Inducible degradation of IkappaBalpha by the proteasome requires interaction with the F-box protein h-betaTrCP.

Activation of NF-kappaB transcription factors requires phosphorylation and ubiquitin-proteasome-dependent degradation of IkappaB proteins. We provide evidence that a human F-box protein, h-betaTrCP, a component of Skp1-Cullin-F-box protein (SCF) complexes, a new class of E3 ubiquitin ligases, is essential for inducible degradation of IkappaBalpha. betaTrCP associates with Ser32-Ser36 phosphorylated, but not with unmodified IkappaBalpha or Ser32-Ser36 phosphorylation-deficient mutants. Expression of a F-box-deleted betaTrCP inhibits IkappaBalpha degradation, promotes accumulation of phosphorylated Ser32-Ser36 IkappaBalpha, and prevents NF-kappaB-dependent transcription. Our findings indicate that betaTrCP is the adaptor protein required for IkappaBalpha recognition by the SCFbetaTrCP E3 complex that ubiquitinates IkappaBalpha and makes it a substrate for the proteasome.

The F-box protein beta-TrCP associates with phosphorylated beta-catenin and regulates its activity in the cell.

Defects in beta-catenin regulation contribute to the neoplastic transformation of mammalian cells. Dysregulation of beta-catenin can result from missense mutations that affect critical sites of phosphorylation by glycogen synthase kinase 3beta (GSK3beta). Given that phosphorylation can regulate targeted degradation of beta-catenin by the proteasome, beta-catenin might interact with an E3 ubiquitin ligase complex containing an F-box protein, as is the case for certain cell cycle regulators. Accordingly, disruption of the Drosophila F-box protein Slimb upregulates the beta-catenin homolog Armadillo. We reasoned that the human homologs of Slimb - beta-TrCP and its isoform beta-TrCP2 (KIAA0696) - might interact with beta-catenin. We found that the binding of beta-TrCP to beta-catenin was direct and dependent upon the WD40 repeat sequences in beta-TrCP and on phosphorylation of the GSK3beta sites in beta-catenin. Endogenous beta-catenin and beta-TrCP could be coimmunoprecipitated from mammalian cells. Overexpression of wild-type beta-TrCP in mammalian cells promoted the downregulation of beta-catenin, whereas overexpression of a dominant-negative deletion mutant upregulated beta-catenin protein levels and activated signaling dependent on the transcription factor Tcf. In contrast, beta-TrCP2 did not associate with beta-catenin. We conclude that beta-TrCP is a component of an E3 ubiquitin ligase that is responsible for the targeted degradation of phosphorylated beta-catenin.

A novel human WD protein, h-beta TrCp, that interacts with HIV-1 Vpu connects CD4 to the ER degradation pathway through an F-box motif.

HIV-1 Vpu interacts with CD4 in the endoplasmic reticulum and triggers CD4 degradation, presumably by proteasomes. Human beta TrCP identified by interaction with Vpu connects CD4 to this proteolytic machinery, and CD4-Vpu-beta TrCP ternary complexes have been detected by coimmunoprecipitation. beta TrCP binding to Vpu and its recruitment to membranes require two phosphoserine residues in Vpu essential for CD4 degradation. In beta TrCP, WD repeats at the C terminus mediate binding to Vpu, and an F box near the N terminus is involved in interaction with Skp1p, a targeting factor for ubiquitin-mediated proteolysis. An F-box deletion mutant of beta TrCP had a dominant-negative effect on Vpu-mediated CD4 degradation. These data suggest that beta TrCP and Skp1p represent components of a novel ER-associated protein degradation pathway that mediates CD4 proteolysis.

Binding of HIV-1 Nef to a novel thioesterase enzyme correlates with Nef-mediated CD4 down-regulation.

Nef is a 27-kDa myristoylated protein conserved in primate lentiviruses. In vivo, simian immunodeficiency virus Nef is required in macaques to produce a high viral load and full pathological effects. Nef has at least three major effects in vitro, induction of CD4 down-regulation, alteration of T cell activation pathways, and enhancement of viral infectivity. We have used the yeast two-hybrid system to identify cellular proteins that interact with HIV-1Lai Nef and could mediate Nef function. A human cDNA was isolated that encodes a new type of thioesterase, an enzyme that cleaves thioester bonds. This novel thioesterase is unlike the animal types I and II thioesterases previously cloned but is homologous to the Escherichia coli thioesterase II. Nef and this thioesterase interact in vitro and are co-immunoprecipitated by anti-Nef antibodies in CEM cells expressing Nef. Nef alleles from human immunodeficiency virus-1 (HIV-1) isolates unable to down-regulate CD4 do not react or react poorly with thioesterase. An HIV-1 NefLai mutant selected for its lack of interaction with thioesterase was also unable to down-regulate CD4 cell-surface expression. These observations suggest that this human thioesterase is a cellular mediator of Nef-induced CD4 down-regulation.

Inhibition of prokaryotic cell growth by HIV1 Vpr.

We have cloned the nef, vif, vpr and vpu genes of HIV1 in the pGEX system to produce auxiliary proteins of HIV1 as N-terminal fusions with glutathione S-transferase (GST). Some GST proteins are difficult to obtain under standard conditions. The synthesis and solubility varied considerably from one protein to another. We investigated the reasons for the poor production of GST-Vpr, GST-Vpu and GST-Vif. Interestingly, using this GST prokaryotic model, we demonstrated that Vpr, which is known to block the cell cycle of mammalian and yeast cells at the G2 phase, is also bacteriostatic for Escherichia coli. The effect on E. coli was specific to Vpr, and was not linked to the expression of the other HIV1 proteins. This suggests that Vpr interferes with components of cell replication that are conserved from prokaryotes to eukaryotes. Thus, E. coli appears to be a convenient model system for studies on the function of Vpr.

Interaction between the cytoplasmic domains of HIV-1 Vpu and CD4: role of Vpu residues involved in CD4 interaction and in vitro CD4 degradation.

The Vpu and CD4 cytoplasmic domains were found, by using a two-hybrid assay in yeast, to interact in the absence of their membrane anchor domains. Studies on several deletion and point mutants revealed that the overall structure of the Vpu cytoplasmic domain is required for this interaction. The Vpu amino acid residues involved in the interaction with CD4 were identified. Deletion of the C-terminal residues of Vpu, required for CD4 degradation, as well as the double mutation on the casein kinase II phosphorylation sites S52N-S56N, also involved in CD4 degradation, resulted in the loss of interaction with CD4 and in the inability to induce CD4 degradation. These results suggest that the ability of Vpu to mediate the degradation of CD4 is linked to its capacity to physically interact with CD4. However, additional mutagenesis on the S52 site revealed that the interaction between the cytoplasmic domains of Vpu and CD4 is not sufficient for in vitro Vpu-mediated CD4 degradation.

Basal promoter and enhancer element of yeast U6 snRNA gene.

The yeast U6 snRNA gene, SNR6, transcribed by RNA polymerase III or C, is shown to have a mixed promoter with upstream, intragenic and downstream elements. The distant downstream B block behaves as a typical enhancer element. Required in vivo, and for transcription of chromatin templates in vitro, it was also active in reversed orientation. As shown by footprinting and electron microscopy, the factor TFIIIC, or tau, bound the B block in an oriented manner and was able to induce DNA looping. The factor TFIIIC appeared to act via a weak A block located at position +21. This A block-related motif was essential in vivo and with chromatin templates. When changed into a consensus A block it favored DNA looping by TFIIIC firmly anchored on the B block, and activated a B block lacking gene in vivo and in vitro. The role of the TATA box at -30 was most apparent using a purified transcription system. With the A block, it appeared to contribute to start site selection. The results suggest a model where three weak promoter elements collaborate to assemble the transcription complex by DNA looping and synergistic protein-DNA interactions.

TFIIIC relieves repression of U6 snRNA transcription by chromatin.

The U6 small nuclear (sn)RNA gene (SNR6) from the yeast Saccharomyces cerevisiae is transcribed by RNA polymerase III in vivo. This gene is unusual in having a TATA box at position -30, and an essential B-block element located downstream of the T-rich termination signal. The B block is one of the two intragenic promoter elements of transfer RNA genes that are recognized by transcription factor (TF)IIIC (ref. 4). But accurate in vitro transcription of yeast U6 snRNA gene by PolIII in a purified system requires only TFIIIB components, including the TATA-box binding protein TBP. Here we report that, after nucleosome reconstitution or chromatin assembly, U6 snRNA synthesis becomes dependent on TFIIIC and on the integrity of the B-block element. This observation resolves an apparent paradox between in vitro and in vivo results concerning the necessity of the downstream B-block element and sheds light on a new role of TFIIIC in gene activation.

Participation of the TATA factor in transcription of the yeast U6 gene by RNA polymerase C.

Fractionation of transcription extracts has led to the identification of multiple transcription factors specific for each form of nuclear RNA polymerase. Accurate transcription in vitro of the yeast U6 RNA gene by RNA polymerase C requires at least two factors. One of them was physically and functionally indistinguishable from transcription factor IID (TFIID or BTF1), a pivotal component of polymerase B transcription complexes, which binds to the TATA element. Purified yeast TFIID (yIID) or bacterial extracts that contained recombinant yIID were equally competent to direct specific transcription of the U6 gene by RNA polymerase C. The results suggest the formation of a hybrid transcription machinery, which may imply an evolutionary relation between class B and class C transcription factors.

The U6 gene of Saccharomyces cerevisiae is transcribed by RNA polymerase C (III) in vivo and in vitro.

Unlike the majority of genes encoding small nuclear RNAs, which are transcribed by RNA polymerase B, the U6 gene contains features found in both class B and class C genes, indicating the involvement of a combination of transcription factors normally specific to each class of genes. We present direct genetic and biochemical evidence that the U6 gene of Saccharomyces cerevisiae is transcribed by RNA polymerase C in vivo as well as in vitro. A mutant strain with a temperature-sensitive defect in the large subunit of RNA polymerase C that results in defective transcription of tRNA and 5S RNA genes shows a corresponding defect in U6 RNA levels. Also, purified RNA polymerase C transcribes the U6 gene when supplemented with partially purified TFIIIB. The other class C transcription factors, TFIIIA and Tau (TFIIIC), are not required in this system.