E3 ubiquitin-protein ligase activity of Parkin is dependent on cooperative interaction of RING finger (TRIAD) elements.

The parkin gene codes for a 465-amino acid protein which, when mutated, results in autosomal recessive juvenile parkinsonism (AR-JP). Symptoms of AR-JP are similar to those of idiopathic Parkinson's disease, with the notable exception being the early onset of AR-JP. We have cloned and expressed human Parkin in Escherichia coli and have examined Parkin-mediated ubiquitination in an in vitro ubiquitination assay using purified recombinant proteins. We found that Parkin has E3 ubiquitin ligase activity in this system, demonstrating for the first time that the E3 activity is an intrinsic function of the Parkin protein and does not require posttranslational modification or association with cellular proteins other than an E2 (human Ubc4 E2 was utilized in this ubiquitination assay). Mutagenesis of individual elements of the conserved RING TRIAD domain indicated that at least two elements were required for ubiquitin ligase activity and suggested a functional cooperation between the RING finger elements. Since the activity assays were conducted with recombinant proteins purified from E. coli, this is the first time TRIAD element interaction has been demonstrated as an intrinsic feature of Parkin E3 activity.

Cancer-predisposing mutations within the RING domain of BRCA1: loss of ubiquitin protein ligase activity and protection from radiation hypersensitivity.

BRCA1 is a breast and ovarian cancer-specific tumor suppressor that seems to be involved in transcription and DNA repair. Here we report that BRCA1 exhibits a bona fide ubiquitin (Ub) protein ligase (E3) activity, and that cancer-predisposing mutations within the BRCA1 RING domain abolish its Ub ligase activity. Furthermore, these mutants are unable to reverse gamma-radiation hypersensitivity of BRCA1-null human breast cancer cells, HCC1937. Additionally, these mutations within the BRCA1 RING domain are not capable of restoring a G(2) + M checkpoint in HCC1937 cells. These results establish a link between Ub protein ligase activity and gamma-radiation protection function of BRCA1, and provide an explanation for why mutations within the BRCA1 RING domain predispose to cancer. Furthermore, we propose that the analysis of the Ub ligase activity of RING-domain mutations identified in patients may constitute an assay to predict predisposition to cancer.

Hrd1p/Der3p is a membrane-anchored ubiquitin ligase required for ER-associated degradation.

In eukaryotes, endoplasmic reticulum-associated degradation (ERAD) functions in cellular quality control and regulation of normal ER-resident proteins. ERAD proceeds by the ubiquitin-proteasome pathway, in which the covalent attachment of ubiquitin to proteins targets them for proteasomal degradation. Ubiquitin-protein ligases (E3s) play a crucial role in this process by recognizing target proteins and initiating their ubiquitination. Here we show that Hrd1p, which is identical to Der3p, is an E3 for ERAD. Hrd1p is required for the degradation and ubiquitination of several ERAD substrates and physically associates with relevant ubiquitin-conjugating enzymes (E2s). A soluble Hrd1 fusion protein shows E3 activity in vitro - catalysing the ubiquitination of itself and test proteins. In this capacity, Hrd1p has an apparent preference for misfolded proteins. We also show that Hrd1p functions as an E3 in vivo, using only Ubc7p or Ubc1p to specifically program the ubiquitination of ERAD substrates.

Biochemistry. Ubiquitination--more than two to tango.

The ubiquitin pathway in the cell is an elegant system for targeting unwanted proteins for degradation. Three enzymes, E1, E2, and E3, are responsible for attaching the ubiquitin tag to proteins destined to be chopped up. In their Perspective, Joazeiro and Hunter discuss new structural findings that reveal the part played by an E3 called c-Cbl in this ubiquitinating process.

Brassinosteroid-insensitive-1 is a ubiquitously expressed leucine-rich repeat receptor serine/threonine kinase.

Brassinosteroid (BR) mutants of Arabidopsis have pleiotropic phenotypes and provide evidence that BRs function throughout the life of the plant from seedling development to senescence. Screens for BR signaling mutants identified one locus, BRI1, which encodes a protein with homology to leucine-rich repeat receptor serine (Ser)/threonine (Thr) kinases. Twenty-seven alleles of this putative BR receptor have been isolated to date, and we present here the identification of the molecular lesions of 14 recessive alleles that represent five new mutations. BR-insensitive-1 (BRI1) is expressed at high levels in the meristem, root, shoot, and hypocotyl of seedlings and at lower levels later in development. Confocal microscopy analysis of full-length BRI1 fused to green fluorescent protein indicates that BRI1 is localized in the plasma membrane, and an in vitro kinase assay indicates that BRI1 is a functional Ser/Thr kinase. Among the bri1 mutants identified are mutants in the kinase domain, and we demonstrate that one of these mutations severely impairs BRI1 kinase activity. Therefore, we conclude that BRI1 is a ubiquitously expressed leucine-rich repeat receptor that plays a role in BR signaling through Ser/Thr phosphorylation.

The APC11 RING-H2 finger mediates E2-dependent ubiquitination.

Polyubiquitination marks proteins for degradation by the 26S proteasome and is carried out by a cascade of enzymes that includes ubiquitin-activating enzymes (E1s), ubiquitin-conjugating enzymes (E2s), and ubiquitin ligases (E3s). The anaphase-promoting complex or cyclosome (APC/C) comprises a multisubunit ubiquitin ligase that mediates mitotic progression. Here, we provide evidence that the Saccharomyces cerevisiae RING-H2 finger protein Apc11 defines the minimal ubiquitin ligase activity of the APC. We found that the integrity of the Apc11p RING-H2 finger was essential for budding yeast cell viability, Using purified, recombinant proteins we showed that Apc11p interacted directly with the Ubc4 ubiquitin conjugating enzyme (E2). Furthermore, purified Apc11p was capable of mediating E1- and E2-dependent ubiquitination of protein substrates, including Clb2p, in vitro. The ability of Apc11p to act as an E3 was dependent on the integrity of the RING-H2 finger, but did not require the presence of the cullin-like APC subunit Apc2p. We suggest that Apc11p is responsible for recruiting E2s to the APC and for mediating the subsequent transfer of ubiquitin to APC substrates in vivo.

The inhibitor of apoptosis, cIAP2, functions as a ubiquitin-protein ligase and promotes in vitro monoubiquitination of caspases 3 and 7.

The inhibitor of apoptosis, cIAP2, contains a putative Ring finger motif at the C terminus. Using in vitro ubiquitination assays, we found that the Ring finger of cIAP2 alone possesses intrinsic ubiquitin ligase activity and promotes substrate-independent ubiquitination. It also promotes ubiquitination of caspases 3 and 7 but not caspase-1. The Ring fingers of c-Cbl and Apc11 failed to promote caspase-7 ubiquitination, suggesting that the Ring finger of cIAP2 itself is involved in substrate recognition.

The tyrosine kinase negative regulator c-Cbl as a RING-type, E2-dependent ubiquitin-protein ligase.

Ubiquitination of receptor protein-tyrosine kinases (RPTKs) terminates signaling by marking active receptors for degradation. c-Cbl, an adapter protein for RPTKs, positively regulates RPTK ubiquitination in a manner dependent on its variant SRC homology 2 (SH2) and RING finger domains. Ubiquitin-protein ligases (or E3s) are the components of ubiquitination pathways that recognize target substrates and promote their ligation to ubiquitin. The c-Cbl protein acted as an E3 that can recognize tyrosine-phosphorylated substrates, such as the activated platelet-derived growth factor receptor, through its SH2 domain and that recruits and allosterically activates an E2 ubiquitin-conjugating enzyme through its RING domain. These results reveal an SH2-containing protein that functions as a ubiquitin-protein ligase and thus provide a distinct mechanism for substrate targeting in the ubiquitin system.

Alternative outcomes in assembly of promoter complexes: the roles of TBP and a flexible linker in placing TFIIIB on tRNA genes.

Saccharomyces cerevisiae transcription factor (TF) IIIB, a TATA-binding protein (TBP)-containing multisubunit factor, recruits RNA polymerase (Pol) III for multiple rounds of transcription. TFIIIC is an assembly factor for TFIIIB on TATA-less tRNA gene promoters. To investigate the role of TBP-DNA interactions in tRNA gene transcription, we generated sequence substitutions in the SUP4 tRNATyr gene TFIIIB binding site. Purified transcription proteins were used to analyze the selection of transcription initiation sites and the physical structures of the protein complexes formed on these mutant genes. We show that the association of TFIIIB with tRNA genes proceeds through an initial step of binding-site selection that is codirected by its TBP subunit and by TFIIIC. TFIIIB is assembled in a predominantly metric manner with regard to box A, the start site-proximal binding site of TFIIIC, but TFIIIC opens a window within which wild-type TBP can select the TFIIIB-binding site. Despite its clear preference for AT-rich sequences, TBP can mediate TFIIIB assembly at diverse DNA sequences, including stretches containing only G and C. However, a mutant TBP, m3, which recognizes TATAAA and TGTAAA and is active for Pol III transcription, utilizes other sequences only poorly. We also show that alternative alignments between DNA-bound TFIIIB and TFIIIC are possible, implying a remarkably flexible linkage, and suggest that Tfc4, the TFIIIB-assembling subunit of TFIIIC, could be responsible for such elasticity. The relevance of these findings to alternative initiation of Pol II- and other Pol III-transcribed genes is discussed.

Yeast RNA polymerase III: transcription complexes and RNA synthesis.

In this article we review and summarize salient features of the mechanism of transcription by eukaryotic RNA polymerase III, focusing a considerable part of the account on the work of our laboratory, dealing with Saccharomyces cerevisiae, but emphasizing properties that are common to all eukaryotic RNA polymerases. The following topics are briefly discussed: promoter structure; transcription factors; internal structure of transcription complexes and DNA-protein interactions; U6 genes; promoter opening and RNA chain elongation; hydrolytic RNA retraction.

Identical components of yeast transcription factor IIIB are required and sufficient for transcription of TATA box-containing and TATA-less genes.

Specific transcription by RNA polymerase III requires recognition of the promoter-bound transcription factor IIIB (TFIIIB), of which the TATA-binding protein (TBP) is a subunit. The recruitment of TFIIIB to TATA-less genes is mediated by protein-protein interactions with transcription factor IIIC (TFIIIC) bound to the box A and box B elements. Here we examine interactions involved in the recruitment of TFIIIB to the TATA element-containing yeast U6 small nuclear RNA gene SNR6. TFIIIC is not required for the formation of TFIIIB-SNR6 gene complexes with purified components. The same three components of TFIIIB that are necessary for TFIIIC-dependent transcription of tRNA genes (recombinant TBP and Brf and the denaturing-gel-purified 90-kDa subunit) are required and sufficient for TATA box-directed U6 transcription. Despite its TFIIIC-independent, DNA sequence-dependent assembly, the TFIIIB-SNR6 complex shares important features with tDNA- and 5S rDNA-TFIIIB complexes, such as extent and location of footprint, stability, and resistance to heparin. These properties are clearly distinct from those of a TBP-SNR6 complex. In the SNR6 gene, box B, the primary binding site for TFIIIC, is suboptimally spaced relative to box A. At limiting TBP concentrations and on bare DNA, TFIIIC stimulates the formation of TFIIIB complexes with SNR6 but contributes poorly, at best, to the formation of properly placed complexes.

The role of the TATA-binding protein in the assembly and function of the multisubunit yeast RNA polymerase III transcription factor, TFIIIB.

The Saccharomyces cerevisiae RNA polymerase III transcription factor (TF)IIIB has been assembled from three components. An assembly pathway of these polypeptides, which specifies their interactions, has been determined. The TATA-binding protein, TBP, and the TFIIB-related BRF1 gene product BRF, together reconstitute the transcription factor activity and TFIIC-dependent DNA-binding activity of the B' component of TFIIIB. BRF alone weakly binds to a TFIIIC-tRNA gene complex; TBP greatly stabilizes this interaction. B" transcription factor activity is recovered with its previously identified 90 kd polypeptide from SDS-polyacrylamide gels. Incorporation of the 90 kd B" protein into the transcription complex requires TBP. The heparin-resistant TFIIIB-DNA complex retains all three of its constituent proteins, TBP, BRF, and B".

Isolation of Drosophila genes encoding G protein-coupled receptor kinases.

G protein-coupled receptors are regulated via phosphorylation by a variety of protein kinases. Recently, termination of the active state of two such receptors, the beta-adrenergic receptor and rhodopsin, has been shown to be mediated by agonist- or light-dependent phosphorylation of the receptor by members of a family of protein-serine/threonine kinases (here referred to as G protein-coupled receptor kinases). We now report the isolation of a family of genes encoding a set of Drosophila protein kinases that appear to code for G protein-coupled receptor kinases. These proteins share a high degree of sequence homology with the bovine beta-adrenergic receptor kinase. The presence of a conserved family of G protein-coupled receptor kinases in vertebrates and invertebrates points to the central role of these kinases in signal transduction cascades.

Generation of cell lines to study the role played by oncogenes and anti-oncogenes in cell proliferation control.

We report the generation of stable transfectant cell lines by DNA-mediated transfection that overexpress viral and/or cellular oncogenes. Expression of heterologous genes (FBJ- and FBR-v-fos, polyoma large and middle T) was confirmed by Northern hybridization, immunofluorescence and immunoprecipitation. We also describe the isolation of two retinoblastoma cell lines from human tumors. Neuronal and glial markers were used to confirm the origin of these cell lines. Oncogene transfectant and retinoblastoma cell lines will be used to assess Rb expression and the possible role of its gene product in cell proliferation control and neoplasia.

Generation of cell lines to study the role played by oncogenes and anti-oncogenes in cell proliferation control

We report the generation of stable transfectant cell lines by DNA-mediated transfection that overexpress viral and/or cellular oncoghenes. Expression of heterologous genes (FBJ- and FBR-v-fos, polyoma large and middle T) was confirmed by Northern hybridization, immunofluorescence and immunoprecipitation. We also describe the isolation of two retinoblastoma cell lines from human tumors. Neuronal and glial markers were used to confirm the origin of these cell lines. Oncogene transfectant and retinoblastoma cell lines will be used to assess Rb expression and the possible role of its gene product in cell proliferation control and neoplasia

Generation of myc/fos transfectant Balb-3T3 cell lines.

Early and transient expression of proto-oncogenes c-fos and c-myc is involved in the mitogenic response to PDGF (platelet-derived growth factor). We used DNA-mediated transfection to approach the role played by these genes in cell growth control by PDGF and in growth deregulation (neoplasia). Cloned pFBJ-2 (v-fos) and glucocorticoid-inducible mouse c-myc were co-transfected with a neo genetic marker to allow a neutral selection on the basis of resistance to the neomycin derivative geneticin G418. pFBJ-2 transfection was found to interfere with the number of G418-resistant (G418r) colonies. By using a v-fos-deleted pFBJ-2 construct, the deleterious effect was attributed to v-fos coding sequences. Cellular fos gene disruption, by homologous recombination with exogenous v-fos, is proposed as the basis for the deleterious effect. Co-transfection with MMTV-H3-c-myc effectively counteracts the negative effects of v-fos. Different from the parental line or single myc or fos transfectants, double myc/fos transfectants are morphologically transformed. Double transfectants still retain the PDGF requirement for growth in monolayer cultures.