Component Of Ubiquitin Ligase Research Articles

The HIV1 Protein Vpr Acts to Promote G2 Cell Cycle Arrest by Engaging a DDB1 and Cullin4A-containing Ubiquitin Ligase Complex Using VprBP/DCAF1 as an Adaptor

The roles of the HIV1 protein Vpr in virus replication and pathogenesis remain unclear. Expression of Vpr in dividing cells causes cell cycle arrest in G(2). Vpr also facilitates low titer infection of terminally differentiated macrophages, enhances transcription, promotes apoptosis, and targets cellular uracil N-glycosylase for degradation. Using co-immunoprecipitation and tandem mass spectroscopy, we found that HIV1 Vpr engages a DDB1- and cullin4A-containing ubiquitin-ligase complex through VprBP/DCAF1. HIV2 Vpr has two Vpr-like proteins, Vpr and Vpx, which cause G(2) arrest and facilitate macrophage infection, respectively. HIV2 Vpr, but not Vpx, engages the same set of proteins. We further demonstrate that the interaction between Vpr and the ubiquitin-ligase components as well as further assembly of the ubiquitin-ligase are necessary for Vpr-mediated G(2) arrest. Our data support a model in which Vpr engages the ubiquitin ligase to deplete a cellular factor that is required for cell cycle progression into mitosis. Vpr, thus, functions like the HIV1 proteins Vif and Vpu to usurp cellular ubiquitin ligases for viral functions.

Renal Damage in Obstructive Nephropathy Is Decreased in Skp2-Deficient Mice

Ubiquitin-dependent degradation of the cyclin-dependent kinase inhibitor p27 mediated by SCF-Skp2 ubiquitin ligase is involved in cell cycle regulation. Proliferation of tubular cells is a characteristic feature in obstructed kidneys of unilateral ureteral obstruction. Comparing Skp2(+/+) mice with Skp2(-/-) mice, we investigated the involvement of Skp2, a component of SCF-Skp2 ubiquitin ligase for p27, in the progression of renal lesions in unilateral ureteral obstructed kidneys. mRNA expression of Skp2 was markedly increased in the obstructed kidneys from Skp2(+/+) mice and peaked 3 days after unilateral ureteral obstruction. Renal atrophy, tubular dilatation, tubulointerstitial fibrosis, and increases in alpha-smooth muscle actin expression, the number of tubular cells, and proliferating tubular cells positive for Ki67 were observed in the obstructed kidneys from Skp2(+/+) mice; however, these findings were significantly attenuated in Skp2(-/-) mice. The p27 protein level was increased in the obstructed kidneys but was significantly greater in Skp2(-/-) mice. The number of Ki67-positive p27-negative cells was lower in obstructed kidneys from Skp2(-/-) mice than Skp2(+/+) mice, whereas that of Ki67-negative p27-positive cells was greater in Skp2(-/-) mice. These findings suggest that p27 accumulation, which results from SCF-Skp2 ubiquitin ligase deficiency in Skp2(-/-) mice, is involved in the amelioration of renal damage induced by obstructive nephropathy.

Adenovirus E4 34k and E1b 55k Oncoproteins Target Host DNA Ligase IV for Proteasomal Degradation

Cells infected by adenovirus E4 mutants accumulate end-to-end concatemers of the viral genome that are assembled from unit-length viral DNAs by nonhomologous end joining (NHEJ). Genome concatenation can be prevented by expression either of E4 11k (product of E4orf3) or of the complex of E4 34k (product of E4orf6) and E1b 55k. Both E4 11k and the E4 34k/E1b 55k complex prevent concatenation at least in part by inactivation of the host protein Mre11: E4 11k sequesters Mre11 in aggresomes, while the E4 34k/E1b 55k complex participates in a virus-specific E3 ubiquitin ligase that mediates ubiquitination and proteasomal degradation. The E4 34k/E1b 55k complex, but not E4 11k, also inhibits NHEJ activity on internal breaks in the viral genome and on V(D)J recombination substrate plasmids, suggesting that it may interfere with NHEJ independently of its effect on Mre11. We show here that DNA ligase IV, which performs the joining step of NHEJ, is degraded as a consequence of adenovirus infection. Degradation is dependent upon E4 34k and E1b 55k, functional proteasomes, and the activity of cellular cullin 5, a component of the adenoviral ubiquitin ligase. DNA ligase IV also interacts physically with E1b 55k. The data demonstrate that DNA ligase IV, like Mre11, is a substrate for the adenovirus-specific E3 ubiquitin ligase; identify an additional viral approach to prevention of genome concatenation; and provide a mechanism for the general inhibition of NHEJ by adenoviruses.

Pollution Disrupts Endocrine Signaling

Dioxins are fat-soluble environmental pollutants that disrupt the endocrine system of humans (see Harper). The aryl hydrocarbon receptor (AhR) binds dioxins; interacts with the AhR nuclear-translocator protein (ARNT) and sex hormone steroid receptors, such as estrogen receptor α (ERα) and androgen receptor (AR); and promotes hormone-independent transcriptional activation. In addition, dioxins also inhibit sex steroid signaling, and Ohtake et al . show that one mechanism for this inhibition is by promoting the proteasome-mediated degradation of the steroid hormone receptors. Exposure of mammary tumor cells (MCF-7) or prostate cancer cells (LNCaP) to various dioxin chemicals decreased the abundance of ERα or AR, respectively. Ubiquitinated ERα was detectable in MCF-7 cells treated with dioxin and in which the proteasome was inhibited. Five dioxin-induced flag-tagged AhR-containing complexes were isolated from HeLa cells, and the "B" complex also contained ubiquitinated components. Complex B, which the authors name CUL4B AhR , contained the ubiquitin ligase components cullin 4B (CUL4B), damaged DNA binding protein (DDB1), and Rbx1; the proteasomal 19 S regulatory particle; ARNT; transducin-β-like 3 (TBL3); and ligand-bound AhR. The complete complex was only formed in the presence of AhR dioxin ligand (in vivo detected by coimmunoprecipitation and in vitro by assembly of isolated constituents). The E3 ubiquitin ligase activity of CUL4B AhR for ERα required dioxin but did not require estrogen. When components of CUL4B AhR were knocked down, dioxin-induced ubiquitination and degradation of ERα were diminished. Injection of dioxin into mice reduced ERα (females) or AR (males) abundance regardless of whether sex steroids were present or absent. Dioxin had no effect in mice deficient for AhR. Thus, AhR appears to trigger the formation of a ligand-induced E3 ubiquitin ligase complex, with AhR serving as the substrate adaptor allowing this complex to target sex steroid receptors. This ligand-dependent E3 ubiquitin ligase is similar to that seen in plants for the auxin receptor, and it will be interesting to see whether there are endogenous ligands for AhR that trigger the formation of this CUL4B AhR E3 ubiquitin ligase. F. Ohtake, A. Baba, I. Takada, M. Okada, K. Iwasaki, H. Miki, S. Takahashi, A. Kouzmenko, K. Nohara, T. Chiba, Y. Fujii-Kuriyama, S. Kato, Dioxin receptor is a ligand-dependent E3 ubiquitin ligase. Nature 446 , 562-566 (2007). [PubMed] J. W. Harper, A degrading solution to pollution. Nature 446 , 499-500 (2007). [PubMed]

HIV-1 Vpr function is mediated by interaction with the damage-specific DNA-binding protein DDB1

The Vpr accessory protein of HIV-1 induces a response similar to that of DNA damage. In cells expressing Vpr, the DNA damage sensing kinase, ATR, is activated, resulting in G(2) arrest and apoptosis. In addition, Vpr causes rapid degradation of the uracil-DNA glycosylases UNG2 and SMUG1. Although several cellular proteins have been reported to bind to Vpr, the mechanism by which Vpr mediates its biological effects is unknown. Using tandem affinity purification and mass spectrometry, we identified a predominant cellular protein that binds to Vpr as the damage-specific DNA-binding protein 1 (DDB1). In addition to its role in the repair of damaged DNA, DDB1 is a component of an E3 ubiquitin ligase that degrades numerous cellular substrates. Interestingly, DDB1 is targeted by specific regulatory proteins of other viruses, including simian virus 5 and hepatitis B. We show that the interaction with DDB1 mediates Vpr-induced apoptosis and UNG2/SMUG1 degradation and impairs the repair of UV-damaged DNA, which could account for G(2) arrest and apoptosis. The interaction with DDB1 may explain several of the diverse biological functions of Vpr and suggests potential roles for Vpr in HIV-1 replication.

P27 degradation by an ellipticinium series of compound via ubiquitin-proteasome pathway

The ellipticinium and its derivatives have been studied as anti-cancer agents with preferentially cyto-toxicity to the brain tumor cell lines. During the course of our study to determine whether an ellipticine derivative, API59-Cl would sensitize radio-resistant U87 glioblastoma cells to radiation, we found that it reduced the level of p27, a cyclin-dependent kinase inhibitor. API59-Cl induced a dose and time dependent p27 reduction in U87 cells. The compound-induced p27 reduction was also seen in three additional glioblastoma lines, T98G, U251 and U118 as well as in mouse embryonic fibroblasts. Mechanistic study of API59-Cl mediated p27 reduction revealed that it was not due to an altered p27 transcription, rather due to a shortened protein half-life as a result of enhanced p27 degradation. Indeed, API59-Cl induced p27 degradation was dependent on ubiquitin-proteasome pathway, particularly E3 ubiquitin ligase component, Skp2, but not Cullin-4A/B, and can be largely blocked by proteasome inhibitors MG132 or PS341. Finally, we demonstrated that API59-Cl inhibited U87 cell growth with an IC50 of 1.7 μM, which is independent of its p27 degrading activity. This is the first report, to our knowledge, that the ellipticinium class of small molecule compounds promotes p27 degradation via ubiquitin-proteasome pathway. The finding could provide a new tool to further understand the mechanism of p27 degradation.

Ubiquitination in Abscisic Acid‐Related Pathway

AbstractUbiquitination is emerging as a tight regulatory mechanism that is necessary for all aspects of development and survival of all eukaryotes. Recent genomic and genetic analysis in Arabidopsis suggests that ubiquitination may also play important roles in plant response to the phytohormone abscisic acid (ABA). Many components of the ubiquitination pathway, such as ubiquitin‐conjugating enzyme E2, ubiquitin ligase E3 and components of the proteasome, have been identified or predicted to be essential in ABA biosynthesis, catabolism and signaling. In addition, the ubiquitination‐related pathway, sumoylation, is also involved in ABA signaling. We summarize in this report recent developments to elucidate their roles in the ABA‐related pathway.

Genetic Characterization and Functional Analysis of the GID1 Gibberellin Receptors inArabidopsis

We investigated the physiological function of three Arabidopsis thaliana homologs of the gibberellin (GA) receptor GIBBERELLIN-INSENSITIVE DWARF1 (GID1) by determining the developmental consequences of GID1 inactivation in insertion mutants. Although single mutants developed normally, gid1a gid1c and gid1a gid1b displayed reduced stem height and lower male fertility, respectively, indicating some functional specificity. The triple mutant displayed a dwarf phenotype more severe than that of the extreme GA-deficient mutant ga1-3. Flower formation occurred in long days but was delayed, with severe defects in floral organ development. The triple mutant did not respond to applied GA. All three GID1 homologs were expressed in most tissues throughout development but differed in expression level. GA treatment reduced transcript abundance for all three GID1 genes, suggesting feedback regulation. The DELLA protein REPRESSOR OF ga1-3 (RGA) accumulated in the triple mutant, whose phenotype could be partially rescued by loss of RGA function. Yeast two-hybrid and in vitro pull-down assays confirmed that GA enhances the interaction between GID1 and DELLA proteins. In addition, the N-terminal sequence containing the DELLA domain is necessary for GID1 binding. Furthermore, yeast three-hybrid assays showed that the GA-GID1 complex promotes the interaction between RGA and the F-box protein SLY1, a component of the SCF(SLY1) E3 ubiquitin ligase that targets the DELLA protein for degradation.

Inhibition of CKS1B Expression Affects Myeloma Cell Growth In Vivo.

CKS1B is the regulatory component of the SCF-Skp2 ubiquitin ligase that ubiquinates the cyclin-dependent kinase inhibitor p27kip1 targeting it for proteasomal degradation. Overexpression of CKS1B is associated with poor prognosis in malignancies such as oral squamous cell carcinomas, colorectal carcinoma, breast cancer, prostate cancer, and others. Recently, high expression levels of CKS1B were associated with an aggressive course of Multiple Myeloma (Shaughnessy J. Hematology . 2005; 10 Suppl 1:117). We investigated whether inhibition of CKS1B in myeloma cell lines with multiple copies of 1q21 and high expression of CKS1B affects in vivo tumor growth. Myeloma cell lines were transduced with lentivirus conditionally expressing (tet on) CKS1B shRNA and lentivirus constitutively expressing luciferase (for in vivo experiments), or with lentivirus constitutively expressing CKS1B shRNA (for in vitro work). Cells with inducible expression were injected into the human bones of SCID-hu mice, and, once tumors established, expression was induced with doxycycline in the drinking water. Changes in tumor sizes were followed by weekly luminescence imaging. Cells with constitutive shRNA expression were used to determine effects on cell cycle by propidium iodide flow cytometry. Effects of shRNA expression on cytoplasmic and nuclear CKS1B and p27kip1 levels were determined by western blot analysis. Induction of CKS1B shRNA expression in JJN3 and OCI-MY5 cells in SCID-hu mice inhibited tumor growth by 57% and 81% within one week, compared to non-targeting construct. As expected, viable cells recovered from these tumors had low levels of CKS1B and higher levels of p27kip1 proteins. Similar to observations in the MM cell lines JJN3 and OCI-MY5 (see Abstract by Zhan and colleagues), constitutive expression of shRNA in CAG and XG1 cells effectively reduced cytoplasmic and nuclear CKS1B protein levels. This was associated with increased levels of p27kip1 protein, marked (51%) increases of cells in the G1 phase of the cell cycle, and reduced S and G2 (33% and 52%, respectively), compatible with a G1 arrest, and a 76% increase in the number of sub G1 (apoptotic) cells. In contrast to these observations, cells constitutively over-expressing p27kip1 had no change in G1, 50% fewer cells in S, and a 150% increase in G2, indicating G1 and G2 arrests, with no change in the sub G1 fraction. Interestingly, cells over-expressing p27kip1 had lower levels of CKS1B proteins, compatible with the reported proteasomal degradation of the molecule during the later phases of the cell cycle (Hattori T et al, Genes Cells. 2003; 8:889). These observations suggest that the poor prognosis associated with 1q21 amplifications and high CKS1B expression in myeloma reflects an elevated proliferative state of the tumor cells that results in rapid regrowth of minimal residual disease.

CD28 Costimulation Induces Transcription of SKP2 and CKS1, the Substrate Recognition Components of the Skp1-Cullin-F-box Ubiquitin Ligase, SCFSkp2.

Cellular immune responses require expansion of antigen-specific T cell clones from the pool of resting T lymphocytes that perform immune surveillance. Regulation of this proliferative potential is critical for defense against pathogens and avoidance of autoimmunity. Costimulation through the T cell receptor and accessory molecule, CD28, triggers replication of T lymphocytes in response to antigen that is mediated via both IL-2-dependent and IL-2-independent mechanisms. This event is associated with decreased abundance of the cyclin-dependent kinase inhibitor p27kip1 and increased cyclin D/cdk4 and cyclin E/cdk2 enzymatic activity. We have previously reported that in contrast to p27kip1 protein, the abundance of P27KIP mRNA transcript is unchanged in response to TCR/CD28 T cell activation. Ubiquitin-targeted degradation by the proteasome regulates the abundance of p27kip1 protein and ubiquitination of p27kip1 occurs in primary human T cells activated through the TCR and CD28. Ubiqutination of p27kip1 is mediated by a Skp1-Cullin-F-box (SCF) family ubiquitin ligase, SCFskp2. The SCFskp2 holoenzyme is comprised of the core components Roc1, Cul1, Skp1, and the substrate recognititon components, an F-box (cyclin F homology) protein, Skp2, that in cooperation with a smaller subunit, Cks1, mediates recognition of p27kip1 substrate. Targeted deletion of the murine Skp2 or Cks1 genes results in accumulation of p27kip1 in multiple cell types. T cell proliferation in response to TCR/CD3-plus-CD28 costimulation is reduced in Skp2−/− mice in comparison to wild-type controls, confirming the role of Skp2 in CD28 costimulation mediated degradation of p27kip1 and T cell proliferation. Here, we examined the mechanisms that regulate Skp2 and Cks1 abundance in primary T lymphocytes in response to TCR/CD3-plus-CD28 costimulation. Using primary human T lymphocytes we observed that the SCFskp2 core components Roc1, Cul1 and Skp1 are constitutively expressed and their levels remain unchanged upon TCR/CD3-plus-CD28 costimulation. In contrast, TCR/CD3-plus-CD28 costimulation lead to striking induction of SKP2 and CKS1 mRNA, and this event is dependent on PI3K/PKB and MEK/ERK signaling pathways. We determined that the SKP2 promoter lies directly upstream of the translation start site and contains binding sites for SP1, Elk-1 and E2F transcription factors. Mutagenesis of SP1 and Elk-1 sites abrogated TCR/CD3-plus-CD28-mediated SKP2 promoter-driven reporter activity, whereas mutagenesis of E2F site enhanced reporter activity, suggesting that SKP2 promoter may act as a node of integration for mitogenic and anti-mitogenic signals. Thus, in primary T lymphocytes CD28 costimulation can directly regulate cell cycle progression by inducing transcription of the substrate recognition components of SCFskp2 ubiquitin ligase that targets p27kip1 for degradation.

Ubiquitin Ligase Component Fbw7 Regulates the Quiescence of Hematopoietic Stem Cells.

Fbw7 is a SCF ubiquitin ligase component that catalyzes the ubiquitination of c-Myc, Cyclin E, and Notch. In several human cancer cell lines and primary cancer cells, Fbw7 is mutated and functions as a tumor suppressor gene. Previously we have reported that Fbw7-deficient mice died at embryonic day 10.5–11.5 with deficiencies in hematopoietic and vascular development, indicating that Fbw7 has a pivotal role in hematopoiesis (Tsunematsu R et al. J Biol Chem. 2004). Fbw7 is widely expressed in various hematopoietic lineages in BM of adult mice, but little has been known about the function of Fbw7 in hematopoiesis. To assess the requirement of Fbw7 in adult hematopoietic cells, we generated Fbw7-deficient mice by conditional gene targeting. Fbw7 was conditionally deleted from Mx-1-Cre;Fbw7fl/− adult mice by injection of pIpC over 1 week to induce Cre expression. We examined Fbw7fl/+ littermates as a control. We found progressive pancytopenia in Fbw7-deficient mice. Furthermore, most Fbw7-deficient mice developed leukemia (mainly ALL) within 3 months after pIpC treatment, suggesting that Fbw7 is essential to maintain normal hematopoiesis and loss of Fbw7 accelerates leukemogenesis. The portion of Fbw7-deficient Lin−Sca-1+c-Kit+CD34− hematopoietic stem cells (HSCs) in the G0 phase was 2.5-fold decreased and the frequency of cell division of Fbw7-deficient HSCs markedly increased in culture. These data suggest that Fbw7 promotes quiescence of HSCs. To examine the function of Fbw7-deficient HSCs, we transplanted 1500 Lin−Sca-1+cKit+ BM cells from Fbw7-dificient mice or littermate controls into lethally irradiated recipient mice with 4×105 normal BM mononuclear cells. In the result, Fbw7-deficient HSCs are impaired in long-term repopulating activity and multipotency. It has been reported that c-Myc controls the self-renewal activity of HSCs through the cell adhesion to the osteoblastic niche (Wilson A et al. Genes Dev. 2004). We found that c-Myc is significantly accumulated in Fbw7-deficient Lin−Sca-1+cKit+ BM cells, suggesting that HSCs leave the niche and show the active cell cycling. We propose that a ubiquitin ligase, Fbw7 is a key mediator of HSC quiescence and self renewal capacity.

DNA damage induces Cdt1 proteolysis in fission yeast through a pathway dependent on Cdt2 and Ddb1

Cdt1 is an essential protein required for licensing of replication origins. Here, we show that in Schizosaccharomyces pombe, Cdt1 is proteolysed in M and G1 phases in response to DNA damage and that this mechanism seems to be conserved from yeast to Metazoa. This degradation does not require Rad3 and Cds1, indicating that it is independent of classic DNA damage and replication checkpoint pathways. Damage-induced degradation of Cdt1 is dependent on Cdt2 and Ddb1, which are components of a Cul4 ubiquitin ligase. We also show that Cdt2 and Ddb1 are needed for cell-cycle changes in Cdt1 levels in the absence of DNA damage. Cdt2 and Ddb1 have been shown to be involved in the degradation of the Spd1 inhibitor of ribonucleotide reductase after DNA damage, and we speculate that Cdt1 downregulation might contribute to genome stability by reducing demand on dNTP pools during DNA repair.

Repression of ergosterol level during oxidative stress by fission yeast F-box protein Pof14 independently of SCF

We describe a new member of the F-box family, Pof14, which forms a canonical, F-box dependent SCF (Skp1, Cullin, F-box protein) ubiquitin ligase complex. The Pof14 protein has intrinsic instability that is abolished by inactivation of its Skp1 interaction motif (the F-box), Skp1 or the proteasome, indicating that Pof14 stability is controlled by an autocatalytic mechanism. Pof14 interacts with the squalene synthase Erg9, a key enzyme in ergosterol metabolism, in a membrane-bound complex that does not contain the core SCF components. pof14 transcription is induced by hydrogen peroxide and requires the Pap1 transcription factor and the Sty1 MAP kinase. Pof14 binds to and decreases Erg9 activity in vitro and a pof14 deletion strain quickly loses viability in the presence of hydrogen peroxide due to its inability to repress ergosterol synthesis. A pof14 mutant lacking the F-box and an skp1-3 ts mutant behave as wild type in the presence of oxidant showing that Pof14 function is independent of SCF. This indicates that modulation of ergosterol level plays a key role in adaptation to oxidative stress.

Expression of mouse Fbxw7 isoforms is regulated in a cell cycle- or p53-dependent manner

Fbxw7 is the F-box protein component of an SCF-type ubiquitin ligase that contributes to the ubiquitin-dependent degradation of cell cycle activators and oncoproteins. Three isoforms (α, β, and γ) of Fbxw7 are produced from mRNAs with distinct 5′ exons. We have now investigated regulation of Fbxw7 expression in mouse tissues. Fbxw7α mRNA was present in all tissues examined, whereas Fbxw7β mRNA was detected only in brain and testis, and Fbxw7γ mRNA in heart and skeletal muscle. The amount of Fbxw7α mRNA was high during quiescence (G 0 phase) in mouse embryonic fibroblasts (MEFs) and T cells, but it decreased markedly as these cells entered the cell cycle. The abundance of Fbxw7α mRNA was unaffected by cell irradiation or p53 status. In contrast, X-irradiation increased the amount of Fbxw7β mRNA in wild-type MEFs but not in those from p53-deficient mice, suggesting that radiation-induced up-regulation of p53 leads to production of Fbxw7β mRNA. Our results thus indicate that expression of Fbxw7 isoforms is differentially regulated in a cell cycle- or p53-dependent manner.

Lysine Residues Lys-19 and Lys-49 of β-Catenin Regulate Its Levels and Function in T Cell Factor Transcriptional Activation and Neoplastic Transformation

Wnt signaling regulates cell fate determination, proliferation, and survival, among other processes. Certain Wnt ligands stabilize the beta-catenin protein, leading to the ability of beta-catenin to activate T cell factor-regulated genes. In the absence of Wnts, beta-catenin is phosphorylated at defined serine and threonine residues in its amino (N) terminus. The phosphorylated beta-catenin is recognized by a beta-transducin repeat-containing protein (betaTrCP) and associated ubiquitin ligase components. The serine/threonine residues and betaTrCP-binding site in the N-terminal region of beta-catenin constitute a key regulatory motif targeted by somatic mutations in human cancers, resulting in constitutive stabilization of the mutant beta-catenin proteins. Structural studies have implicated beta-catenin lysine 19 as the major target for betaTrCP-dependent ubiquitination, but Lys-19 mutations in cancer have not been reported. We studied the consequences of single amino acid substitutions of the only 2 lysine residues in the N-terminal 130 amino acids of beta-catenin. Mutation of Lys-19 minimally affected beta-catenin levels and functional activity, and mutation of Lys-49 led to reduced beta-catenin levels and function. In contrast, beta-catenin proteins with substitutions at both Lys-19 and Lys-49 positions were present at elevated levels and had the ability to potently activate T cell factor-dependent transcription and promote neoplastic transformation. We furthermore demonstrate that the K19/K49 double mutant forms of beta-catenin are stabilized as a result of reduced betaTrCP-dependent ubiquitination. Our findings suggest that Lys-19 is a primary in vivo site of betaTrCP-dependent ubiquitination and Lys-49 may be a secondary or cryptic site. Moreover, our results inform understanding of why single amino acid substitutions at lysine 19 or 49 have not been reported in human cancer.

CK2 phosphorylation of SAG at Thr10 regulates SAG stability, but not its E3 ligase activity

Sensitive to Apoptosis Gene (SAG), a RING component of SCF E3 ubiquitin ligase, was shown to be phosphorylated by protein kinase CK2 at the Thr10 residue. It is, however, unknown whether this phosphorylation is stress-responsive or whether the phosphorylation changes its E3 ubiquitin ligase activity. To address these, we made a specific antibody against the phosphor-SAG(Thr10). Transient transfection experiment showed that SAG was phosphorylated at Thr10 which can be significantly inhibited by TBB, a relatively specific inhibitor of protein kinase CK2. To determine whether this SAG phosphorylation is stress-responsive, we defined a chemical-hypoxia condition in which SAG and CK2 were both induced. Under this condition, we failed to detect SAG phosphorylation at Thr10, which was readily detected, however, in the presence of MG132, a proteasome inhibitor, suggesting that the phosphorylated SAG has undergone a rapid degradation. To further define this, we made two SAG mutants, SAG-T10A which abolishes the SAG phosphorylation and SAG-T10E, which mimics the constitutive SAG phosphorylation. The half-life study revealed that indeed, SAG-T10E has a much shorter protein half-life (2 h), as compared to wild-type SAG (10 h). Again, rapid degradation of SAG-T10E in cells can be blocked by MG132. Thus, it appears that CK2-induced SAG phosphorylation at Thr10 regulates its stability through a proteasome-dependent pathway. Immunocytochemistry study showed that SAG as well as its phosphorylation mutants, was mainly localized in nucleus and lightly in cytoplasm. Hypoxia condition did not change their sub-cellular localization. Finally, an in vitro ubiqutination assay showed that SAG mutation at Thr10 did not change its E3 ligase activity when complexed with cullin-1. These studies suggested that CK2 might regulate SAG-SCF E3 ligase activity through modulating SAG's stability, rather than its enzymatic activity directly.

LMP1 Strain Variants: Biological and Molecular Properties

The ubiquitous herpesvirus Epstein-Barr virus (EBV) is linked to the development of several malignancies, including nasopharyngeal carcinoma. Latent membrane protein 1 (LMP1) is considered the EBV oncogene as it is necessary for EBV-induced transformation of B lymphocytes and is able to transform Rat-1 fibroblasts. LMP1 can activate a wide array of signaling pathways, including phosphatidylinositol 3-kinase (PI3K)-Akt and NF-kappaB. Six sequence variants of LMP1, termed Alaskan, China 1, China 2, Med+, Med-, and NC, have been identified, and individuals can be infected with multiple variants. The frequencies of detection of these variants differ for various EBV-associated malignancies from different geographic regions. In this study, the biological and signaling properties of the LMP1 variants have been characterized. All of the LMP1 variants transformed Rat-1 fibroblasts, induced increased motility of HFK cells, and induced increased homotypic adhesion of BJAB cells. While all the variants activated the PI3K-Akt signaling pathway to similar extents, the Alaskan, China 1, and Med+ variants had limited binding to the E3 ubiquitin ligase component homologue of Slimb and had slightly enhanced NF-kappaB signaling. These findings indicate that the signature amino acid changes of the LMP1 variants do not hinder or enhance their in vitro transforming potentials or affect their signaling properties.

The GRR1 gene of Candida albicans is involved in the negative control of pseudohyphal morphogenesis

The opportunistic fungal pathogen Candida albicans can grow as yeast, pseudohyphae or true hyphae. C. albicans can switch between these morphologies in response to various environmental stimuli and this ability to switch is thought to be an important virulence trait. In Saccharomyces cerevisiae, the Grr1 protein is the substrate recognition component of an SCF ubiquitin ligase that regulates cell cycle progression, cell polarity and nutrient signaling. In this study, we have characterized the GRR1 gene of C. albicans. Deletion of GRR1 from the C. albicans genome results in a highly filamentous, pseudohyphal morphology under conditions that normally promote the yeast form of growth. Under hypha-inducing conditions, most cells lacking GRR1 retain a pseudohyphal morphology, but some cells appear to switch to hyphal-like growth and express the hypha-specific genes HWP1 and ECE1. The C. albicans GRR1 gene also complements the elongated cell morphology phenotype of an S. cerevisiae grr1Δ mutant, indicating that C. albicans GRR1 encodes a true orthologue of S. cerevisaie Grr1. These results support the hypothesis that the Grr1 protein of C. albicans, presumably as the F-box subunit of an SCF ubiquitin ligase, has an essential role in preventing the switch from the yeast cell morphology to a pseudohyphal morphology.

Elucidation of the Substrate Binding Site of Siah Ubiquitin Ligase

The Siah family of RING proteins function as ubiquitin ligase components, contributing to the degradation of multiple targets involved in cell growth, differentiation, angiogenesis, oncogenesis, and inflammation. Previously, a binding motif (degron) was recognized in many of the Siah degradation targets, suggesting that Siah itself may facilitate substrate recognition. We report the crystal structure of the Siah in complex with a peptide containing the degron motif. Binding is within a groove formed in part by the zinc fingers and the first two beta strands of the TRAF-C domain of Siah. We show that residues in the degron, previously described to facilitate binding to Siah, interact with the protein. Mutagenesis of Siah at sites of interaction also abrogates both in vitro peptide binding and destabilization of a known Siah target.

The U-box protein CMPG1 is required for efficient activation of defense mechanisms triggered by multiple resistance genes in tobacco and tomato.

We previously identified three Avr9/Cf-9 Rapidly Elicited (ACRE) genes essential for Cf-9- and Cf-4-dependent hypersensitive response (HR) production in Nicotiana benthamiana. Two of them encode putative E3 ubiquitin ligase components. This led us to investigate other ACRE genes associated with the ubiquitination pathway. ACRE74 encodes a U-box E3 ligase homolog, highly related to parsley (Petroselinum crispum) CMPG1 and Arabidopsis thaliana PLANT U-BOX20 (PUB20) and PUB21 proteins, and was called Nt CMPG1. Transcript levels of Nt CMPG1 and the homologous tomato (Solanum lycopersicum) Cmpg1 are induced in Cf9 tobacco (Nicotiana tabacum) and Cf9 tomato after Avr9 elicitation. Tobacco CMPG1 possesses in vitro E3 ligase activity. N. benthamiana plants silenced for Nt CMPG1 show reduced HR after Cf-9/Avr9 elicitation, while overexpression of Nt CMPG1 induces a stronger HR in Cf9 tobacco plants after Avr9 infiltration. In tomato, silencing of Cmpg1 decreased resistance to Cladosporium fulvum. Overexpression of epitope-tagged tobacco CMPG1 mutated in the U-box domain confers a dominant-negative phenotype. We also show that Nt CMPG1 is involved in the Pto/AvrPto and Inf1 responses. In summary, we show that the E3 ligase Nt CMPG1 is essential for plant defense and disease resistance.