Analysis of the expression and localisation of a LAP protein, human scribble, in the normal and neoplastic epithelium of uterine cervix.

Recently, a LAP protein, scribble, was identified in Drosophila epithelia as a basolateral protein that controls the apical-basolateral polarity. Loss of scribble causes disorganisation and overgrowth of the epithelia. Scribble has a human homologue, human scribble (hScrib), which is a substrate of ubiquitin-mediated degradation by human papillomavirus E6 and the E6AP ubiquitin-protein ligase. In the present study, we revealed that hScrib localised to the basolateral regions of the epithelial cell line MDCK and human uterine cervical epithelial tissues by immunofluorescence. Human scribble colocalised rather with the adherens junction protein E-cadherin, but not with the tight junction protein ZO-1. Histochemical analysis showed a dramatic decrease in the expression of hScrib with the progression of disease from normal uterine cervical tissues to invasive cervical cancers through the precursor lesions. In contrast, the expression of hScrib was retained in the throughout epithelial layer of the HPV-negative cervical high-grade squamous intraepithelial lesions (H-SIL). Although quantitative RT-PCR revealed no significant downregulation of hScrib mRNA expression in the H-SIL, it revealed a clear downregulation in the invasive cancers. These results suggest the possibility that degradation by HPV E6 is one of the causal roles for the progressive decrease of hScrib expression during the disease progression from low-grade squamous intraepithelial lesions to H-SIL, and a cooperative role of downregulation of hScrib mRNA expression and ubiquitin-mediated degradation of hScrib by E6 and E6AP led to the complete decrease of hScrib expression during the process of carcinogenesis from H-SIL to invasive cancer. These data underscore the importance of hScrib in the construction of tissue architecture and prevention of cancer development.

Rhabdoviruses and the cellular ubiquitin-proteasome system: a budding interaction.

The matrix (M) proteins of vesicular stomatitis virus (VSV) and rabies virus (RV) play a key role in both assembly and budding of progeny virions. A PPPY motif (PY motif or late-budding domain) is conserved in the M proteins of VSV and RV. These PY motifs are important for virus budding and for mediating interactions with specific cellular proteins containing WW domains. The PY motif and flanking sequences of the M protein of VSV were used as bait to screen a mouse embryo cDNA library for cellular interactors. The mouse Nedd4 protein, a membrane-localized ubiquitin ligase containing multiple WW domains, was identified from this screen. Ubiquitin ligase Rsp5, the yeast homolog of Nedd4, was able to interact both physically and functionally with full-length VSV M protein in a PY-dependent manner. Indeed, the VSV M protein was multiubiquitinated by Rsp5 in an in vitro ubiquitination assay. To demonstrate further that ubiquitin may be involved in the budding process of rhabdoviruses, proteasome inhibitors (e.g., MG132) were used to decrease the level of free ubiquitin in VSV- and RV-infected cells. Viral titers measured from MG132-treated cells were reproducibly 10- to 20-fold lower than those measured from untreated control cells, suggesting that free ubiquitin is important for efficient virus budding. Last, release of a VSV PY mutant was not inhibited in the presence of MG132, signifying that the functional L domain of VSV is required for the inhibitory effect exhibited by MG132. These data suggest that the cellular ubiquitin-proteasome machinery is involved in the budding process of VSV and RV.

Nuclear import/export of hRPF1/Nedd4 regulates the ubiquitin-dependent degradation of its nuclear substrates.

The ubiquitin-protein ligase (E3), hRPF1/Nedd4, is a component of the ubiquitin-proteasome pathway responsible for substrate recognition and specificity. Although previously characterized as a regulator of the stability of cytoplasmic proteins, hRPF1/Nedd4 has also been suggested to have a role in the nucleus. However, in light of the cytoplasmic localization of hRPF1/Nedd4, it is unclear whether bona fide nuclear substrates of hRPF1/Nedd4 exist, and if so, what mechanism may allow a cytoplasmic ubiquitin ligase to manifest nuclear activity. Our search for nuclear substrates led to the identification of the human proline-rich transcript, brain-expressed (hPRTB) protein, the ubiquitination and degradation of which is regulated by hRPF1/Nedd4. Interestingly, hPRTB colocalizes with the splicing factor SC35 in nuclear speckles. Finally, we demonstrate that hRPF1/Nedd4 is indeed capable of entering the nucleus; however, the presence of a functional Rev-like nuclear export sequence in hRPF1/Nedd4 ensures a predominant cytoplasmic localization. Cumulatively, these findings highlight a nuclear role for the ubiquitin ligase hRPF1/Nedd4 and underscore cytoplasmic/nuclear localization as an important regulatory component of hRPF1/Nedd4-substrate recognition.

Localization of the Rsp5p ubiquitin-protein ligase at multiple sites within the endocytic pathway.

The Saccharomyces cerevisiae RSP5 gene encodes an essential HECT E3 ubiquitin-protein ligase. Rsp5p contains an N-terminal C2 domain, three WW domains in the central portion of the molecule, and a C-terminal catalytic HECT domain. A diverse group of substrates of Rsp5p and vertebrate C2 WW-domain-containing HECT E3s have been identified, including both nuclear and membrane-associated proteins. We determined the intracellular localization of Rsp5p and the determinants necessary for localization, in order to better understand how Rsp5p activities are coordinated. Using both green fluorescent protein fusions to Rsp5p and immunogold electron microscopy, we found that Rsp5p was distributed in a punctate pattern at the plasma membrane, corresponding to membrane invaginations that are likely sites of endosome formation, as well as at perivacuolar sites. The latter appeared to correspond to endocytic intermediates, as these structures were not seen in a sla2/end4-1 mutant, and double-immunogold labeling demonstrated colocalization of Rsp5p with the endosomal markers Pep12p and Vps32p. The C2 domain was an important determinant of localization; however, mutations that disrupted HECT domain function also caused mislocalization of Rsp5p, indicating that enzymatic activity is linked to localization. Deletion of the C2 domain partially stabilized Fur4p, a protein previously shown to undergo Rsp5p- and ubiquitin-mediated endocytosis; however, Fur4p was still ubiquitinated at the plasma membrane when the C2 domain was deleted from the protein. Together, these results indicate that Rsp5p is located at multiple sites within the endocytic pathway and suggest that Rsp5p may function at multiple steps in the ubiquitin-mediated endocytosis pathway.

Human scribble (Vartul) is targeted for ubiquitin-mediated degradation by the high-risk papillomavirus E6 proteins and the E6AP ubiquitin-protein ligase.

The high-risk human papillomavirus (HPV) E6 proteins stimulate the ubiquitination and degradation of p53, dependent on the E6AP ubiquitin-protein ligase. Other proteins have also been shown to be targeted for degradation by E6, including hDlg, the human homolog of the Drosophila melanogaster Discs large (Dlg) tumor suppressor. We show here that the human homolog of the Drosophila Scribble (Vartul) (hScrib) tumor suppressor protein is also targeted for ubiquitination by the E6-E6AP complex in vitro and that expression of E6 induces degradation of hScrib in vivo. Characterization of the E6AP-E6-hScrib complex indicated that hScrib binds directly to E6 and that the binding is mediated by the PDZ domains of hScrib and a carboxyl-terminal epitope conserved among the high-risk HPV E6 proteins. Green fluorescent protein-hScrib was localized to the periphery of MDCK cells, where it colocalized with ZO-1, a component of tight junctions. E6 expression resulted in loss of integrity of tight junctions, as measured by ZO-1 localization, and this effect was dependent on the PDZ binding epitope of E6. Thus, the high-risk HPV E6 proteins induce the degradation of the human homologs of two Drosophila PDZ domain-containing tumor suppressor proteins, hDlg and hScrib, both of which are associated with cell junction complexes. The fact that Scrib/Vart and Dlg appear to cooperate in a pathway that controls Drosophila epithelial cell growth suggests that the combined targeting of hScrib and hDlg is an important component of the biologic activity of high-risk HPV E6 proteins.

Human papillomavirus type 16 E6 induces self-ubiquitination of the E6AP ubiquitin-protein ligase.

The E6 protein of the high-risk human papillomaviruses (HPVs) and the cellular ubiquitin-protein ligase E6AP form a complex which causes the ubiquitination and degradation of p53. We show here that HPV16 E6 promotes the ubiquitination and degradation of E6AP itself. The half-life of E6AP is shorter in HPV-positive cervical cancer cells than in HPV-negative cervical cancer cells, and E6AP is stabilized in HPV-positive cancer cells when expression of the viral oncoproteins is repressed. Expression of HPV16 E6 in cells results in a threefold decrease in the half-life of transfected E6AP. E6-mediated degradation of E6AP requires (i) the binding of E6 to E6AP, (ii) the catalytic activity of E6AP, and (iii) activity of the 26S proteasome, suggesting that E6-E6AP interaction results in E6AP self-ubiquitination and degradation. In addition, both in vitro and in vivo experiments indicate that E6AP self-ubiquitination results primarily from an intramolecular transfer of ubiquitin from the active-site cysteine to one or more lysine residues; however, intermolecular transfer can also occur in the context of an E6-mediated E6AP multimer. Finally, we demonstrate that an E6 mutant that is able to immortalize human mammary epithelial cells but is unable to degrade p53 retains its ability to bind and degrade E6AP, raising the possibility that E6-mediated degradation of E6AP contributes to its ability to transform mammalian cells.

Structure of an E6AP-UbcH7 complex: insights into ubiquitination by the E2-E3 enzyme cascade.

The E6AP ubiquitin-protein ligase (E3) mediates the human papillomavirus-induced degradation of the p53 tumor suppressor in cervical cancer and is mutated in Angelman syndrome, a neurological disorder. The crystal structure of the catalytic hect domain of E6AP reveals a bilobal structure with a broad catalytic cleft at the junction of the two lobes. The cleft consists of conserved residues whose mutation interferes with ubiquitin-thioester bond formation and is the site of Angelman syndrome mutations. The crystal structure of the E6AP hect domain bound to the UbcH7 ubiquitin-conjugating enzyme (E2) reveals the determinants of E2-E3 specificity and provides insights into the transfer of ubiquitin from the E2 to the E3.

Rsp5 ubiquitin-protein ligase mediates DNA damage-induced degradation of the large subunit of RNA polymerase II in Saccharomyces cerevisiae.

Rsp5 is an E3 ubiquitin-protein ligase of Saccharomyces cerevisiae that belongs to the hect domain family of E3 proteins. We have previously shown that Rsp5 binds and ubiquitinates the largest subunit of RNA polymerase II, Rpb1, in vitro. We show here that Rpb1 ubiquitination and degradation are induced in vivo by UV irradiation and by the UV-mimetic compound 4-nitroquinoline-1-oxide (4-NQO) and that a functional RSP5 gene product is required for this effect. The 26S proteasome is also required; a mutation of SEN3/RPN2 (sen3-1), which encodes an essential regulatory subunit of the 26S proteasome, partially blocks 4-NQO-induced degradation of Rpb1. These results suggest that Rsp5-mediated ubiquitination and degradation of Rpb1 are components of the response to DNA damage. A human WW domain-containing hect (WW-hect) E3 protein closely related to Rsp5, Rpf1/hNedd4, also binds and ubiquitinates both yeast and human Rpb1 in vitro, suggesting that Rpf1 and/or another WW-hect E3 protein mediates UV-induced degradation of the large subunit of polymerase II in human cells.

Functional domains of the Rsp5 ubiquitin-protein ligase.

RSP5, an essential gene of Saccharomyces cerevisiae, encodes a hect domain E3 ubiquitin-protein ligase. Hect E3 proteins have been proposed to consist of two broad functional domains: a conserved catalytic carboxyl-terminal domain of approximately 350 amino acids (the hect domain) and a large, nonconserved amino-terminal domain containing determinants of substrate specificity. We report here the mapping of the minimal region of Rsp5 necessary for its essential in vivo function, the minimal region necessary to stably interact with a substrate of Rsp5 (Rpb1, the large subunit of RNA polymerase II), and the finding that the hect domain, by itself, is sufficient for formation of the ubiquitin-thioester intermediate. Mutations within the hect domain that affect either the ability to form a ubiquitin-thioester or to catalyze substrate ubiquitination abrogate in vivo function, strongly suggesting that the ubiquitin-protein ligase activity of Rsp5 is intrinsically linked to its essential function. The amino-terminal region of Rsp5 contains three WW domains and a C2 calcium-binding domain. Two of the three WW domains are required for the essential in vivo function, while the C2 domain is not, and requirements for Rpb1 binding and ubiquitination lie within the region required for in vivo function. Together, these results support the two-domain model for hect E3 function and indicate that the WW domains play a role in the recognition of at least some of the substrates of Rsp5, including those related to its essential function. In addition, we show that haploid yeast strains bearing complete disruptions of either of two other hect E3 genes of yeast, designated HUL4 (YJR036C) and HUL5 (YGL141W), are viable.

The role of E6AP in the regulation of p53 protein levels in human papillomavirus (HPV)-positive and HPV-negative cells.

The E6 protein encoded by the oncogenic human papillomaviruses (HPVs) targets p53 for ubiquitin-dependent proteolysis. E6-mediated p53 degradation requires the 100-kDa cellular protein E6-associated protein (E6AP). E6AP and E6 together provide the E3-ubiquitin protein ligase activity in the transfer of ubiquitin to p53. In vitro studies have shown that E6AP can form a high energy thiolester bond with ubiquitin and, in the presence of E6, transfer ubiquitin to p53. In this study we have addressed the role of E6AP in vivo in the degradation of p53. Overexpression of wild-type E6AP in HeLa cells, which are HPV18-positive and express E6, resulted in a decreased steady state level of p53 and a decrease in the half-life of p53. Mutant forms of E6AP proteins were identified that were catalytically incapable of participating in E6-dependent ubiquitination of p53 and functioned in a dominant-negative manner in that they inhibited the E6-mediated ubiquitination of p53 by the wild-type E6AP in vitro. Transient transfection of one of these dominant negative (dn) mutants resulted in an increase in both the steady state level and half-life of p53 in vivo in HeLa cells. Consistent with this observation, overexpression of the dn E6AP resulted in a marked G1 shift in the cell cycle profile. In contrast, dn E6AP had no effect on p53 levels in U2OS cells, an HPV-negative cell line that contains wild-type p53. These studies provide evidence for the involvement of E6AP in E6-mediated p53 degradation in vivo and also indicate that E6AP may not be involved in the regulation of p53 ubiquitination in the absence of E6.

The large subunit of RNA polymerase II is a substrate of the Rsp5 ubiquitin-protein ligase.

The E3 ubiquitin-protein ligases play an important role in controlling substrate specificity of the ubiquitin proteolysis system. A biochemical approach was taken to identify substrates of Rsp5, an essential hect (homologous to E6-AP carboxyl terminus) E3 of Saccharomyces cerevisiae. We show here that Rsp5 binds and ubiquitinates the largest subunit of RNA polymerase II (Rpb1) in vitro. Stable complex formation between Rsp5 and Rpb1 was also detected in yeast cell extracts, and repression of RSP5 expression in vivo led to an elevated steady-state level of Rpb1. The amino-terminal domain of Rsp5 mediates binding to Rpb1, while the carboxyl-terminal domain of Rpb1, containing the heptapeptide repeats characteristic of polymerase II, is necessary and sufficient for binding to Rsp5. Fusion of the Rpb1 carboxyl-terminal domain to another protein also causes that protein to be ubiquitinated by Rsp5. These findings indicate that Rsp5 targets at least a subset of cellular Rpb1 molecules for ubiquitin-dependent degradation and may therefore play a role in regulating polymerase II activities. In addition, the results support a model for hect E3 function in which the amino-terminal domain mediates substrate binding, while the carboxyl-terminal hect domain catalyzes ubiquitination of bound substrates.

The human E6-AP gene (UBE3A) encodes three potential protein isoforms generated by differential splicing.

The E6-AP gene (UBE3A) encodes an E3 ubiquitin-protein ligase that binds the human papillomavirus E6 oncoprotein and catalyzes the ubiquitination of p53. Recent studies have also established that mutations in E6-AP are the genetic basis of the Angelman syndrome in humans. In this study we present the genomic structure of the coding region of E6-AP and an analysis of a set of five E6-AP mRNAs with the potential to encode three protein isoforms of the E6-AP protein (isoforms I, II, and III) that differ at their extreme amino-termini. These transcripts were expressed in a variety of different cell lines examined.

Mechanism of HPV E6 proteins in cellular transformation.

The E6 protein is a major transforming protein of many types of papillomaviruses. Mechanistically, the best characterized E6 proteins are those of the high-risk genital HPVs (e.g. HPV-16 and 18 E6), which function, at least in part, by inactivating the p53 tumor suppressor protein. Biochemical studies have shown that this occurs by targeted degradation of p53, dependent on the E6-AP ubiquitin-protein ligase. The model that has emerged from E6/E6-AP-dependent p53 degradation has provided insight into both HPV-associated carcinogenesis and the problem of substrate specificity of the ubiquitin system. Several observations suggest that the high-risk HPV E6 proteins may also have activities in addition to inactivation of p53.

In vivo ubiquitination and proteasome-mediated degradation of p53(1).

The levels of the tumor suppressor protein p53 are generally quite low in normal cells, due in part to its rapid turnover. Previous studies have implicated ubiquitin-dependent proteolysis in the turnover of wild-type p53 but have not established whether or not p53 is itself a substrate of the ubiquitin system. In this study, inhibitors of the 26S proteasome have been used to further explore the role of ubiquitin proteolysis in regulating p53 turnover. Increased levels of the tumor suppressor protein p53 were observed in normal cells, as well as in cells expressing the human papillomavirus 16 E6 oncoprotein, on exposure of the cells to proteasome inhibitors. Pulse-chase experiments indicated that the increased p53 levels resulted from stabilization of the protein. Furthermore, ubiquitin-p53 conjugates were detected in untreated as well as gamma-irradiated cells, indicating that ubiquitin-dependent proteolysis plays a role in the normal turnover of p53. Increased levels of the cyclin:cyclin-dependent kinase inhibitor p21, a downstream effector of p53 function, were also observed in proteasome inhibitor-treated cells, and this increase was due in part to an increase in p2l mRNA.

A family of proteins structurally and functionally related to the E6-AP ubiquitin-protein ligase.

E6-AP is a 100-kDa cellular protein that interacts with the E6 protein of the cancer-associated human papillomavirus types 16 and 18. The E6/E6-AP complex binds to and targets the p53 tumor-suppressor protein for ubiquitin-mediated proteolysis. E6-AP is an E3 ubiquitin-protein ligase which accepts ubiquitin from an E2 ubiquitin-conjugating enzyme in the form of a thioester and then directly transfers the ubiquitin to targeted substrates. The amino acid sequence of E6-AP shows similarity to a number of protein sequences over an approximately 350-aa region corresponding to the carboxyl termini of both E6-AP and the E6-AP-related proteins. Of particular note is a conserved cysteine residue within the last 32-34 aa, which in E6-AP is likely to be the site of ubiquitin thioester formation. Two of the E6-AP-related proteins, a rat 100-kDa protein and a yeast 95-kDa protein (RSP5), both of previously unknown function, are shown here to form thioesters with ubiquitin. Mutation of the conserved cysteine residue of these proteins destroys their ability to accept ubiquitin. These data strongly suggest that the rat 100-kDa protein and RSP5, as well as the other E6-AP-related proteins, belong to a class of functionally related E3 ubiquitin-protein ligases, defined by a domain homologous to the E6-AP carboxyl terminus (hect domain).

Protein ubiquitination involving an E1-E2-E3 enzyme ubiquitin thioester cascade.

Ubiquitination of proteins involves the concerted action of the E1 ubiquitin-activating enzyme, E2 ubiquitin-conjugating enzymes and E3 ubiquitin-protein ligases. It has been proposed that E3s function as 'docking proteins', specifically binding substrate proteins and specific E2s, and that ubiquitin is then transferred directly from E2s to substrates. We show here that formation of a ubiquitin thioester on E6-AP, an E3 involved in the human papillomavirus E6-induced ubiquitination of p53 (refs 6-10), is an intermediate step in E6-AP-dependent ubiquitination. The order of ubiquitin transfer is from E1 to E2, from E2 to E6-AP, and finally from E6-AP to a substrate. This cascade of ubiquitin thioester complexes suggests that E3s have a defined enzymatic activity and do not function simply as docking proteins. The cysteine residue of E6-AP responsible for ubiquitin thioester formation was mapped to a region that is highly conserved among several proteins of unknown function, suggesting that these proteins share the ability to form thioesters with ubiquitin.

Identification of a human ubiquitin-conjugating enzyme that mediates the E6-AP-dependent ubiquitination of p53.

The E6 protein of the oncogenic human papillomavirus types 16 and 18 facilitates the rapid degradation of the tumor-suppressor protein p53 via the ubiquitin-dependent proteolytic pathway. The E6 protein binds to a cellular protein of 100 kDa termed E6-AP. The complex of E6 and E6-AP specifically interacts with p53 and induces the ubiquitination of p53 in a reaction which requires the ubiquitin-activating enzyme (E1) and a cellular fraction thought to contain a mammalian ubiquitin-conjugating enzyme (E2). This mammalian E2 activity could be replaced with bacterially expressed UBC8 from Arabidopsis thaliana, which belongs to a subfamily of E2s including yeast UBC4 and UBC5 which are highly conserved at the amino acid level. In this paper we describe the cloning of a human cDNA encoding a human E2 that we have designated UbcH5 and that is related to Arabidopsis UBC8 and the other members of this subfamily. We demonstrate that UbcH5 can function in the E6/E6-AP-induced ubiquitination of p53.