) homozygous deletions (HDs) in both 3p21.3 regions in the biopsies and lung cancer cell lines. In addition, we discovered that amplification of 3p is a very common (15-42.5%) event in these cancers and probably in other epithelial malignancies. QPCR showed that aberrations of either NLJ-003 or NL3-001 were detected in more than 90% of all studied cases. HDs were frequently detected simultaneously both in NLJ-003 or NL3-001 loci in the same tumour (P<3-10(-7)). This observation suggests that tumour suppressor genes (TSG) in these regions could have a synergistic effect. The exceptionally high frequency of chromosome aberrations in NLJ-003 and NL3-001 loci suggests that multiple TSG(s) involved in different malignancies are located very near to these markers. Precise mapping of 15 independent HDs in the LUCA region allowed us to establish the smallest HD region in 3p21.3C located between D3S1568 (CACNA2D2 gene) and D3S4604 (SEMA3F gene). This region contains 17 genes. Mapping of 19 HDs in the AP20 region resulted in the localization of the minimal region to the interval flanked by D3S1298 and D3S3623 markers. Only four genes were discovered in this interval, namely, APRG1, ITGA9, HYA22 and VILL.

• Sundquist WI, Schubert HL, Kelly BN, Hill GC, Holton JM, Hill CP
• Ubiquitin recognition by the human TSG101 protein.
• Mol Cell. 2004; 13: 783-9
• Display abstract

• The UEV domain of the TSG101 protein functions in both HIV-1 budding and the vacuolar protein sorting (VPS) pathway, where it binds ubiquitylated proteins as they are sorted into vesicles that bud into late endosomal compartments called multivesicular bodies (MVBs). TSG101 UEV-ubiquitin interactions are therefore important for delivery of both substrates and hydrolytic enzymes to lysosomes, which receive proteins via fusion with MVBs. Here, we report the crystal structure of the TSG101 UEV domain in complex with ubiquitin at 2.0 A resolution. TSG101 UEV contacts the Ile44 surface and an adjacent loop of ubiquitin through a highly solvated interface. Mutations that disrupt the interface inhibit MVB sorting, and the structure also explains how the TSG101 UEV can independently bind its ubiquitin and Pro-Thr/Ser-Ala-Pro peptide ligands. Remarkably, comparison with mapping data from other UEV and related E2 proteins indicates that although the different E2/UEV domains share the same structure and have conserved ubiquitin binding activity, they bind through very different interfaces.

• Yahiro K et al.
• Essential domain of receptor tyrosine phosphatase beta (RPTPbeta) for interaction with Helicobacter pylori vacuolating cytotoxin.
• J Biol Chem. 2004; 279: 51013-21
• Display abstract

• Helicobacter pylori produces a potent exotoxin, VacA, which causes progressive vacuolation as well as gastric injury. Although VacA was able to interact with two receptor-like protein tyrosine phosphatases, RPTPbeta and RPTPalpha, RPTPbeta was found to be responsible for gastric damage caused by VacA. To define the region of RPTPbeta involved in VacA binding, we made mutants of human cDNA RPTPbeta-B, a short receptor form of RPTPbeta. Immunoprecipitation experiments to assess VacA binding to RPTPbeta-B mutants indicated that five residues (QTTQP) at positions 747-751 of the extracellular domain of RPTPbeta-B (which is commonly retained in RPTPbeta-A, a long form of RPTPbeta) play a crucial role in its interaction with VacA, resulting in vacuolation as well as Git-1 phosphorylation. Transfected cells expressing deletion mutant Delta752, which lacks QTTQP, or the double point mutant Delta747 (T748A,T749A) had diminished vacuolation in response to VacA. Treatment of RPTPbeta-B and Delta747 (which have QTTQP at 747-751) with neuraminidase and O-glycosidase diminished their VacA binding, whereas chondroitinase ABC did not have an effect. No inhibitory effect of pleiotrophin, a natural RPTPbeta ligand, on VacA binding to RPTPbeta-B or Delta747 was observed, supporting the conclusion that the extracellular region of RPTPbeta-B responsible for VacA binding is different from that involved in binding pleiotrophin. These data define the region in the RPTPbeta extracellular domain critical for VacA binding, in particular the sequence QTTQP at positions 747-751 with crucial threonines at positions 748 and 749 and are consistent with a role for terminal sialic acids possibly because of threonine glycosylation.

• Teo H, Perisic O, Gonzalez B, Williams RL
• ESCRT-II, an endosome-associated complex required for protein sorting: crystal structure and interactions with ESCRT-III and membranes.
• Dev Cell. 2004; 7: 559-69
• Display abstract

• ESCRT-I, -II, and -III protein complexes are sequentially recruited to endosomal membranes, where they orchestrate protein sorting and MVB biogenesis. In addition, they play a critical role in retrovirus budding. Structural understanding of ESCRT interaction networks is largely lacking. The 3.6 A structure of the yeast ESCRT-II core presented here reveals a trilobal complex containing two copies of Vps25, one copy of Vps22, and the C-terminal region of Vps36. Unexpectedly, the entire ESCRT-II core consists of eight repeats of a common building block, a "winged helix" domain. Two PPXY-motifs from Vps25 are involved in contacts with Vps22 and Vps36, and their mutation leads to ESCRT-II disruption. We show that purified ESCRT-II binds directly to the Vps20 component of ESCRT-III. Surprisingly, this binding does not require the protruding N-terminal coiled-coil of Vps22. Vps25 is the chief subunit responsible for Vps20 recruitment. This interaction dramatically increases binding of both components to lipid vesicles in vitro.

• Schmidt MH et al.
• Alix/AIP1 antagonizes epidermal growth factor receptor downregulation by the Cbl-SETA/CIN85 complex.
• Mol Cell Biol. 2004; 24: 8981-93
• Display abstract

• The assembly of the Cbl-SETA/CIN85-endophilin complex at the C terminus of the epidermal growth factor receptor (EGFR) following ligand activation mediates its internalization and ubiquitination. We found that the SETA/CIN85-interacting protein Alix/AIP1, which also binds endophilins, modulates this complex. Alix was found to associate indirectly with EGFR, regardless of its activation state, and with DeltaEGFR, which signals at low intensity and does not bind Cbls or SETA/CIN85. In agreement with this, Alix interaction did not occur via SETA/CIN85. However, SETA/CIN85 and Alix were capable of mutually promoting their interaction with the EGFR. Increasing the level of Alix weakened the interaction between SETA/CIN85 and Cbl and reduced the tyrosine phosphorylation of c-Cbl and the level of ubiquitination of EGFR, SETA/CIN85, and Cbls. This antagonism of the Cbl-SETA/CIN85 complex by Alix was reflected in its diminution of EGFR internalization. In agreement with this, small interfering RNA-mediated knockdown of Alix promoted EGFR internalization and downregulation. It has been suggested that SETA/CIN85 promotes receptor internalization by recruiting endophilins. However, Alix was also capable of increasing the level of endophilin associated with EGFR, implying that this is not sufficient to promote receptor internalization. We propose that Alix inhibits EGFR internalization by attenuating the interaction between Cbl and SETA/CIN85 and by inhibiting Cbl-mediated ubiquitination of the EGFR.

• Vincent O, Rainbow L, Tilburn J, Arst HN Jr, Penalva MA
• YPXL/I is a protein interaction motif recognized by aspergillus PalA and its human homologue, AIP1/Alix.
• Mol Cell Biol. 2003; 23: 1647-55
• Display abstract

• The zinc finger transcription factor PacC undergoes two-step proteolytic activation in response to alkaline ambient pH. PalA is a component of the fungal ambient pH signal transduction pathway. Its mammalian homologue AIP1/Alix interacts with the apoptosis-linked protein ALG-2. We show that both PalA and AIP1/Alix recognize a protein-protein binding motif that we denote YPXL/I, where Tyr, Pro, and Leu/Ile are crucial for its interactive properties. Two such motifs flanking the signaling protease cleavage site mediate direct binding of PalA to PacC, required for the first and only pH-regulated cleavage of this transcription factor. PalA can bind the "closed" (i.e., wild-type full-length) conformer of PacC, suggesting that PalA binding constitutes the first stage in the two-step proteolytic cascade, recruiting or facilitating access of the signaling protease, presumably PalB. In addition to recognizing YPXL/I motifs, both PalA and AIP1/Alix interact with the Aspergillus class E Vps protein Vps32 homologue, a member of a protein complex involved in the early steps of the multivesicular body pathway, suggesting that this interaction is an additional feature of proteins of the PalA/AIP1/Alix family.

• Yu MY et al.
• Hypermethylation of the tumor suppressor gene RASSFIA and frequent concomitant loss of heterozygosity at 3p21 in cervical cancers.
• Int J Cancer. 2003; 105: 204-9
• Display abstract

• Loss of heterozygosity (LOH) at chromosome 3p21 is frequent in cervical cancers. The candidate tumor suppressor gene, RASSF1A located at 3p21.3, is found to be inactivated in several major human cancers, implicating its significance in carcinogenesis. We aimed to investigate the status of RASSF1A in cervical cancers. The mutation and methylation status of RASSF1A were analysed in 4 cervical cancer cell lines, 50 primary cervical cancers including 33 squamous cell carcinoma (SCC), 17 adenocarcinoma (AC) and 11 normal controls. The primary cancer samples were also detected for LOH at 3p21 and human papillomavirus (HPV). Hypermethylation of RASSF1A was detected in 30% of SCC, 12% of AC and in 1 of the 4 cancer cell lines but was absent in all normal cases. Methylation of the cancer cell line was associated with loss of gene expression, which was restored by demethylation. About 67% (8 of 12) of hypermethylated primary cancers showed concomitant LOH at 3p21. No somatic mutation was found in all primary cancer samples or cell lines but 2 cases showed germline polymorphism at codon 133. Oncogenic HPV DNAs were found in most cancer samples. No correlation was detected between RASSF1A-hypermethylation or LOH at 3p21 and age of patient, HPV genotype, tumor grade and stage. Hypermethylation of RASSF1A occurs in a subset of cervical cancers, among which concomitant LOH at 3p21 is common. The results supported that RASSF1A may be one of the cervical cancer-related tumor suppressor genes located at 3p21 regions.

• Yeo SC et al.
• Vps20p and Vta1p interact with Vps4p and function in multivesicular body sorting and endosomal transport in Saccharomyces cerevisiae.
• J Cell Sci. 2003; 116: 3957-70
• Display abstract

• Vps4p (End13p) is an AAA-family ATPase that functions in membrane transport through endosomes, sorting of soluble vacuolar proteins to the vacuole, and multivesicular body (MVB) sorting of membrane proteins to the vacuole lumen. In a yeast two-hybrid screen with Vps4p as bait we isolated VPS20 (YMR077c) and the novel open reading frame YLR181c, for which the name VTA1 has recently been assigned (Saccharomyces Genome Database). Vps4p directly binds Vps20p and Vta1p in vitro and binding is not dependent on ATP - conversely, Vps4p binding to Vps20p is partially sensitive to ATP hydrolysis. Both ATP binding [Vps4p-(K179A)] and ATP hydrolysis [Vps4p-(E233Q)] mutant proteins exhibit enhanced binding to Vps20p and Vta1p in vitro. The Vps4p-Vps20p interaction involves the coiled-coil domain of each protein, whereas the Vps4p-Vta1p interaction involves the (non-coiled-coil) C-terminus of each protein. Deletion of either VPS20 (vps20Delta) or VTA1 (vta1Delta) leads to similar class E Vps- phenotypes resembling those of vps4Delta, including carboxypeptidase Y (CPY) secretion, a block in ubiquitin-dependent MVB sorting, and a delay in both post-internalisation endocytic transport and biosynthetic transport to the vacuole. The vacuole resident membrane protein Sna3p (whose MVB sorting is ubiquitin-independent) does not appear to exit the class E compartment or reach the vacuole in cells lacking Vps20p, Vta1p or Vps4p, in contrast to other proteins whose delivery to the vacuole is only delayed. We propose that Vps20p and Vta1p regulate Vps4p function in vivo.

• Katzmann DJ, Stefan CJ, Babst M, Emr SD
• Vps27 recruits ESCRT machinery to endosomes during MVB sorting.
• J Cell Biol. 2003; 162: 413-23
• Display abstract

• Down-regulation (degradation) of cell surface proteins within the lysosomal lumen depends on the function of the multivesicular body (MVB) sorting pathway. The function of this pathway requires the class E vacuolar protein sorting (Vps) proteins. Of the class E Vps proteins, both the ESCRT-I complex (composed of the class E proteins Vps23, 28, and 37) and Vps27 (mammalian hepatocyte receptor tyrosine kinase substrate, Hrs) have been shown to interact with ubiquitin, a signal for entry into the MVB pathway. We demonstrate that activation of the MVB sorting reaction is dictated largely through interactions between Vps27 and the endosomally enriched lipid species phosphatidylinositol 3-phosphate via the FYVE domain (Fab1, YGL023, Vps27, and EEA1) of Vps27. ESCRT-I then physically binds to Vps27 on endosomal membranes via a domain within the COOH terminus of Vps27. A peptide sequence in this domain, PTVP, is involved in the function of Vps27 in the MVB pathway, the efficient endosomal recruitment of ESCRT-I, and is related to a motif in HIV-1 Gag protein that is capable of interacting with Tsg101, the mammalian homologue of Vps23. We propose that compartmental specificity for the MVB sorting reaction is the result of interactions of Vps27 with phosphatidylinositol 3-phosphate and ubiquitin. Vps27 subsequently recruits/activates ESCRT-I on endosomes, thereby facilitating sorting of ubiquitinated MVB cargoes.

• Hoornaert I, Marynen P, Goris J, Sciot R, Baens M
• MAPK phosphatase DUSP16/MKP-7, a candidate tumor suppressor for chromosome region 12p12-13, reduces BCR-ABL-induced transformation.
• Oncogene. 2003; 22: 7728-36
• Display abstract

• Recurrent chromosome 12p deletions are associated with distinct tumor types and suggest the presence of a tumor suppressor gene (TSG). Previously, we mapped an EST with similarity to a protein tyrosine phosphatase to the minimally deleted region for all these neoplasms. The corresponding gene, DUSP16/MKP-7, was recently shown to code for a mitogen-activated protein kinase phosphatase, suggestive for a function as tumor suppressor. Overexpression of DUSP16 in BCR-ABL-transformed Rat-1 fibroblasts reduces their transforming capacity in vitro and in vivo via downregulation of BCR-ABL-induced JNK activation. A role for DUSP16 as a regulator of JNK signaling was further demonstrated via overexpression in Ba/F3 cells, which increased their antiapoptosis. However, no inactivating mutations could be detected in leukemia patients hemizygous for DUSP16, and the effect of hemizygosity on DUSP16 expression level could not be assessed due to the variability of DUSP16 transcript levels observed in leukaemia cell lines and in patients. Taken together, the functional data point to a context-dependent role for DUSP16 on cell transformation and apoptosis, reflecting the dual role of JNK, and therefore suggest that DUSP16 might be haploinsufficient for tumor suppression.

• Raiborg C, Rusten TE, Stenmark H
• Protein sorting into multivesicular endosomes.
• Curr Opin Cell Biol. 2003; 15: 446-55
• Display abstract

• Multivesicular endosomes are important as compartments for receptor downregulation and as intermediates in the formation of secretory lysosomes. Work during the past year has shed light on the molecular mechanisms of protein sorting into multivesicular endosomes and yielded information about the machinery involved in multivesicular endosome formation. Monoubiquitination functions as a signal for sorting transmembrane proteins into intraluminal vesicles of multivesicular endosomes and subsequent delivery to lysosomes. A molecular machinery that contains the ubiquitin-binding protein Hrs/Vps27 appears to be central in this sorting process. Three conserved multisubunit complexes, ESCRT-I, -II and -III, are essential for both sorting and multivesicular endosomes formation. Enveloped RNA viruses such as HIV can redirect these complexes from multivesicular endosomes to the plasma membrane to facilitate viral budding.

• Chen YY et al.
• Genomic structure and mutational analysis of the human KIF1Balpha gene located at 1p36.2 in neuroblastoma.
• Int J Oncol. 2003; 23: 737-44
• Display abstract

• KIF1a is a member of the kinesin superfamily proteins that are microtubule-dependent molecular motors involved in important intracellular functions such as organelle transport and cell division. We previously determined the structure of the human KIF1Bbeta gene, which was found to be a homologue of the murine Kif1bbeta, and demonstrated that the human KIF1Bbeta is a causative gene of Charcot-Marie-Tooth disease type 2A although we did not prove that it is a tumor suppressor gene of neuroblastoma. Here, we identified another isoform of the human KIF1B gene, KIF1Balpha. The KIF1Balpha and KIF1Bbeta are alternative splicing products of the KIF1B gene located on 1p36.2. The KIF1Balpha is distinct from KIF1Bbeta in the C-terminal cargo-binding domain; however, they have the same N-terminal motor domain. We found that the transcript of approximately 7.8 kb of KIF1Balpha was expressed in several tissues, especially in skeletal muscle, by Northern blot analysis. To determine whether this gene is one of the candidate tumor suppressor genes for neuroblastoma (NB) or other pediatric solid tumors, we performed mutational screening of KIF1Balpha in 25 NB, 9 rhabdomyosarcoma, 12 Ewing sarcoma and 24 other pediatric solid tumor cell lines. Using RT-PCR single-strand conformation polymorphism analysis and direct sequencing we detected a missense mutation (M807I) in 1 NB cell line (SK-N-SH), 3 silent mutations in 2 NB cell lines and 1 primitive neuroectodermal tumor cell line, respectively. RT-PCR analysis revealed that KIF1Balpha was obviously expressed in almost all of the tumor cell lines examined except NB-1. Furthermore, real-time quantitative RT-PCR showed that there was no significant difference in KIF1Balpha expression between 14 early-stage (stage I and II) and 14 advanced-stage (stage III and IV) NB fresh tumor specimens. These results suggest that KIF1Ba in addition to KIF1Bbeta may not be a candidate tumor suppressor gene for NB.

• Katoh K et al.
• The ALG-2-interacting protein Alix associates with CHMP4b, a human homologue of yeast Snf7 that is involved in multivesicular body sorting.
• J Biol Chem. 2003; 278: 39104-13
• Display abstract

• Alix (ALG-2-interacting protein X) is a 95-kDa protein that interacts with an EF-hand type Ca(2+)-binding protein, ALG-2 (apoptosis-linked gene 2), through its C-terminal proline-rich region. In this study, we searched for proteins that interact with human AlixDeltaC (a truncated form not containing the C-terminal region) by using a yeast two-hybrid screen, and we identified two similar human proteins, CHMP4a and CHMP4b (chromatin-modifying protein; charged multivesicular body protein), as novel binding partners of Alix. The interaction of Alix with CHMP4b was confirmed by a glutathione S-transferase pull-down assay and by co-immunoprecipitation experiments. Fluorescence microscopic analysis revealed that CHMP4b transiently expressed in HeLa cells mainly exhibited a punctate distribution in the perinuclear area and co-localized with co-expressed Alix. The distribution of CHMP4b partly overlapped the distributions of early and late endosomal marker proteins, EEA1 (early endosome antigen 1) and Lamp-1 (lysosomal membrane protein-1), respectively. Transient overexpression of CHMP4b induced the accumulation of ubiquitinated proteins as punctate patterns that were partly overlapped with the distribution of CHMP4b and inhibited the disappearance of endocytosed epidermal growth factor. In contrast, stably expressed CHMP4b in HEK293 cells was observed diffusely in the cytoplasm. Transient overexpression of AlixDeltaC in stably CHMP4b-expressing cells, however, induced formation of vesicle-like structures in which CHMP4b and AlixDeltaC were co-localized. SKD1(E235Q), a dominant negative form of the AAA type ATPase SKD1 that plays critical roles in the endocytic pathway, was co-immunoprecipitated with CHMP4b. Furthermore, CHMP4b co-localized with SKD1(E235Q) as punctate patterns in the perinuclear area, and Alix was induced to exhibit dot-like distributions overlapped with SKD1(E235Q) in HeLa cells. These results suggest that CHMP4b and Alix participate in formation of multivesicular bodies by cooperating with SKD1.

• Wang J, Stuckey JA, Wishart MJ, Dixon JE
• A unique carbohydrate binding domain targets the lafora disease phosphatase to glycogen.
• J Biol Chem. 2002; 277: 2377-80
• Display abstract

• Lafora disease (progressive myoclonus epilepsy of Lafora type) is an autosomal recessive neurodegenerative disorder resulting from defects in the EPM2A gene. EPM2A encodes a 331-amino acid protein containing a carboxyl-terminal phosphatase catalytic domain. We demonstrate that the EPM2A gene product also contains an amino-terminal carbohydrate binding domain (CBD) and that the CBD is critical for association with glycogen both in vitro and in vivo. The CBD domain localizes the phosphatase to specific subcellular compartments that correspond to the expression pattern of glycogen processing enzyme, glycogen synthase. Mutations in the CBD result in mis-localization of the phosphatase and thereby suggest that the CBD targets laforin to intracellular glycogen particles where it is likely to function. Thus naturally occurring mutations within the CBD of laforin likely result in progressive myoclonus epilepsy due to mis-localization of phosphatase expression.

• Katzmann DJ, Odorizzi G, Emr SD
• Receptor downregulation and multivesicular-body sorting.
• Nat Rev Mol Cell Biol. 2002; 3: 893-905
• Display abstract

• The sorting of proteins into the inner vesicles of multivesicular bodies is required for many key cellular processes, which range from the downregulation of activated signalling receptors to the proper stimulation of the immune response. Recent advances in our understanding of the multivesicular-body sorting pathway have resulted from the identification of ubiquitin as a signal for the efficient sorting of proteins into this transport route, and from the discovery of components of the sorting and regulatory machinery that directs this complex process.

• Horikawa I, Barrett JC
• cDNA cloning of the human polybromo-1 gene on chromosome 3p21.
• DNA Seq. 2002; 13: 211-5
• Display abstract

• The bromodomain is suggested as a chromatin-targeting domain involved in regulation of gene expression and chromatin structure. We here report the cDNA cloning of the human polybromo-1 (hPB1) gene that encodes a 1634-amino acid polypeptide containing six bromodomains. The hPB1 polypeptide shows 91% identity to the previously identified chicken polybromo-1 with conservation of the bromodomains and other characteristic features. Northern blot analysis detected the ubiquitous expression of hPB1 mRNA in a variety of human tissues. Four alternative splicings were found within the hPB1 coding region: three making in-frame deletions and one resulting in a C-terminal truncation. The hPB1 gene is located on chromosome 3p21, where the tumor suppressor genes for breast, lung and kidney cancers have been mapped.

• Oh JJ, West AR, Fishbein MC, Slamon DJ
• A candidate tumor suppressor gene, H37, from the human lung cancer tumor suppressor locus 3p21.3.
• Cancer Res. 2002; 62: 3207-13
• Display abstract

• Frequent allelic loss and homozygous deletions within chromosome 3p in human lung cancers have suggested that the 3p21.3 (370-kb) region contains a critical tumor suppressor gene(s) (TSG). With the exact identity/characteristics of such a gene(s) still unconfirmed, a lack of inactivating structural mutations in the expressed genes contained within this region may indicate that the 3p TSG(s) do not fit into the classical "two-mutation" model. This report characterizes a candidate 3p TSG, H37, located within the 370-kb region. Reduced expression of the H37 transcript was found in 9 of 11 (82%) of primary non-small cell lung cancers (NSCLCs) when compared with adjacent normal tissues. Generation of an H37 antibody followed by immunohistochemical analysis of primary NSCLC specimens demonstrated that 46 of 62 (73%) of these cancers contain reduced levels of H37 protein when compared with adjacent normal bronchial cells. Moreover, introduction of the H37 cDNA into human breast cancer cells deleted of 3p21-22 reduced both anchorage-dependent and -independent cell growth in vitro. Subsequent transfection of H37 cDNA into one of the human lung cancer cell lines homozygously deleted in this region resulted in a very low yield of H37-expressing clones. H37 also suppressed anchorage-dependent and -independent growth of A9 mouse fibrosarcoma cells and inhibited tumor formation in nude mice. These data indicate a potential role for H37 as one of the 3p TSGs in human lung cancer.

• Bateman A et al.
• The Pfam protein families database.
• Nucleic Acids Res. 2002; 30: 276-80
• Display abstract

• Pfam is a large collection of protein multiple sequence alignments and profile hidden Markov models. Pfam is available on the World Wide Web in the UK at http://www.sanger.ac.uk/Software/Pfam/, in Sweden at http://www.cgb.ki.se/Pfam/, in France at http://pfam.jouy.inra.fr/ and in the US at http://pfam.wustl.edu/. The latest version (6.6) of Pfam contains 3071 families, which match 69% of proteins in SWISS-PROT 39 and TrEMBL 14. Structural data, where available, have been utilised to ensure that Pfam families correspond with structural domains, and to improve domain-based annotation. Predictions of non-domain regions are now also included. In addition to secondary structure, Pfam multiple sequence alignments now contain active site residue mark-up. New search tools, including taxonomy search and domain query, greatly add to the functionality and usability of the Pfam resource.

• Babst M, Katzmann DJ, Snyder WB, Wendland B, Emr SD
• Endosome-associated complex, ESCRT-II, recruits transport machinery for protein sorting at the multivesicular body.
• Dev Cell. 2002; 3: 283-9
• Display abstract

• Sorting of ubiquitinated endosomal membrane proteins into the MVB pathway is executed by the class E Vps protein complexes ESCRT-I, -II, and -III, and the AAA-type ATPase Vps4. This study characterizes ESCRT-II, a soluble approximately 155 kDa protein complex formed by the class E Vps proteins Vps22, Vps25, and Vps36. This protein complex transiently associates with the endosomal membrane and thereby initiates the formation of ESCRT-III, a membrane-associated protein complex that functions immediately downstream of ESCRT-II during sorting of MVB cargo. ESCRT-II in turn functions downstream of ESCRT-I, a protein complex that binds to ubiquitinated endosomal cargo. We propose that the ESCRT complexes perform a coordinated cascade of events to select and sort MVB cargoes for delivery to the lumen of the vacuole/lysosome.

• Zhao R, Qi Y, Chen J, Zhao ZJ
• FYVE-DSP2, a FYVE domain-containing dual specificity protein phosphatase that dephosphorylates phosphotidylinositol 3-phosphate.
• Exp Cell Res. 2001; 265: 329-38
• Display abstract

• We have recently isolated FYVE-DSP1, a FYVE domain-containing dual specificity protein phosphatase (R. Zhao, Y. Qi, and Z. J. Zhao, Biochem. Biophys. Res. Commun. 270, 222--229 (2000)). Here, we report a novel isozyme that we designated FYVE-DSP2. FYVE-2 contains a single FYVE domain at the C-terminus, and it shares approximately 47% overall sequence identity with FYBE-DSP1. Genomic sequence analyses revealed that the FYVE-DSP1 and FYVE-DSP2 genes share similar intron/exon organization. They are localizedon human chromosome 22q12 and chromosome 17, respectively. Like FYVE-DSP1, recombinant FYVE-DSP2 dephosphorylated low-molecular-weight phosphatase substrate para-nitrophenylphosphate, and its activity was inhibited by sodium vanadate. More importantly, our study also revealed that both FYVE-DSP1 and FYVE-DSP2 efficiently and specifically dephosphorylated phosphotidylinositol 3-phosphate. Subcellular fractionation demonstrated partition of FYVE-DSP1 and FYVE-DSP2 in membrane fractions, and immunofluorescent cell staining showed perinuclear localization of the enzymes. FYVE-DSP2 is expressed in many human tissues with an alternatively spliced isoform expressed in the kidney. Together with two homologous hypothetical proteins found in Caenorhabditis elegans and Drosophila, FYVE-DSP1 and FYVE-DSP2 form a subfamilyof phosphatases that may have an importantrole in cellular processes.

• Chen C et al.
• Defining a common region of deletion at 13q21 in human cancers.
• Genes Chromosomes Cancer. 2001; 31: 333-44
• Display abstract

• Previous molecular genetic analyses identified a region of deletion at 13q21 in a variety of human cancers, suggesting the existence of a tumor suppressor gene(s) at this locus. In our earlier study on prostate cancer, the region of deletion was confined to a 3.1 cM interval between D13S152 and D13S162. At present, however, no known gene located in this interval has been firmly implicated in cancer, and the region remains too large for gene identification. To fine-map the area of interest, we established a contig of bacterial artificial chromosome (BAC) clones, narrowed the region of deletion by loss of heterozygosity (LOH) and homozygosity-mapping-of-deletion (HOMOD) analyses in different types of cancers, and tested a candidate gene from the region for mutation and alteration of expression in prostate cancers. The contig consisted of 75 overlapping BAC clones. In addition to the generation of 47 new sequence-tagged-site (STS) markers from the ends of BAC inserts, 76 known STS and expressed sequence tag markers were mapped to the contig (25 kb per marker on average). The minimal region of deletion was further defined to be about 700 kb between markers D13S791 and D13S166 by LOH analysis of 42 cases of prostate cancer, and by HOMOD analysis of eight prostate cancer cell lines/xenografts and 49 cell lines from cancers of the breast, ovary, endometrium, and cervix, using 18 microsatellite markers encompassing the deletion region. A gene that is homologous to the WT1 tumor suppressor gene, AP-2rep (KLF12), was mapped in this region and was analyzed for its expression and genetic mutation. In addition to low levels of expression in both normal and neoplastic cells of the prostate, this gene did not have any mutations in a group of aggressive prostate cancers and cell lines/xenografts, as assessed by the methods of polymerase chain reaction-single strand conformational polymorphism analysis and direct sequencing. These studies suggest that a 700 kb interval at 13q21 harbors a tumor suppressor gene(s) that seems to be involved in multiple types of cancer, and that the AP-2rep gene is unlikely to be an important tumor suppressor gene in prostate cancer. The BAC contig and high-resolution physical map of the defined region of deletion should facilitate the cloning of a tumor suppressor gene(s) at 13q21.

• Wu Y, Dowbenko D, Pisabarro MT, Dillard-Telm L, Koeppen H, Lasky LA
• PTEN 2, a Golgi-associated testis-specific homologue of the PTEN tumor suppressor lipid phosphatase.
• J Biol Chem. 2001; 276: 21745-53
• Display abstract

• The tumor suppressor PTEN is a phosphatidylinositol phospholipid phosphatase, which indirectly down-regulates the activity of the protein kinase B/Akt survival kinases. Examination of sequence data bases revealed the existence of a highly conserved homologue of PTEN. This homologue, termed PTEN 2, contained an extended amino-terminal domain having four potential transmembrane motifs, a lipid phosphatase domain, and a potential lipid-binding C2 domain. Transcript analysis demonstrated that PTEN 2 is expressed only in testis and specifically in secondary spermatocytes. In contrast to PTEN, PTEN 2 was localized to the Golgi apparatus via the amino-terminal membrane-spanning regions. Molecular modeling suggested that PTEN 2 is a phospholipid phosphatase with potential specificity for the phosphate at the 3 position of inositol phosphates. Enzymatic analysis of PTEN 2 revealed substrate specificity that is similar to PTEN, with a preference for the dephosphorylation of the phosphatidylinositol 3,5-phosphate phospholipid, a known mediator of vesicular trafficking. Together, these data suggest that PTEN 2 is a Golgi-localized, testis-specific phospholipid phosphatase, which may contribute to the terminal stages of spermatocyte differentiation.

• Hicke L
• A new ticket for entry into budding vesicles-ubiquitin.
• Cell. 2001; 106: 527-30

• Gross C, Heumann R, Erdmann KS
• The protein kinase C-related kinase PRK2 interacts with the protein tyrosine phosphatase PTP-BL via a novel PDZ domain binding motif.
• FEBS Lett. 2001; 496: 101-4
• Display abstract

• Protein tyrosine phosphatase-basophil like (PTP-BL) is a large non-transmembrane protein tyrosine phosphatase implicated in the modulation of the cytoskeleton. Here we describe a novel interaction of PTP-BL with the protein kinase C-related kinase 2 (PRK2), a serine/threonine kinase regulated by the G-protein rho. This interaction is mediated by the PSD-95, Drosophila discs large, zonula occludens (PDZ)3 domain of PTP-BL and the extreme C-terminus of PRK2 as shown by yeast two-hybrid assays and coimmunoprecipitation experiments from transfected HeLa cells. In particular, we demonstrate that a conserved C-terminal cysteine of PRK2 is indispensable for the interaction with PTP-BL. In HeLa cells we demonstrate colocalization of both proteins in lamellipodia like structures. Interaction of PTP-BL with the rho effector kinase PRK2 gives further evidence for a possible function of PTP-BL in the regulation of the actin cytoskeleton.

• Kumar A et al.
• An unexpected extended conformation for the third TPR motif of the peroxin PEX5 from Trypanosoma brucei.
• J Mol Biol. 2001; 307: 271-82
• Display abstract

• A number of helix-rich protein motifs are involved in a variety of critical protein-protein interactions in living cells. One of these is the tetratrico peptide repeat (TPR) motif that is involved, amongst others, in cell cycle regulation, chaperone function and post-translation modifications. So far, these helix-rich TPR motifs have always been observed to be a compact unit of two helices interacting with each other in antiparallel fashion. Here, we describe the structure of the first three TPR-motifs of the peroxin PEX5 from Trypanosoma brucei, the causative agent of sleeping sickness. Peroxins are proteins involved in peroxisome, glycosome and glyoxysome biogenesis. PEX5 is the receptor of the proteins targeted to these organelles by the "peroxisomal targeting signal-1", a C-terminal tripeptide called PTS-1. The first two of the three TPR-motifs of T. brucei PEX5 appear to adopt the canonical antiparallel helix hairpin structure. In contrast, the third TPR motif of PEX5 has a dramatically different conformation in our crystals: the two helices that were supposed to form a hairpin are folded into one single 44 A long continuous helix. Such a conformation has never been observed before for a TPR motif. This raises interesting questions including the potential functional importance of a "jack-knife" conformational change in TPR motifs.

• Davidson D, Veillette A
• PTP-PEST, a scaffold protein tyrosine phosphatase, negatively regulates lymphocyte activation by targeting a unique set of substrates.
• EMBO J. 2001; 20: 3414-26
• Display abstract

• There is increasing interest in elucidating the mechanisms involved in the negative regulation of lymphocyte activation. Herein, we show that the cytosolic protein tyrosine phosphatase PTP-PEST is expressed abundantly in a wide variety of haemopoietic cell types, including B cells and T cells. In a model B-cell line, PTP-PEST was found to be constitutively associated with several signalling molecules, including Shc, paxillin, Csk and Cas. The interaction between Shc and PTP-PEST was augmented further by antigen receptor stimulation. Overexpression studies, antisense experiments and structure-function analyses provided evidence that PTP-PEST is an efficient negative regulator of lymphocyte activation. This function correlated with the ability of PTP-PEST to induce dephosphorylation of Shc, Pyk2, Fak and Cas, and inactivate the Ras pathway. Taken together, these data suggest that PTP-PEST is a novel and unique component of the inhibitory signalling machinery in lymphocytes.

• Angeloni D, Wei MH, Duh FM, Johnson BE, Lerman MI
• A G-to-A single nucleotide polymorphism in the human alpha 2 delta 2 calcium channel subunit gene that maps at chromosome 3p21.3.
• Mol Cell Probes. 2000; 14: 53-4

• Lemmon SK, Traub LM
• Sorting in the endosomal system in yeast and animal cells.
• Curr Opin Cell Biol. 2000; 12: 457-66
• Display abstract

• The endosomal system is a major membrane-sorting apparatus. New evidence reveals that novel coat proteins assist specific sorting steps and docking factors ensure the vectorial nature of trafficking in the endosomal compartment. There is also good evidence for ubiquitin regulating passage of certain proteins into multivesicular late endosomes, which mature by accumulating invaginated membrane. Lipids play a central role in this involution process, as do the class E vacuolar protein-sorting proteins.

• Muda M, Manning ER, Orth K, Dixon JE
• Identification of the human YVH1 protein-tyrosine phosphatase orthologue reveals a novel zinc binding domain essential for in vivo function.
• J Biol Chem. 1999; 274: 23991-5
• Display abstract

• A human orthologue of the Saccharomyces cerevisiae YVH1 protein-tyrosine phosphatase is able to rescue the slow growth defect caused by the disruption of the S. cerevisiae YVH1 gene. The human YVH1 gene is located on chromosome 1q21-q22, which falls in a region amplified in human liposarcomas. The evolutionary conserved COOH-terminal noncatalytic domain of human YVH1 is essential for in vivo function. The cysteine-rich COOH-terminal domain is capable of coordinating 2 mol of zinc/mol of protein, defining it as a novel zinc finger domain. Human YVH1 is the first protein-tyrosine phosphatase that contains and is regulated by a zinc finger domain.

• Carter DA
• Expression of a novel rat protein tyrosine phosphatase gene.
• Biochim Biophys Acta. 1998; 1442: 405-8
• Display abstract

• The cDNA sequence and expression of a novel rat protein tyrosine phosphatase (PTP) gene is reported. The predicted amino acid sequence is similar to rat PRL-1, but is more closely related to human PTP4A, another member of the recently identified fourth group of PTPs. Therefore, multiple PTPs of this group are expressed in mammalian species. The novel rat PTP gene is expressed in the anterior pituitary gland in a sexually dimorphic pattern which is indicative of a specialized role in endocrine function.

• Cao L et al.
• A novel putative protein-tyrosine phosphatase contains a BRO1-like domain and suppresses Ha-ras-mediated transformation.
• J Biol Chem. 1998; 273: 21077-83
• Display abstract

• To investigate a potential role of protein-tyrosine phosphatases (PTPases) in myocardial growth and signaling, a degenerate primer-based reverse transcription-polymerase chain reaction approach was used to isolate cDNAs for proteins that contain a PTPase catalytic domain. Among the 16 cDNA clones isolated by reverse transcription-polymerase chain reaction from total neonatal rat cardiomyocyte RNA, one, designated PTP-TD14, was unique. Subsequent isolation and sequencing of a full-length PTP-TD14 cDNA confirmed that it encodes a novel 164-kDa protein, p164(PTP-TD14). The C-terminal region contains the PTP-like domain, whereas the N-terminal region shows no homology to any known mammalian protein. However, this region is homologous to a yeast protein, BRO1, that is involved in the mitogen-activated protein kinase signaling pathway. Like BRO1, p164(PTP-TD14) contains a proline-rich region with two putative SH3-domain binding sites. By Northern blot analysis, PTP-TD14 is expressed as a 5.3-kilobase pair transcript, not only in neonatal heart but also in many adult rat tissues. When expressed in either COS-7 or NIH-3T3 cells, p164(PTP-TD14) localizes to the cytoplasm in association with vesicle-like structures. Expression of p164(PTP-TD14) in NIH-3T3 cells inhibits Ha-ras-mediated transformation more than 3-fold. This inhibitory activity is localized to the C-terminal PTPase homology domain, since no inhibition of Ha-ras-mediated focus formation was observed with a PTP-TD14 mutant, in which the putative catalytic activity was presumably inactivated by a point mutation. These findings indicate that PTP-TD14 encodes a novel protein that may be critically involved in regulating Ha-ras-dependent cell growth.

• Kuramochi S, Matsuda S, Matsuda Y, Saitoh T, Ohsugi M, Yamamoto T
• Molecular cloning and characterization of Byp, a murine receptor-type tyrosine phosphatase similar to human DEP-1.
• FEBS Lett. 1996; 378: 7-14
• Display abstract

• Novel murine cDNAs encoding a receptor-like protein tyrosine phosphatase, termed Byp (HPTP beta-like tyrosine phosphatase) were cloned. The putative Byp protein consists of 1238 amino acids, which possesses a single catalytic domain in the cytoplasmic region. The extracellular region comprises eight repeats of a fibronectin type III module and contains multiple N-glycosylation sites. The byp mRNA was 7.7-kb long and expressed in every tissue examined, its level being high in the brain and kidney. Transfection of the byp cDNA expression plasmid into COS7 cells resulted in the expression of a 220-kDa tyrosine phosphorylated protein. Furthermore, co-expression of Byp and the Src family kinase Fyn increased the level of tyrosine phosphorylation of Byp, suggesting that Byp was tyrosine-phosphorylated by Fyn. Finally, the byp gene was located to mouse chromosome 2E1-2 and rat chromosome 3q32-33.

• Kim YW, Wang H, Sures I, Lammers R, Martell KJ, Ullrich A
• Characterization of the PEST family protein tyrosine phosphatase BDP1.
• Oncogene. 1996; 13: 2275-9
• Display abstract

• Using a polymerase chain reaction (PCR) amplification strategy, we identified a novel protein tyrosine phosphatase (PTPase) designated Brain Derived Phosphatase (BDP1). The full length sequence encoded an open reading frame of 459 amino acids with no transmembrane domain and had a calculated molecular weight of 50 kDa. The predicted amino acid sequence contained a PEST motif and accordingly, BDP1 shared the greatest homology with members of the PTP-PEST family. When transiently expressed in 293 cells BDP1 hydrolyzed p-Nitrophenylphosphate, confirming it as a functional protein tyrosine phosphatase. Northern blot analysis indicated that BDP1 was expressed not only in brain, but also in colon and several different tumor-derived cell lines. Furthermore, BDP1 was found to differentially dephosphorylate autophosphorylated tyrosine kinases which are known to be overexpressed in tumor tissues.

• Hendriks W et al.
• Molecular cloning of a mouse epithelial protein-tyrosine phosphatase with similarities to submembranous proteins.
• J Cell Biochem. 1995; 59: 418-30
• Display abstract

• Protein-tyrosine phosphatases (PTPases) form an important class of cell regulatory proteins. We have isolated overlapping cDNA clones that together comprise an 8 kb transcript encoding a novel murine PTPase which is expressed in various organs. Sequence analysis revealed an open reading frame of 2,460 amino acid residues. The predicted protein, PTP-BL, is a large non-transmembrane PTPase that exhibits 80% homology with PTP-BAS, a recently described human PTPase. PTP-BL shares some intriguing sequence homologies with submembranous proteins. It contains a band 4.1-like motif also present in the tumor suppressors neurofibromatosis 2 and expanded, five 80 amino acid repeats also present in the discs-large tumor suppressor, and a single catalytic phosphatase domain. No obvious homologies to other proteins were found for the N-terminal region of the protein other than human PTP-BAS. RNA in situ hybridization experiments show that the PTP-BL gene is expressed in epithelial cells, predominantly in kidney, lung, and skin. These data suggest a cell cortical localization for PTP-BL in epithelial cells and a possible role in the morphology and motility of epithelial tissues.

• Harder KW, Saw J, Miki N, Jirik F
• Coexisting amplifications of the chromosome 1p32 genes (PTPRF and MYCL1) encoding protein tyrosine phosphatase LAR and L-myc in a small cell lung cancer line.
• Genomics. 1995; 27: 552-3

• Ariyama T et al.
• Assignment of the human protein tyrosine phosphatase, receptor-type, zeta (PTPRZ) gene to chromosome band 7q31.3.
• Cytogenet Cell Genet. 1995; 70: 52-4
• Display abstract

• Human protein tyrosine phosphatase, receptor-type, zeta (PTPRZ; also denoted HPTP zeta or RPTP beta) has a large extracellular region with the N-terminal carbonic anhydrase-like domain and a cytoplasmic region with two tandemly located protein tyrosine phosphatase domains. One of the characteristics of PTPRZ is its preferential expression in the central nervous system. We localized the human PTPRZ gene to chromosome band 7q31.3 by somatic cell hybrid mapping and fluorescence in situ hybridization.

• Yang Q, Co D, Sommercorn J, Tonks NK
• Cloning and expression of PTP-PEST. A novel, human, nontransmembrane protein tyrosine phosphatase.
• J Biol Chem. 1993; 268: 17650-17650

• Jirik FR, Anderson LL, Duncan AM
• The human protein-tyrosine phosphatase PTP alpha/LRP gene (PTPA) is assigned to chromosome 20p13.
• Cytogenet Cell Genet. 1992; 60: 117-8
• Display abstract

• In situ hybridization was employed to localize a cDNA probe from the human protein-tyrosine phosphatase PTP alpha/LRP to human metaphase chromosomes. The PTPA gene has been localized to chromosome 20p13.

• Tsukamoto T, Takahashi T, Ueda R, Hibi K, Saito H, Takahashi T
• Molecular analysis of the protein tyrosine phosphatase gamma gene in human lung cancer cell lines.
• Cancer Res. 1992; 52: 3506-9
• Display abstract

• The protein tyrosine phosphatase gamma (PTP gamma) gene has recently been suggested as a candidate tumor suppressor gene involved in the oncogenesis of human lung and renal cancers, although no direct evidence for PTP gamma mutations has been demonstrated thus far. We explored the status of PTP gamma in 31 human lung cancer cell lines as well as in various other types of human tumor cell lines. Northern blot analysis revealed that two independent cell lines expressed PTP gamma mRNAs with sizes distinct from those in human fetal and adult normal lung. However, our extensive search for mutations in the PTP gamma gene failed to identify any abnormalities in the cytoplasmic region, which contains two protein tyrosine phosphatase-like domains. These results warrant further examination of genetic alterations in the extracellular and transmembrane domains of PTP gamma, which had not been cloned at the time of the present study.

• LaForgia S et al.
• Receptor protein-tyrosine phosphatase gamma is a candidate tumor suppressor gene at human chromosome region 3p21.
• Proc Natl Acad Sci U S A. 1991; 88: 5036-40
• Display abstract

• PTPG, the gene for protein-tyrosine phosphatase gamma (PTP gamma), maps to a region of human chromosome 3, 3p21, that is frequently deleted in renal cell carcinoma and lung carcinoma. One of the functions of protein-tyrosine phosphatases is to reverse the effect of protein-tyrosine kinases, many of which are oncogenes, suggesting that some protein-tyrosine phosphatase genes may act as tumor suppressor genes. A hallmark of tumor suppressor genes is that they are deleted in tumors in which their inactivation contributes to the malignant phenotype. In this study, one PTP gamma allele was lost in 3 of 5 renal carcinoma cell lines and 5 of 10 lung carcinoma tumor samples tested. Importantly, one PTP gamma allele was lost in three lung tumors that had not lost flanking loci. PTP gamma mRNA was expressed in kidney cell lines and lung cell lines but not expressed in several hematopoietic cell lines tested. Thus, the PTP gamma gene has characteristics that suggest it as a candidate tumor suppressor gene at 3p21.

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