Prolyl hydroxylation by EglN2 destabilizes FOXO3a by blocking its interaction with the USP9x deubiquitinase.

The three EglN prolyl hydroxylases (EglN1, EglN2, and EglN3) regulate the stability of the HIF transcription factor. We recently showed that loss of EglN2, however, also leads to down-regulation of Cyclin D1 and decreased cell proliferation in a HIF-independent manner. Here we report that EglN2 can hydroxylate FOXO3a on two specific prolyl residues in vitro and in vivo. Hydroxylation of these sites prevents the binding of USP9x deubiquitinase, thereby promoting the proteasomal degradation of FOXO3a. FOXO transcription factors can repress Cyclin D1 transcription. Failure to hydroxylate FOXO3a promotes its accumulation in cells, which in turn suppresses Cyclin D1 expression. These findings provide new insights into post-transcriptional control of FOXO3a and provide a new avenue for pharmacologically altering Cyclin D1 activity.

Peptidic degron in EID1 is recognized by an SCF E3 ligase complex containing the orphan F-box protein FBXO21.

EP300-interacting inhibitor of differentiation 1 (EID1) belongs to a protein family implicated in the control of transcription, differentiation, DNA repair, and chromosomal maintenance. EID1 has a very short half-life, especially in G0 cells. We discovered that EID1 contains a peptidic, modular degron that is necessary and sufficient for its polyubiquitylation and proteasomal degradation. We found that this degron is recognized by an Skp1, Cullin, and F-box (SCF)-containing ubiquitin ligase complex that uses the F-box Only Protein 21 (FBXO21) as its substrate recognition subunit. SCF(FBXO21) polyubiquitylates EID1 both in vitro and in vivo and is required for the efficient degradation of EID1 in both cycling and quiescent cells. The EID1 degron partially overlaps with its retinoblastoma tumor suppressor protein-binding domain and is congruent with a previously defined melanoma-associated antigen-binding motif shared by EID family members, suggesting that binding to retinoblastoma tumor suppressor and melanoma-associated antigen family proteins could affect the polyubiquitylation and turnover of EID family members in cells.

Loss of the retinoblastoma binding protein 2 (RBP2) histone demethylase suppresses tumorigenesis in mice lacking Rb1 or Men1.

Aberrations in epigenetic processes, such as histone methylation, can cause cancer. Retinoblastoma binding protein 2 (RBP2; also called JARID1A or KDM5A) can demethylate tri- and dimethylated lysine 4 in histone H3, which are epigenetic marks for transcriptionally active chromatin, whereas the multiple endocrine neoplasia type 1 (MEN1) tumor suppressor promotes H3K4 methylation. Previous studies suggested that inhibition of RBP2 contributed to tumor suppression by the retinoblastoma protein (pRB). Here, we show that genetic ablation of Rbp2 decreases tumor formation and prolongs survival in Rb1(+/-) mice and Men1-defective mice. These studies link RBP2 histone demethylase activity to tumorigenesis and nominate RBP2 as a potential target for cancer therapy.

Kinase requirements in human cells: III. Altered kinase requirements in VHL-/- cancer cells detected in a pilot synthetic lethal screen.

Clear cell renal carcinomas are the most common form of kidney cancer and frequently are linked to biallelic inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene. The VHL gene product, pVHL, has multiple functions including directing the polyubiquitylation of the HIF transcription factor. We screened 100 shRNA vectors, directed against 88 kinases, for their ability to inhibit the viability of VHL-/- renal carcinoma cells preferentially compared with isogenic cells in which pVHL function was restored. shRNAs for "hits" identified in the primary screen were interrogated in secondary screens that included shRNA titration studies. Multiple shRNAs against CDK6, MET, and MAP2K1 (also known as MEK1) preferentially inhibited the viability of 786-O and RCC4 VHL-/- cells compared with their wild-type pVHL-reconstituted counterparts. The sensitivity of pVHL-proficient cells to these shRNAs was not restored upon HIF activation, suggesting that loss of an hypoxia-inducible factor (HIF)-independent pVHL function formed the basis for selectivity. A small-molecule Cdk4/6 inhibitor displayed enhanced activity against VHL-/- renal carcinoma cells, suggesting that in some cases hits from shRNA screens such as described here might translate into therapeutic targets.

Loss of p27(Kip1) cooperates with cyclin E in T-cell lymphomagenesis.

Cyclin E and p27(Kip1) are key regulators for cyclin-dependent kinases (Cdks) acting at the G1-/S-phase transition of the cell cycle. Whereas cyclin E is required for the activation of Cdk2, p27(Kip1) is a specific Cdk inhibitor and can block cell division. High levels of cyclin E and low levels of p27(Kip1) expression have been associated with malignant lymphomas in humans; the level of p27(Kip1) is even considered of prognostic significance. However, mice that lack p27(Kip1) do not develop any malignant lymphomas despite a pronounced lymphoid hyperplasia in thymus and spleen. We have previously described transgenic mice that carry a construct in which the cyclin E cDNA is under the control of the CD2 promoter/enhancer region and thus express high levels of cyclin E in the T-cell compartment (CD2-cyclin E). These animals are not predisposed for T-cell lymphomas in the absence of other cooperating events. Here we show that T-cells from CD2-cyclin E mice that at the same time are deficient for p27(Kip1) show a significantly higher Cdk2 activity than cells from wild-type or single mutant animals. Accordingly, a higher percentage of T cells in S/G2/M phase is found in CD2-cyclin E/p27(Kip1-/-) mice. After a long latency period of over 200 days, these animals develop spontaneous monoclonal T cell lymphoma whereas none of the single CD2-cyclin E transgenic or the p27(Kip1)-deficient mice showed any sign of lymphoid malignancies. Our findings demonstrate that a deregulation of control mechanisms at the G1/S transition by the combination of high cyclin E levels in the absence of p27(Kip1) is sufficient to predispose mice to develop lymphoid malignancies and further support a role of p27(Kip1) as a tumor suppressor and of cyclin E as a dominant oncogene.

Cyclin E.

E-type cyclins (cyclin E1 and cyclin E2) are expressed during the late G1 phase of the cell cycle until the end of the S-phase. The activity of cyclin E is limiting for the passage of cells through the restriction point "R" which marks a "point of no return" for cells entering the division cycle from a resting state or passing from G1 into S-phase. Expression of cyclin E is regulated on the level of gene transcription mainly by members of the E2F trrnscription factor family and by its degradation via the proteasome pathway. Cyclin E binds and activates the kinase Cdk2 and by phosphorylating its substrates, the so-called "pocket proteins", the cyclic/Cdk2 complexes initiate a cascade of events that leads to the expression of S-phase specific genes. Aside from this specific function as a regulator of S-phase-entry, cyclin E plays a direct role in the initiation of DNA replication, the control of genomic stability, and the centrosome cycle. Surprisingly, recent studies have shown that the once thought essential cyclin E is dispensable for the development of higher eukaryotes and for the mitotic division of eukaryotic cells. Nevertheless, high level cyclin E expression has been associated with the initiation or progression of different human cancers, in particular breast cancer but also leukemia, lymphoma and others. Transgenic mouse models in which cyclin E is constitutively expressed develop malignant diseases, supporting the notion of cyclin E as a dominant onco-protein.

Yaf2 inhibits Myc biological function.

The proto-oncogenes of the myelocytomatosis viral oncogene homolog (MYC) family, including MYC, MYCN and MYCL, encode nuclear proteins that act as transcription factors. The Myc protein is the best studied member of this family and is involved in cell cycle regulation, differentiation and cell death. We have previously demonstrated that the zinc-finger protein Yaf2 interacts with the central region of MycN and enhances MycN dependent transcriptional activation. Here we show that Yaf2 also binds to the Myc protein in vivo and in vitro. In contrast to the activating effect on MycN function, Yaf2 inhibits Myc mediated transactivation and transformation. This differential influence on two members of the Myc family gives insight into a new mechanism to modulate the biological activities of Myc transcription factors.

Comparison of three dsDNA-ELISAs with regard to their efficiency in the diagnosis of systemic lupus erythematosus.

We compared the performance of three commercially available anti-double-stranded DNA antibody ELISA kits with respect to their precision, accuracy, linearity, and the detection limit. We tested six sets of patients (lupus erythematosus, scleroderma, rheumatoid arthritis, psoriatic arthritis, hepatitis C, malignancies) to assess specificity, sensitivity, and diagnostic efficiency of the three assays. The diagnostic efficiency of the Crithidia luciliae immunofluorescence test was analyzed as a reference. The sensitivity of the evaluated enzyme immunoassays ranged from 55.6 to 66.7%, the specificity from 98.4 to 100%, and the diagnostic efficiency from 90.2-92.7%. All ELISAs were superior to the immunofluorescence test, both with respect to sensitivity, specificity, and diagnostic efficiency. The commercial kits tested were comparable with respect to their performance characteristics.

DRE-1/FBXO11-dependent degradation of BLMP-1/BLIMP-1 governs C. elegans developmental timing and maturation.

Developmental timing genes catalyze stem cell progression and animal maturation programs across taxa. Caenorhabditis elegans DRE-1/FBXO11 functions in an SCF E3-ubiquitin ligase complex to regulate the transition to adult programs, but its cognate proteolytic substrates are unknown. Here, we identify the conserved transcription factor BLMP-1 as a substrate of the SCF(DRE-1/FBXO11) complex. blmp-1 deletion suppressed dre-1 mutant phenotypes and exhibited developmental timing defects opposite to dre-1. blmp-1 also opposed dre-1 for other life history traits, including entry into the dauer diapause and longevity. BLMP-1 protein was strikingly elevated upon dre-1 depletion and dysregulated in a stage- and tissue-specific manner. The role of DRE-1 in regulating BLMP-1 stability is evolutionary conserved, as we observed direct protein interaction and degradation function for worm and human counterparts. Taken together, posttranslational regulation of BLMP-1/BLIMP-1 by DRE-1/FBXO11 coordinates C. elegans developmental timing and other life history traits, suggesting that this two-protein module mediates metazoan maturation processes.

Control of cyclin D1 and breast tumorigenesis by the EglN2 prolyl hydroxylase.

2-Oxoglutarate-dependent dioxygenases, including the EglN prolyl hydroxylases that regulate HIF, can be inhibited with drug-like molecules. EglN2 is estrogen inducible in breast carcinoma cells and the lone Drosophila EglN interacts genetically with Cyclin D1. Although EglN2 is a nonessential gene, we found that EglN2 inactivation decreases Cyclin D1 levels and suppresses mammary gland proliferation in vivo. Regulation of Cyclin D1 is a specific attribute of EglN2 among the EglN proteins and is HIF independent. Loss of EglN2 catalytic activity inhibits estrogen-dependent breast cancer tumorigenesis and can be rescued by exogenous Cyclin D1. EglN2 depletion also impairs the fitness of lung, brain, and hematopoietic cancer lines. These findings support the exploration of EglN2 inhibitors as therapeutics for estrogen-dependent breast cancer and other malignancies.

A protective role for the human SMG-1 kinase against tumor necrosis factor-alpha-induced apoptosis.

The human suppressor of morphogenesis in genitalia-1 (hSMG-1) protein kinase plays dual roles in mRNA surveillance and genotoxic stress response pathways in human cells. Here, we report that small interfering RNA-mediated depletion of hSMG-1, but not ATM, ATR, hUpf1, or hUpf2, in human U2OS osteosarcoma cells markedly increases the magnitude and accelerates the rate of apoptosis induced by tumor necrosis factor-alpha (TNFalpha) stimulation. The increase in TNFalpha-mediated cell killing observed in hSMG-1-depleted cells is not related to the suppression of nonsense-mediated mRNA decay or to the inhibition of TNFalpha-induced NF-kappaB activation. Rather, we observed that loss of hSMG-1 accelerates the degradation of the long form of the FLICE-inhibitory protein (FLIP(L)), an inhibitor of death-inducing signaling complex-mediated caspase-8 activation, in TNFalpha-treated cells. These results suggest that hSMG-1 plays an important role in cell survival during TNFalpha-induced stress.

The oncogenic activity of cyclin E is not confined to Cdk2 activation alone but relies on several other, distinct functions of the protein.

We have previously shown that cyclin E can malignantly transform primary rat embryo fibroblasts in cooperation with constitutively active Ha-Ras. In addition, we demonstrated that high level cyclin E expression potentiates the development of methyl-nitroso-urea-induced T-cell lymphomas in mice. To further investigate the mechanism underlying cyclin E-mediated malignant transformation, we have performed a mutational analysis of cyclin E function. Here we show that cyclin E mutants defective to form an active kinase complex with Cdk2 are unable to drive cells from G(1) into S phase but can still malignantly transform rat embryo fibroblasts in cooperation with Ha-Ras. In addition, Cdk2 activation is not a prerequisite for the ability of cyclin E to rescue yeast triple cln mutations. We also find that the oncogenic properties of cyclin E did not entirely correspond with its ability to interact with the negative cell cycle regulator p27(Kip1) or the pocket protein p130. These findings suggest that the oncogenic activity of cyclin E does not exclusively rely on its ability as a positive regulator of G(1) progression. Rather, we propose that cyclin E harbors other functions, independent of Cdk2 activation and p27(Kip1) binding, that contribute significantly to its oncogenic activity.

Interaction between human MCM7 and Rad17 proteins is required for replication checkpoint signaling.

Human Rad17 (hRad17) is centrally involved in the activation of cell-cycle checkpoints by genotoxic agents or replication stress. Here we identify hMCM7, a core component of the DNA replication apparatus, as a novel hRad17-interacting protein. In HeLa cells, depletion of either hRad17 or hMCM7 with small-interfering RNA suppressed ultraviolet (UV) light- or aphidicolin-induced hChk1 phosphorylation, and abolished UV-induced S-phase checkpoint activation. Similar results were obtained after transfection of these cells with a fusion protein containing the hMCM7-binding region of hRad17. The hMCM7-depleted cells were also defective for the formation of ATR-containing nuclear foci after UV irradiation, suggesting that hMCM7 is required for stable recruitment of ATR to damaged DNA. These results demonstrate that hMCM7 plays a direct role in the transmission of DNA damage signals from active replication forks to the S-phase checkpoint machinery in human cells.

Identification of a novel Krüppel-associated box domain protein, Krim-1, that interacts with c-Myc and inhibits its oncogenic activity.

We have used the Ras recruitment system to screen for proteins that interact with the N-terminally located transactivation domain of c-Myc. The Ras recruitment system is based on the activation of the mitogenic RAS signaling pathway in yeast by the mammalian GTPase Ha-Ras. This screen led to the identification of two novel nuclear proteins termed Krim-1A and Krim-1B that both contain an N-terminal KRAB box domain and 12 or 9 Krüppel C2H2 type zinc fingers at the C terminus, respectively. We found that sequences covering the Myc box II homology region are essential for the interaction with the Krim-1 proteins and that the second N-terminal zinc finger of Krim-1 is essential for Myc binding. Both Krim-1A and -B genes appear to be expressed ubiquitously with highest levels in spleen and lymph nodes. In particular, Krim-1B and, to a lesser extent, Krim-1A are able to decrease E-box-dependent transcriptional transactivation by c-Myc-Max complexes and also the ability of Myc to malignantly transform primary rat embryo fibroblasts, which is consistent with the functional repressive properties of their KRAB domains. The transcriptional corepressor Tif-1beta is a binding partner for Krim-1 and stabilizes the protein. Our findings suggest that Myc-mediated functions can be negatively regulated by Krim-1, potentially in a complex with Tif-1beta.

New indene-derivatives with anti-proliferative properties.

Metabolites of the non-steroidal anti-inflammatory drug Sulindac inhibit cell proliferation by affecting several intracellular signaling pathways including the tumorigenic Ras/Raf/MAPK pathway. Here, we report the synthesis of eight new indene derivatives derived from the Sulindac structure, and present data on their anti-proliferative properties and their effects on the p21ras protein.

The mRNA surveillance protein hSMG-1 functions in genotoxic stress response pathways in mammalian cells.

Members of the PI 3-kinase-related kinase (PIKK) family function in mitogenic and stress-induced signaling pathways in eukaryotic cells. Here, we characterize the newest PIKK family member, hSMG-1, as a genotoxic stress-activated protein kinase that displays some functional overlap with the related kinase, ATM, in human cells. Both ATM and hSMG-1 phosphorylate Ser/Thr-Gln-containing target sequences in the checkpoint protein p53 and the nonsense-mediated mRNA decay (NMD) protein hUpf1. Expression of hSMG-1 is required for optimal p53 activation after cellular exposure to genotoxic stress, and depletion of hSMG-1 leads to spontaneous DNA damage and increased sensitivity to ionizing radiation (IR). Moreover, IR exposure triggers hUpf1 phosphorylation at Ser/Thr-Gln motifs, and both ATM and hSMG-1 contribute to these phosphorylation events. Finally, NMD is suppressed in hSMG-1- but not ATM-deficient cells. These results indicate that hSMG-1 plays important roles in the maintenance of both genome and transcriptome integrity in human cells.