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PubMed2006

Hgs physically interacts with Smad5 and attenuates BMP signaling.

Signaling by members of the bone morphogenetic protein family plays a critical role in cartilage development and differentiation. Recently, the potential involvement of BMPs in the maintenance and repair of damaged adult articular cartilage has initiated an interest in the role of BMP signaling and the involved signaling pathways in the adult tissue. In this study, we identified Hgs as a novel Smad5 interactor using a cDNA expression library constructed from human adult cartilage. This interaction was confirmed by coimmunoprecipitation experiments in 293 EBNA cells and the chondrocytic cell line T/C-28a2. Overexpression of Hgs resulted in an attenuation of BMP-dependent transcriptional responses suggesting that Hgs acts as an inhibitor of BMP signaling. Of note, osteoarthritic chondrocytes which have been suggested previously to show increased reactivity to BMP-stimulation showed less expression of Hgs. Thus, it is tempting to speculate that both might be related to each other given the suppressive effect of BMP signaling on Hgs shown in this study.

Adult↗

PubMed2006

UBPy/MSJ-1 system during male germ cell progression in the frog, Rana esculenta.

mUBPy (mouse ubiquitin specific processing protease) is a de-ubiquitinating enzyme expressed in mouse testis and brain. In testis, it interacts with the DnaJ protein MSJ-1 (mouse sperm cell specific DnaJ first homologue), a molecular chaperone expressed in spermatids and spermatozoa. Since MSJ-1 is conserved among vertebrates, to demonstrate an evolutionarily conserved function of UBPy/MSJ-1 system, we assayed mUBPy presence in the anuran amphibian, the frog, Rana esculenta, during the annual sexual cycle. By Western blot we have detected a specific signal of 126kDa in testis and isolated spermatozoa. During the annual sexual cycle, the signal gradually increases as soon as spermatogenesis resumes after the winter stasis. Using immunocytochemistry, we have localized the protein in spermatids and spermatozoa. In conclusion, UBPy/MSJ-1 system is available in R. esculenta testis suggesting a conserved fundamental function in spermatogenesis and sperm formation.

Animals↗

PubMed2003

Characterization of the interactions between Nedd4-2, ENaC, and sgk-1 using surface plasmon resonance.

Previous studies have characterized interactions between the ubiquitin ligase Nedd4-1 and the epithelial Na(+) channel (ENaC). Such interactions control the channel cell surface expression and activity. Recently, evidence has been provided that a related protein, termed Nedd4-2, is likely to be the true physiological regulator of the channel. Unlike Nedd4-1, Nedd4-2 also interacts with the aldosterone-induced channel activating kinase sgk-1. The current study uses surface plasmon resonance to quantify the binding of the four WW domains of Nedd4-2 to synthetic peptides corresponding to the PY motifs of ENaC and sgk-1. The measurements demonstrate that WW3 and WW4 are the only Nedd4-2 domains interacting with both ENaC and sgk-1 and that their binding constants are in the 1-6 microM range.

Amino Acid Motifs↗

PubMed2003

The role of ubiquitin in down-regulation and intracellular sorting of membrane proteins: insights from yeast.

Ubiquitination is a versatile tool used by all eukaryotic organisms for controlling the stability, function, and intracellular localization of a wide variety of proteins. Two of the best characterized functions of protein ubiquitination are to mark proteins for degradation by cytosolic proteasome and to promote the internalization of certain plasma membrane proteins via the endocytotic pathway, followed by their degradation in the vacuole. Recent studies of membrane proteins both in yeast and mammalian cells suggest that the role of ubiquitin may extend beyond its function as an internalization signal in that it also may be required for modification of some component(s) of the endocytotic machinery, and for cargo protein sorting at the late endosome and the Golgi apparatus level. In this review, I will attempt to bring together what is currently known about the role of ubiquitination in controlling protein trafficking between the yeast plasma membrane, the trans-Golgi network, and the vacuole/lysosome.

Down-Regulation↗

PubMed2002

A ubiquitin-interacting motif from Hrs binds to and occludes the ubiquitin surface necessary for polyubiquitination in monoubiquitinated proteins.

The ubiquitin-interacting motif (UIM) is a short, approximately 20 residue, structural element, which is present in, but not limited to, the proteins involved in endocytotic and proteasomal degradation. UIMs facilitate endocytotic vesicular sorting of the monoubiquitinated proteins and may be important for the targeting of the polyubiquitinated proteins to the proteasome. Using heteronuclear NMR backbone and side-chain chemical shift mapping of the ubiquitin interaction surface, the UIM from the hepatocyte growth factor-regulated tyrosine kinase substrate, Hrs, specifically binds to the ubiquitin hydrophobic surface using UIM's well-conserved central helical LALAL motif. Molecular modeling of the ubiquitin:UIM_Hrs complex suggests that binding occurs through a specific interaction of Leu263 and Leu267 of the UIM_Hrs with two ubiquitin hydrophobic patches located in close proximity to the ubiquitin major polyubiquitination site, Lys48. Intramolecular binding of ubiquitin to a UIM in monoubiquitinated proteins would render Lys48 unavailable for further ubiquitination, thus, explaining the absolute requirement of UIMs for monoubiquitination. Two leucines, Leu265 and Leu269, located on the opposite face of UIM_Hrs can also interact, albeit less favorably than Leu263 and Leu267, with the ubiquitin hydrophobic patches, suggesting a possible mode for polyubiquitin:UIM binding and apparent preference of UIMs for polyubiquitins.

Amino Acid Motifs↗

PubMed2003

Identification of AMSH-LP containing a Jab1/MPN domain metalloenzyme motif.

We have isolated a cDNA clone encoding a new AMSH (associated molecule with the SH3 domain of STAM) family protein, termed AMSH-like protein (AMSH-LP). AMSH-LP has similar characteristics to AMSH; both AMSH-LP and AMSH are expressed ubiquitously in various human tissues, contain a putative nuclear localization signal (NLS), an Mpr/Pad1/N-terminal (MPN) domain, and a Jab1/MPN domain metalloenzyme (JAMM) motif in their structures, and are excluded from the nucleus when lacking either the NLS or MPN domain. Moreover, we observed an enhancement of interleukin 2 (IL-2)-mediated c-myc induction in AMSH-LP-transfected cells similar to that seen in AMSH-transfected cells, suggesting a functional similarity between AMSH-LP and AMSH. However, the present study demonstrated that AMSH-LP, unlike AMSH, fails to bind to the SH3 domains of STAM1 (signal transducing adaptor molecule 1) and Grb2. These results suggest that AMSH-LP and AMSH may have different functions.

Amino Acid Sequence↗

PubMed2003

Rsp5-Bul1/2 complex is necessary for the HSE-mediated gene expression in budding yeast.

Rsp5 is an essential ubiquitin ligase in Saccharomyces cerevisiae and is concerned with many functions such as endocytosis and transcription through ubiquitination of various substrates. Bul1 or its homologue Bul2 binds to Rsp5 through the PY-motif and the bul1 bul2 double mutant is sensitive to various stresses. We demonstrate here that heat shock element (HSE)-mediated gene expression was defective in both rsp5-101 and bul1 bul2 mutants under high temperature condition. The bul1 gene containing mutations in the PY motif region did not recover this defective gene expression of the bul1 bul2 mutant. The protein level and phosphorylation state of the HSE-binding transcription factor, Hsf1, was not affected by these mutations. Thus, the Rsp5-Bul1/2 complex has a new function for the HSE-mediated gene expression and may regulate it through other factors than Hsf1.

Adaptor Proteins, Signal Transducing↗

PubMed2003

Enhanced degradation of MDM2 by a nuclear envelope component, mouse germ cell-less.

A mouse homologue of Drosophila germ cell less, mouse germ cell less-1 (mgcl-1), encodes a nuclear envelope component essential for nuclear integrity. To analyze the molecular function of mGCL-1, we carried out two hybrid screening and found that mGCL-1 bound to the gene product of tumor susceptibility gene 101 (tsg101). Effects of mGCL-1 on the expression of MDM2-p53 axis were examined, since TSG101 has been shown to elevate the amount of MDM2 by inhibiting the ubiquitination. mGCL-1 significantly reduced the amount of MDM2 probably by changing the sub-cellular localization of the MDM2 and facilitating the ubiquitination of MDM2. In addition, the amount of p53 was increased and transactivation by p53 was enhanced by mGCL-1. Thus, mGCL-1 turned out to be a factor modulating MDM2-p53 axis by enhanced degradation of MDM2.

Animals↗

PubMed2003

A newly identified AMSH-family protein is specifically expressed in haploid stages of testicular germ cells.

Associated Molecule with SH3 domain of STAM (AMSH) plays a critical role in the cytokine-mediated intracellular signal transduction downstream of the Jak2/Jak3-STAM complex. We newly identified a family molecule of AMSH, AMSH-FP (AMSH-Family Protein) in the mouse brain. AMSH-FP encodes the intracellular protein and has a highly conserved JAB1 Subdomain Homologous (JSH) region, suggesting that AMSH-FP may act as adaptor of gene transcription and/or regulation system. AMSH-FP has two splicing forms, one is expressed in various tissues, whereas the other one is restricted to expression in testis. We named the abundant type AMSH-FPalpha and the testis type AMSH-FPbeta. AMSH-FPbeta is a variant lacking N-terminal 166 amino acid residues of AMSH-FPalpha. Analysis of the 5(')-untranslated regions in AMSH-FPalpha and AMSH-FPbeta mRNAs and exon-intron structure of AMSH-FP gene suggests that testis-specific transcripts are generated due to alternative promoter usage and/or alternative splicing. Importantly, AMSH-FPbeta mRNA was not detected in juvenile and infertile mouse testis but was restrictively expressed in the haploid stage of testicular germ cells in the normal mature testis. We suggested that AMSH-FPbeta had a functional role in the spermiogenesis.

5' Untranslated Regions↗

PubMed2000

Regulation of the cardiac voltage-gated Na+ channel (H1) by the ubiquitin-protein ligase Nedd4.

The cardiac voltage-gated Na+ channel H1, involved in the generation of cardiac action potential, contains a C-terminal PY motif (xPPxY). Since PY motifs are known ligands to WW domains, we investigated their role for H1 regulation and the possible involvement of the WW domain containing ubiquitin-protein ligase Nedd4, taking advantage of the Xenopus oocyte system. Mutation of the PY motif leads to higher peak currents when compared to wild-type channel. Moreover, co-expression of Nedd4 reduced the peak currents, whereas an enzymatically inactive Nedd4 mutant increased them, likely by competing with endogenous Nedd4. The effect of Nedd4 was not observed in the PY motif mutated channel or in the skeletal muscle voltage-gated Na+ channel, which lacks a PY motif. We conclude that H1 may be regulated by Nedd4 depending on WW-PY interaction, and on an active ubiquitination site.

Amino Acid Motifs↗

PubMed2000

STAM2, a new member of the STAM family, binding to the Janus kinases.

We here cloned a cDNA encoding STAM2, a new member of the STAM family, which contains an SH3 domain and ITAM. STAM2 like STAM1 is associated with Jak2 and Jak3, and involved in the signaling for DNA synthesis and c-myc induction mediated by IL-2 and GM-CSF. Co-expression of the SH3 deletion mutants of STAM1 and STAM2 induces an additive effect on suppressing DNA synthesis upon stimulation with IL-2 and GM-CSF, suggesting that STAM1 and STAM2 exhibit compensatory effects on the signaling pathways downstream of Jak2 and Jak3 upon stimulation with GM-SCF and IL-2, respectively.

Adaptor Proteins, Signal Transducing↗

PubMed2001

STAM/EAST/Hbp adapter proteins--integrators of signalling pathways.

STAM/EAST/Hbp family of proteins consists of eight members well conserved from yeast to mammals. The basic domain architecture is comprised of an N-terminal Vps27, Hrs and STAM homology domain, a ubiquitin-interacting motif and a central Src homology-3 domain. Vertebrate members also carry an immunoreceptor tyrosine-based activation motif. STAM/EAST/Hbp proteins become tyrosine-phosphorylated by a variety of cytokines and growth factors. STAM 1 and STAM 2A are involved in cytokine-mediated signalling for DNA synthesis and c-myc induction. EAST and STAM 2A/Hbp play a role in receptor-mediated endo- and exocytosis and probably also in the regulation of actin cytoskeleton. Knockout experiments implicate a role for STAM 1 in neural cell survival. A picture is emerging of STAM/EAST/Hbp proteins acting as integrators of thus far mechanistically disparate cellular signalling events.

Adaptor Proteins, Signal Transducing↗

PubMed2002

VHS domain -- a longshoreman of vesicle lines.

The VHS (Vps-27, Hrs and STAM) domain is a 140 residue long domain present in the very NH2-terminus of at least 60 proteins. Based on their functional characteristics and on recent data on the involvement of VHS in cargo recognition in trans-Golgi, VHS domains are considered to have a general membrane targeting/cargo recognition role in vesicular trafficking. Structurally, VHS is a right-handed superhelix of eight helices with charged surface patches probably serving as sites of protein-protein recognition and docking.

Amino Acid Sequence↗

PubMed2002

PY motifs of Rod1 are required for binding to Rsp5 and for drug resistance.

In Saccharomyces cerevisiae, the overexpression of ROD1 confers resistance to o-dinitrobenzene (o-DNB), a representative of target drugs of glutathione S-transferase. The roles of Rod1 in drug resistance have remained to be determined. We isolated the rog3 mutation as a suppressor mutation of the temperature sensitivity of the strain, in that two of the total four glycogen synthase kinase 3 homologs were deleted. Rog3 is homologous to Rod1, and its overexpression also conferred resistance to o-DNB. Furthermore, these two proteins have PY-motifs, and bound to Rsp5, a hect-type ubiquitin ligase. The rsp5-101 mutant showed sensitivity to o-DNB as did the rod1 mutant, a mutant Rod1 containing altered PY motifs was defective in ability to bind to Rsp5 and in conferring o-DNB resistance. These results suggest that interaction of Rod1 and Rsp5 is important for drug resistance.

Amino Acid Motifs↗

PubMed1997

Glucose activation of the yeast plasma membrane H+-ATPase requires the ubiquitin-proteasome proteolytic pathway.

Glucose triggers transcriptional and post-transcriptional mechanisms that increase the level and activity of Saccharomyces cerevisiae plasma membrane H+-ATPase. We have studied the post-transcriptional activation of the enzyme by glucose and have found that Rsp5, a ubiquitin-protein ligase enzyme, Ubc4, a ubiquitin-conjugating enzyme, and the 26S proteasome complex are implicated in this activation. These results suggest that ATPase activation by glucose requires the ubiquitin-proteasome proteolytic pathway. This is supported by the fact that over-expression of the ubiquitin-specific protease Ubp2, which cleaves ubiquitin from its branched conjugates, inhibits this activation. We propose that glucose triggers degradation of an inhibitory protein resulting in enzyme activation.

Cell Membrane↗

PubMed1998

Role of UEV-1A, a homologue of the tumor suppressor protein TSG101, in protection from DNA damage.

The open reading frame YGL087c in the budding yeast Saccharomyces cerevisiae genome encodes a polypeptide highly similar to the human UEV (ubiquitin-conjugating E2 enzyme variant) proteins, which have been proposed to belong to a family of putative dominant negative ubiquitin regulators. Deletion of the YGL087c open reading frame yields viable cells which are sensitive to UV irradiation or methyl methanesulfonate, but not to hydroxyurea. This phenotype is reminiscent of that of rad mutants and suggests that the YGL087c-encoded protein functions in a process related to tolerance to DNA damage. We also show that the mutant phenotype is fully complemented by expression of the human UEV-1A cDNA and we propose that UEV-1 proteins could also have a role in protecting higher eukaryotic cells from DNA damaging agents.

Amino Acid Sequence↗

PubMed2002

ATF/CREB sites present in sub-telomeric regions of Saccharomyces cerevisiae chromosomes are part of promoters and act as UAS/URS of highly conserved COS genes.

A highly conserved 48 bp DNA element was identified present at 26 chromosome ends of Saccharomyces cerevisiae. Each element harbours an ideal or a mutated ATF/CREB site, which is a well-known target sequence for bZip transcription factors. In all cases, the sub-telomeric ATF/CREB site element (SACE) is a direct extension of the respective sub-telomeric coreX element. Eight SACEs are part of very long quasi-identical regions of several kilobases, including a sub-telomeric COS open reading frame. Three of these eight SACEs harbour an ideal ATF/CREB site, four a triple-exchange variant (5'-ATGGTATCAT-3'; GTA variant), and one a single exchange variant with a C to G exchange at the left side of the center of symmetry. We analyzed the function of the SACE of the left arm of chromosome VIII in vivo and found its ATF/CREB site to act as UAS/URS of the COS8 promoter, effected by the yeast bZip proteins Sko1p, Aca1p, and Aca2p. Cos8 protein was found in proximity to the nuclear membrane, where it accumulated, especially during cell division. When the ATF/CREB site of the COS8 promoter was exchanged with the GTA variant, the regulation was changed. COS8 was then regulated by Hac1p, a bZip protein known to be involved in the unfolded protein response of S. cerevisiae, indicating, for the first time, a possible functional category for the Cos proteins of S. cerevisiae.

Adenosine Triphosphatases↗

PubMed2003

Ebola virus matrix protein VP40 interaction with human cellular factors Tsg101 and Nedd4.

The Ebola virus matrix protein VP40 is a major viral structural protein and plays a central role in virus assembly and budding at the plasma membrane of infected cells. For efficient budding, a full amino terminus of VP40 is required, which includes a PPXY and a PT/SAP motif, both of which have been proposed to interact with cellular proteins. Here, we report that Ebola VP40 can interact with cellular factors human Nedd4 and Tsg101 in vitro. We show that WW domain 3 of human Nedd4 is necessary and sufficient for binding to the PPXY motif of VP40, which requires an oligomeric conformation of VP40. Single particle electron microscopy reconstructions indicate that WW3 of Nedd4 is in close contact with the N-terminal domain of hexameric VP40. In contrast, the ubiquitin enzyme variant domain of Tsg101 was sufficient for binding to the PT/SAP motif of VP40, regardless of the oligomeric state of the matrix protein. These results suggest that hNedd4 and Tsg101 may play complimentary roles at a late stage of the assembly process, by recruiting cellular factors of two independent pathways to the site of budding at the plasma membrane.

Amino Acid Motifs↗