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PubMed2006

Regulation of functional diversity within the Nedd4 family by accessory and adaptor proteins.

Ubiquitination is essential in mediating diverse cellular functions including protein degradation and trafficking. Ubiquitin-protein (E3) ligases determine the substrate specificity of the ubiquitination process. The Nedd4 family of E3 ligases is an evolutionarily conserved family of proteins required for the ubiquitination of a large number of cellular targets. As a result, this family regulates a wide variety of cellular processes including transcription, stability and trafficking of plasma membrane proteins, and the degradation of misfolded proteins. The modular architecture of the proteins, comprising a C2 domain, multiple WW domains and a catalytic domain, enables diverse intermolecular interactions and recruitment to various subcellular locations. The WW domains commonly mediate interaction with substrate proteins; however, an increasing number of Nedd4 targets do not contain obvious WW domain-interaction motifs suggesting the involvement of accessory proteins. This review discusses recent insights into how accessory and adaptor proteins modulate the activities of Nedd4 family members, including recruitment of novel substrates, alteration of subcellular localisation and effects on ubiquitination.

Adaptor Proteins, Signal Transducing↗

PubMed2006

Targeting early apoptotic genes in batch and fed-batch CHO cell cultures.

Based on the transcriptional profiling of CHO cell culture using microarray, four key early apoptosis signaling genes, Fadd, Faim, Alg-2, and Requiem, were identified and CHO GT (Gene Targeted) cell lines were developed by targeting these four genes. Two were CHO GT(O) cell lines overexpressing anti-apoptotic genes, Faim and Fadd DN and two were CHO GT(KD) cell lines involving knockdown of Alg-2 and Requiem which are pro-apoptotic genes using small interfering RNA (siRNA) technology. Comparisons of these CHO GT cell lines with the parental cell line in batch culture (BC) and fed-batch culture (FBC) were performed. Compared to parental cells, the CHO GT cell lines showed apoptosis resistance as they significantly delayed and/or suppressed initiator caspase-8 and -9 and executioner caspase-3 activities during culture. FBC of CHO GT cell lines reached significantly higher maximum viable cell densities (up to 9 x 10(6) cells/mL) compared with the parental cell line (5 x 10(6) cells/mL). The recombinant interferon gamma (IFN-gamma) yields were increased by up to 2.5-fold. Furthermore, it was observed that the IFN-gamma was more highly sialylated.

Adaptor Proteins, Signal Transducing↗

PubMed2003

Prognostic implications of neuroendocrine differentiation and hormone production in patients with Stage I nonsmall cell lung carcinoma.

BACKGROUND: Approximately 10-20% of nonsmall cell lung carcinomas (NSCLC) show neuroendocrine (NE) differentiation, as evaluated by panendocrine markers or ultrastructural evidence of dense-core secretory granules. However, little is known regarding the prevalence and clinical implications of NE differentiation in patients with Stage I NSCLC. METHODS: The authors analyzed 220 consecutive patients with Stage I NSCLC (pT1-T2N0M0) among 2100 patients with primary lung carcinoma who underwent surgical treatment between 1987 and 1993. Using light microscopy and immunohistochemical staining for synaptophysin, chromogranin A, and respiratory tract-related hormones, 28 NSCLC specimens with NE differentiation (NSCLC-ND) and 11 large cell neuroendocrine carcinoma (LCNEC) specimens were identified. RESULTS: The 28 NSCLC-ND specimens included 15 adenocarcinomas and 13 squamous cell carcinomas. Neoplastic cells with NE features never exceeded 20% in NSCLC-ND specimens, whereas neoplastic cells amounted to 20-90% in LCNEC specimens. NSCLC-ND specimens with > 5% NE-differentiated tumor cells showed increased Ki-67 labeling index (P = 0.007) and invasive phenotype, as evaluated by fascin immunoreactivity (P = 0.021). Patients with adenocarcinoma, but not with squamous cell carcinoma, who had > 5% NE-differentiated cells had a worse clinical course compared with patients who had ordinary NSCLC, with reduced overall survival (P = 0.017) and disease free survival (P = 0.049). In multivariate analysis, NE differentiation > 5% neoplastic cells in patients with adenocarcinoma independently predicted a poorer prognosis (hazard ratio, 2.61; 95% confidence interval, 0.99-6.85). Hormone production was restricted to chromogranin positive NSCLC-ND but did not affect prognosis. CONCLUSIONS: Stage I adenocarcinomas with >or= 5% NE tumor cells are clinically aggressive tumors, similar to LCNEC. Hormone production identifies a more fully developed neuroendocrine phenotype but is not relevant to prognosis. The identification of NE-differentiated cells in patients with NSCLC may have clinical relevance.

Adenocarcinoma↗

PubMed2004

Reduction of TSG101 protein has a negative impact on tumor cell growth.

TSG101 was defined originally as a tumor-suppressor gene, raising the expectation that absence of the encoded protein should lead to increased tumor cell growth and, perhaps, increased tumor cell aggressiveness. We have used the RNA interference (RNAi) technique to downregulate TSG101 in PC3 (prostate cancer) and MDA-MB-231 (breast cancer) cells. An approximately 85% selective downregulation at the protein level was achieved in both cell lines over a period of 12 days as detected by Western blotting. This treatment resulted in inhibition of tumor cell growth, with a decreased level of TSG101 causing partial cell cycle arrest at the G(1)/S boundary and a reduction in the rate at which cells passed from G(2) through mitosis and back into G(1). In both cell lines, the percentage of cells in S-phase was reduced significantly at day 4 after the TSG101 siRNA transfection (27% vs. 41% in MDA-MB-231 cells; 22% vs. 39% in PC3 cells). Additionally, RNAi-mediated downregulation of TSG101 reduced the colony formation capacities of both cancer cell lines. Rather more surprisingly, TSG101 downregulation affected the migratory activity of the MDA-MB-231 cells, independent of any effect on proliferation. Thus, in a Transwell assay, after 4-hr incubation, 36.0% of control MDA-MB-231 cells had migrated to the lower chamber vs. 7.3% of TSG101-downregulated cells (p < 0.001; scrambled control, 36.5%). These results show that the TSG101 gene does not comply with the usual characteristics of a tumor-suppressor gene; rather, its expression may be necessary for activities associated with aspects of tumor progression.

Apoptosis↗

PubMed1995

A human phosphatidylinositol 3-kinase complex related to the yeast Vps34p-Vps15p protein sorting system.

Phosphoinositide (PI) 3-kinases have been characterized as enzymes involved in receptor signal transduction in mammalian cells and in a complex which mediates protein trafficking in yeast. PI 3-kinases linked to receptors with intrinsic or associated tyrosine kinase activity are heterodimeric proteins, consisting of p85 adaptor and p110 catalytic subunits, which can generate the 3-phosphorylated forms of phosphatidylinositol (PtdIns), PtdIns4P and PtdIns(4,5)P2 as potential second messengers. Yeast Vps34p kinase, however, has a substrate specificity restricted to PtdIns and is a PtdIns 3-kinase. Here the molecular characterization of a new human PtdIns 3-kinase with extensive sequence homology to Vps34p is described. PtdIns 3-kinase does not associate with p85 and phosphorylates PtdIns, but not PtdIns4P or PtdIns(4,5)P2. In vivo PtdIns 3-kinase is in a complex with a cellular protein of 150 kDa, as detected by immunoprecipitation from human cells. Protein sequence analysis and cDNA cloning show that this 150 kDa protein is highly homologous to Vps15p, a 160 kDa protein serine/threonine kinase associated with yeast Vps34p. These results suggest that the major components of the yeast Vps intracellular trafficking complex are conserved in humans.

Amino Acid Sequence↗

PubMed2004

Regulation of the voltage gated K+ channel Kv1.3 by the ubiquitin ligase Nedd4-2 and the serum and glucocorticoid inducible kinase SGK1.

The stimulation of cell proliferation by insulin like growth factor IGF-1 has previously been shown to depend on activation of voltage gated K(+) channels. The signaling involved in activation of voltage gated K(+) channel Kv1.3 includes the phosphatidylinositol-3 (PI3) protein kinase, 3-phosphoinositide dependent protein kinase PDK1 and the serum and glucocorticoid inducible kinase SGK1. However, nothing is known about mechanisms mediating the stimulation of Kv1.3 by SGK1. Most recently, SGK1 has been shown to phosphorylate and thus inactivate the ubiquitin ligase Nedd4-2. The present study has been performed to explore whether the regulation of Kv1.3 involves Nedd4-2. To this end Kv1.3 has been expressed in Xenopus oocytes with or without coexpression of Nedd4-2 and/or constitutively active (S422D)SGK1. In oocytes expressing Kv1.3 but not in water injected oocytes, depolarization from a holding potential of -80 mV to +20 mV triggers rapidly inactivating currents typical for Kv1.3. Coexpression of Nedd4-2 decreases, coexpression of (S422D)SGK1 enhances the currents significantly. The effects of either Nedd4-2 or of SGK1 are abrogated by destruction of the respective catalytic subunits ((C938S)Nedd4-2 or (K127N)SGK1). Further experiments revealed that wild type SGK1 and SGK3 and to a lesser extent SGK2 are similarly effective in stimulating Kv1.3 in both, presence and absence of Nedd4-2. It is concluded that Kv1.3 is downregulated by Nedd4-2 and stimulates by SGK1, SGK2, and SGK3. The data thus disclose a novel mechanism of Kv1.3 channel regulation.

Animals↗

PubMed1995

Molecular analysis of the SNF8 gene of Saccharomyces cerevisiae.

Mutations in the SNF8 gene impair derepression of the SUC2 gene, encoding invertase, in response to glucose limitation of Saccharomyces cerevisiae. We report here the cloning of the SNF8 gene by complementation. Sequence analysis predicts a 26,936-dalton product. Disruption of the chromosomal locus caused a five-fold decrease in invertase derepression, defective growth on raffinose, and a sporulation defect in homozygous diploids. Genetic analysis of the interactions of the snf8 null mutation with spt6/ssn20 and ssn6 suppressors distinguished SNF8 from the groups, SNF1, SNF4 and SNF2, SNF5, SNF6. Notably, the snf8 ssn6 double mutants were extremely sick. Mutations of SNF8 and SNF7 showed similar phenotypes and genetic interactions, and the double mutant combination caused no additional phenotypic impairment. These findings suggest that SNF7 and SNF8 are functionally related.

Amino Acid Sequence↗

PubMed1996

Cloning of a novel signal-transducing adaptor molecule containing an SH3 domain and ITAM.

We molecularly cloned a cDNA coding for a novel phosphotyrosine molecule with a 70 kDa molecular mass, named STAM (signal transducing adaptor molecule), which is tyrosine-phosphorylated rapidly after stimulation with various cytokines such as IL-2, IL-3, IL-4, IL-7, GM-CSF, EGF and PDGF. STAM contains an SH3 (Src-homology 3) domain and the ITAM (immunoreceptor tyrosine-based activation motif), suggesting that STAM acts as an adaptor molecule involved in signal transducing pathways from the cytokine receptors.

Adaptor Proteins, Signal Transducing↗

PubMed1998

A putative tumor suppressor, TSG101, acts as a transcriptional suppressor through its coiled-coil domain.

TSG101 is thought as a putative tumor suppressor gene, and mutations of this gene were recently found in 7 of 15 breast cancer patients, though the physiological function remains to be elucidated. In this report, we showed that TSG101 protein acts as a transcriptional suppressor for estrogen receptor (ER) as well as other members of the nuclear hormone receptor super-family, VP16, and on its own. The basal promoter activity was also inhibited by TSG101. The suppression of transcription by TSG101 protein required its coiled-coil domain, which is also shown to be required for the tumor suppressive function. Expressed TSG101 protein did not have any histone acetylase nor deacetylase activity, which certain transcriptional co-factors have. The requirement of the same domain in the TSG101 protein for transcriptional suppression and in the tumor suppression indicates a possibility that transcriptional suppression of TSG101 is related to its tumor suppression.

Acetyltransferases↗

PubMed1998

Evidence that TSG101 aberrant transcripts are PCR artifacts.

Critical analysis of the data published so far concerning the TSG101 gene revealed some inconsistencies leading us to its re-evaluation in 80 breast, brain, colon, and neuroectodermal tumors and 37 normal tissue specimens. In this study, the occurrence of TSG101 cDNA aberrant transcripts was verified, but in addition we made observations that are in apparent conflict with the aberrant splicing theory supposed as the underlying mechanism for transcript formation: the location of most deletion breakpoints within exons and nonconformity of these putative splice sites with the highly conserved GT-AG rule, detection of insertions as well as nonreproducible and highly variable results in repeated RT-nested PCRs. Furthermore, we found that reamplification of full-length TSG101 cDNA products leads to the generation of deleted transcripts. In summary, for the first time we provide evidence that the acquired TSG101 transcripts are caused by PCR artifacts.

Artifacts↗

PubMed2000

Involvement of hrs binding protein in IgE receptor-triggered exocytosis in RBL-2H3 mast cells.

Hrs binding protein (Hbp) tightly associated with Hrs is thought to play a regulatory role in vesicular trafficking during endocytosis and exocytosis. In this study, we have expressed dominant-negative mutants of Hbp to evaluate their effects on the degranulation of secretory granules in RBL-2H3 mast cells. The dominant-negative mutants of Hbp significantly inhibited IgE receptor (FcepsilonRI)-triggered secretory response as tested by beta-hexosaminidase release. These results suggest that Hbp functions as a regulator in the FcepsilonRI-triggered degranulation of secretory granules in mast cells.

3T3 Cells↗

PubMed2001

Hrs and hbp: possible regulators of endocytosis and exocytosis.

The molecular mechanisms of endocytosis and exocytosis are not yet fully understood. Hrs and Hbp, two tightly associated proteins in eukaryotic cells, have been implicated in these cellular processes. Hrs is homologous to Vps27p, an endosomal protein required for vacuolar and endocytic trafficking in yeast. Hrs is localized to early endosomes and is required for the normal morphology of early endosomes in mammalian cells. Hrs also associates with proteins implicated in endocytosis and exocytosis such as SNAP-25 and Eps15. Hrs treatment inhibits neurotransmitter release in permeabilized neuronal cells and its overexpression inhibits internalization of transferrin. Overexpression of dominant-negative Hbp mutants inhibits ligand-induced downregulation of growth factor/receptor complexes and immunoglobulin E receptor-triggered degranulation of secretory granules in mast cells. These observations suggest an important role for the Hrs/Hbp protein complex in vesicular trafficking during endocytosis and exocytosis.

Adaptor Proteins, Signal Transducing↗

PubMed2001

Characterization of interactions between Nedd4 and beta and gammaENaC using surface plasmon resonance.

Cell surface expression of the epithelial Na(+) channel ENaC is regulated by the ubiquitin ligase Nedd4. Binding of the WW domains of Nedd4 to the PY region in the carboxy tails of beta and gammaENaC, results in channel ubiquitination and degradation. Kinetic analysis of these interactions has been done using surface plasmon resonance. Synthetic peptides corresponding to the PY regions of beta and gammaENaC were immobilized on a sensor chip and "real-time" kinetics of their binding to recombinant WW proteins was determined. Specificity of the interactions was established by competition experiment, as well as by monitoring effects of a point mutation known to impair Nedd4/ENaC binding. These data provides the first determination of association, dissociation and equilibrium constants for the interactions between WW2 and beta or gammaENaC.

Animals↗

PubMed2002

Alternative splicing determines the domain structure of WWP1, a Nedd4 family protein.

Nedd-4-like proteins are E3 ubiquitin-ligase molecules which regulate key trafficking decisions, including targeting of proteins to proteosomes or lysosomes. Here we show that a human Nedd4 family gene, WWP1, is localized on 8q21 and generates at least six isoforms through alternative splicing. We show that alternative splicing affects the domain structure of WWP1, with forms that contain or lack an N-terminal C2 domain. Interestingly, the relative ratio of these forms varies in a tissue-specific manner. Other splice forms were also identified which may disrupt the structure of the C2 domain by removing its predicted C-terminal beta-strands. One splice form generates, through the introduction of a reading frame shift, a C2 domain-only form of WWP1. We discuss the hypothesis that regulation of splice site usage may modulate the activity of WWP1 and possibly other Nedd4 family proteins.

Alternative Splicing↗

PubMed1999

Role of TSG101 in uterine cervix cancer.

OBJECTIVE: TSG101 was first described as a possible tumor suppressor gene in breast cancer. To determine whether TSG101 might play a role in cervical carcinogenesis, we examined a panel of cervical cancer cell lines and primary tumor specimens for transcript abnormalities and mutations in TSG101. METHODS: Total RNA was derived from cell line cultures or primary tumor specimens. We performed nested reverse transcription polymerase chain reaction (RT-PCR) with eight overlapping primer sets, followed by single-strand conformational polymorphism (SSCP) analysis, to screen for mutations in the TSG101 open reading frame. Representative normal and shifted SSCP bands were sequenced. To identify abnormal-sized transcripts, we performed RT-PCR with primers flanking the open reading frame followed by gel electrophoresis. RESULTS: Mutational analysis was performed on cDNAs from 20 primary cervical tumors and 8 cervical carcinoma cell lines. Two polymorphisms were identified, neither of which resulted in an altered amino acid sequence. Transcript analysis was performed on a subset of 16 primary cervix tumors and 6 cervix carcinoma cell lines. The wild-type transcript (1228 bp) was the dominant transcript expressed in all samples. A transcript measuring 330 bp was detected in 5 of 6 cell lines and 11 of 16 primary tumor specimens. CONCLUSION: Our results suggest that mutations in TSG101 rarely occur in carcinomas of the uterine cervix. However, the presence of minor aberrant TSG101 transcripts is a common feature. The relationship between aberrant transcription and carcinogenesis should be further investigated.

Acid Anhydride Hydrolases↗

PubMed2006

Polymorphisms in eight host genes associated with control of HIV replication do not mediate elite control of viral replication in SIV-infected Indian rhesus macaques.

Polymorphisms in several host genes in HIV-infected individuals facilitate slow progression to AIDS. We have identified several SIV-infected Indian rhesus macaques that naturally control viral replication. We investigated whether spontaneous control of SIV in any of these animals could be explained by mutations in host genes. Such variables could confound studies of associations between MHC class I alleles and control of viral replication. We searched for polymorphisms in CCR5, CXCR6, GPR15, RANTES, IL-10, APOBEC3G, TNF-alpha, and TSG101 and looked for associations with decreased viral replication. We did not detect any correlations between plasma viral concentration and polymorphisms in host genes examined in this study. In addition, we did not find the polymorphisms present in humans in any of our macaques.

Animals↗

PubMed2001

Genetic interactions between the ESS1 prolyl-isomerase and the RSP5 ubiquitin ligase reveal opposing effects on RNA polymerase II function.

Transcription of protein-coding genes by RNA polymerase II (pol II) is a highly coordinated process that requires the stepwise association of distinct protein complexes with the C-terminal domain (CTD) of Rpbl, the largest subunit of RNA pol II. Interaction of these complexes with the CTD might be subject to regulation by proteins such as Ess1 and Rsp5. Ess1, a prolyl-isomerase, binds the CTD and is thought to play a positive role in pol II transcription by generating conformational isomers of the CTD. Rsp5, a ubiquitin ligase, binds the CTD and is thought to play a negative role in transcription by mediating Rpbl ubiquitination and degradation. In this paper, we demonstrate that ESS1 and RSP5 interact genetically and that these interactions occur via RPBI. We show that over-expression of RSP5 enhances the growth defect of ess1ts cells and this effect is reversed by introducing extra copies of RPB1. Over-expression of RSP5 also mimics the sensitivity of ess1ts mutant cells to the toxicity of plasmids carrying dominant-negative CTD mutations, whereas mutations in RSP5 suppress this effect. Using a modified two-hybrid assay, we also demonstrate that Essl and Rsp5 compete directly for binding to the CTD. The results suggest a model in which Essl and Rsp5 act opposingly on pol II function to control the level of pol II available for transcription.

Binding, Competitive↗

PubMed1999

A Pichia pastoris VPS15 homologue is required in selective peroxisome autophagy.

Methylotrophic yeasts contain large peroxisomes during growth on methanol. Upon exposure to excess glucose or ethanol these organelles are selectively degraded by autophagy. Here we describe the cloning of a Pichia pastoris gene (PpVPS15) involved in peroxisome degradation, which is homologous to Saccharomyces cerevisiae VPS15. In methanol-grown cells of a P. pastoris VPS15 deletion strain, the levels of peroxisomal marker enzymes remained high after addition of excess glucose or ethanol. Electron microscopic studies revealed that the organelles were not taken up by vacuoles, suggesting that PpVPS15 is required at an early stage in peroxisome degradation.

Amino Acid Sequence↗