RNF5 is a RING finger protein found to be important in the growth and development of Caenorhabditis elegans. The search for RNF5-associated proteins via a yeast two-hybrid screen identified a LIM-containing protein in C. elegans which shows homology with human paxillin. Here we demonstrate that the human homologue of RNF5 associates with the amino-terminal domain of paxillin, resulting in its ubiquitination. RNF5 requires intact RING and C-terminal domains to mediate paxillin ubiquitination. Whereas RNF5 mediates efficient ubiquitination of paxillin in vivo, protein extracts were required for in vitro ubiquitination, suggesting that additional modifications and/or an associated E3 ligase assist RNF5 targeting of paxillin ubiquitination. Mutant Ubc13 efficiently inhibits RNF5 ubiquitination, suggesting that RNF5 generates polychain ubiquitin of the K63 topology. Expression of RNF5 increases the cytoplasmic distribution of paxillin while decreasing its localization within focal adhesions, where it is primarily seen under normal growth. Concomitantly, RNF5 expression results in inhibition of cell motility. Via targeting of paxillin ubiquitination, which alters its localization, RNF5 emerges as a novel regulator of cell motility.A growing number of RING (really interesting new gene) finger proteins have been implicated as key regulators of cell growth and in organ and tissue development (1). RINGs are cysteine-rich, zinc-binding domains defined by a pattern of conserved cysteine and histidine residues (reviewed in reference 1) that exert their regulatory function through association with and effects on signal transduction and cell cycle control proteins. In many cases RINGs elicit E3 ligase activity, which serves to limit their availability and to alter the stability and/or localization of their associated proteins (1,4,18,20).RINGs that directly target ubiquitination of their associated substrates via their E3 ligase activity include the mammalian homologues of seven in absentia (Siah), AO7, BRCA1, and Mdm2 (1,12,18,20); Parkin, which is associated with autosomal juvenile parkinsonism (36); and the Cbl protein family, which regulates cell signaling (18, 37). In the process of searching for RING finger proteins resembling mammalian RINGs known to exhibit E3 ligase activities in Caenorhabditis elegans, we identified and characterized RNF5 (C16C10.7), the C. elegans homologue of the human RING-finger protein 5 (RNF5) (17) and Arabidopsis thaliana RMA1 (17, 22). RNF5 shows structural homology within the RING domain with BRCA1, Cbl, and Mdm2 and is important to development in C. elegans (Broday et al., unpublished data). RNF5 expression is reduced in human tumors (our unpublished observations), partly due to transcriptional suppression mediated by the polycomb group protein EZH2, which is involved in prostate cancer progression (44). The search for RNF5-associated proteins identified a LIM domain-containing protein in C. elegans which shows homology with human paxillin. Here we demonstrate that through its association ...

The small ubiquitin-like modifier (SUMO) modification alters the subcellular distribution and function of its substrates. Here we show the major role of SUMO during the development of the Caenorhabditis elegans reproductive system. smo-1 deletion mutants develop into sterile adults with abnormal somatic gonad, germ line, and vulva. SMO-1ϻGFP reporter is highly expressed in the somatic reproductive system. smo-1 animals lack a vulval-uterine connection as a result of impaired ventral uterine -cell differentiation and anchor cell fusion. Mutations in the LIN-11 LIM domain transcription factor lead to a uterine phenotype that resembles the smo-1 phenotype. LIN-11 is sumoylated, and its sumoylation is required for its activity during uterine morphogenesis. Expression of a SUMO-modified LIN-11 in the smo-1 background partially rescued -cell differentiation and retained LIN-11 in nuclear bodies. Thus, our results identify the reproductive system as the major SUMO target during postembryonic development and highlight LIN-11 as a physiological substrate whose sumoylation is associated with the formation of a functional vulval-uterine connection.[Keywords: SUMO; somatic gonad; Supplemental material is available at http://www.genesdev.org. (Schwarz et al. 1998). SUMO conjugation has been shown to affect subcellular localization of the modified substrate, thereby affecting its activity and stability (Matunis et al. 1996;Mahajan et al. 1997; Muller at al. 1998). Several transcription factors are modified by sumoylation. Whereas SUMO modification negatively regulates the androgen receptor, SP3, c-Jun, and p53 (Gostissa et al. 1999; Muller et al. 2000;Poukka et al. 2000;Schmidt and Muller 2002), sumoylation of the glucocorticoid receptor increases its transcriptional activities (LeDrean et al. 2002). Sumoylation also affects transcriptional activities indirectly. For example, SUMO conjugation to class II histone deacetylase impairs its transcription-repressing function (Kirsch et al. 2002). Alternatively, sumoylation has also been shown to affect nuclear and subnuclear (nucleolar or PML nuclear body) localization of regulatory proteins primarily implicated in transcriptional control (Sternsdorf et al. 1997;Pichler et al. 2002).The SUMO conjugation system is essential for viability in Saccharomyces cerevisiae (Melchoir 2000). Phenotypes observed upon aberrant sumoylation in S. cerevisiae include impaired septin ring formation, chromosomal segregation, and progression of the cell cycle through G 2 -M (Johnson and Blobel 1999). Studies in Arabidopsis suggest that the SUMO conjugation system has a role in protection against stress and/or repair of stressrelated damage (Kurepa et al. 2002). In Drosophila melanogaster, the loss-of-function mutation of semushi, the UBC9 (SUMO-conjugating enzyme) ortholog, prevents nuclear import of the transcription factor Bicoid (Bcd) and results in impaired embryogenesis (Epps and Tanda 1998).

The nucleoside analog cytarabine, an inhibitor of DNA replication fork progression that results in DNA damage, is currently used in the treatment of acute myeloid leukemia (AML). We explored the prognostic value of the expression of 72 genes involved in various aspects of DNA replication in a set of 198 AML patients treated by cytarabine-based chemotherapy. We unveiled that high expression of the DNA replication checkpoint gene CHEK1 is a prognostic marker associated with shorter overall, event-free, and relapse-free survivals and determined that the expression of CHEK1 can predict more frequent and earlier postremission relapse. CHEK1 encodes checkpoint kinase 1 (CHK1), which is activated by the kinase ATR when DNA replication is impaired by DNA damage. High abundance of CHK1 in AML patient cells correlated with higher clonogenic ability and more efficient DNA replication fork progression upon cytarabine treatment. Exposing the patient cells with the high abundance of CHK1 to SCH900776, an inhibitor of the kinase activity of CHK1, reduced clonogenic ability and progression of DNA replication in the presence of cytarabine. These results indicated that some AML cells rely on an efficient CHK1-mediated replication stress response for viability and that therapeutic strategies that inhibit CHK1 could extend current cytarabine-based treatments and overcome drug resistance. Furthermore, monitoring CHEK1 expression could be used both as a predictor of outcome and as a marker to select AML patients for CHK1 inhibitor treatments.

Genomic instability in solid tumors participates in the oncogenetic process and is associated with the activation of the DNA damage response pathway. Here, we report the activation of the constitutive DNA damage and checkpoint pathway associated with complex karyotypes in samples from patients with acute myeloid leukemia (AML). We show that antagonizing CHK1 kinase with a small inhibitory compound or by RNA interference strongly reduces the clonogenic properties of high-DNA damage level AML samples, particularly those with complex karyotypes. Moreover, we observe a beneficial effect of CHK1 inhibition in high-DNA damage level AML samples treated with 1-β-D-arabinofuranosylcytosine. In contrast, CHK1 inhibition has no effect on the clonogenic properties of normal hematopoietic progenitors. All together, our results indicate that CHK1 inhibition may represent an attractive therapeutic opportunity in AML with complex karyotype.

Here, we describe a new muscle LIM domain protein, UNC-95, and identify it as a novel target for the RING finger protein RNF-5 in the Caenorhabditis elegans body wall muscle. unc-95(su33) animals have disorganized muscle actin and myosin-containing filaments as a result of a failure to assemble normal muscle adhesion structures. UNC-95 is active downstream of PAT-3/β-integrin in the assembly pathways of the muscle dense body and M-line attachments, and upstream of DEB-1/vinculin in the dense body assembly pathway. The translational UNC-95::GFP fusion construct is expressed in dense bodies, M-lines, and muscle–muscle cell boundaries as well as in muscle cell bodies. UNC-95 is partially colocalized with RNF-5 in muscle dense bodies and its expression and localization are regulated by RNF-5. rnf-5(RNAi) or a RING domain deleted mutant, rnf-5(tm794), exhibit structural defects of the muscle attachment sites. Together, our data demonstrate that UNC-95 constitutes an essential component of muscle adhesion sites that is regulated by RNF-5.

Centrosomes are dynamic organelles that consist of a pair of cylindrical centrioles, surrounded by pericentriolar material. The pericentriolar material contains factors that are involved in microtubule nucleation and organization, and its recruitment varies during the cell cycle. We report here that proteasome inhibition in HeLa cells induces the accumulation of several proteins at the pericentriolar material, including gamma-tubulin, GCP4, NEDD1, ninein, pericentrin, dynactin, and PCM-1. The effect of proteasome inhibition on centrosome proteins does not require intact microtubules and is reversed after removal of proteasome inhibitors. This accrual of centrosome proteins is paralleled by accumulation of ubiquitin in the same area and increased polyubiquitylation of nonsoluble gamma-tubulin. Cells that have accumulated centrosome proteins in response to proteasome inhibition are impaired in microtubule aster formation. Our data point toward a role of the proteasome in the turnover of centrosome proteins, to maintain proper centrosome function.

The effects of cell adhesion on leukemia cell proliferation remain poorly documented and somehow controversial. In this work, we investigated the effect of adhesion to fibronectin on the proliferation of acute myeloid leukemia (AML) cell lines (U937 and KG1a) and CD34 + normal or leukemic primary cells. We observed an increased rate of proliferation of AML cells when adhered to fibronectin, concomitant with accelerated S-phase entry and accumulation of CDC25A. Conversely, normal CD34+ cell proliferation was decreased by adhesion to fibronectin with a concomitant drop in CDC25A expression. Importantly, we showed that both small interfering RNA (siRNA)-mediated CDC25A down-regulation and a recently developed CDC25 pharmacologic inhibitor impaired this adhesion-dependent proliferation, establishing a functional link between CDC25A accumulation and adhesion-dependent proliferation in leukemic cells. CDC25A accumulation was found only slightly dependent on transcriptional regulation and essentially due to modifications of the proteasomal degradation of the protein as shown using proteasome inhibitors and reverse transcription-PCR. Interestingly, CDC25A regulation was Chk1 dependent in these cells as suggested by siRNA-mediated down-regulation of this protein. Finally, we identified activation of the phosphatidylinositol 3-kinase/ Akt pathway as an adhesion-dependent regulation mechanism of CDC25A protein expression. Altogether, our data show that in leukemic cells adhesion to fibronectin increases CDC25A expression through proteasome-and Chk1-dependent mechanisms, resulting in enhanced proliferation. They also suggest that these adhesion-dependent proliferation properties of hematopoietic cells may be modified during leukemogenesis.

Acute myeloid leukemia (AML) cells exposed to genotoxic agents arrest their cell cycle at the G2/M checkpoint and are inherently chemoresistant. To understand the mechanism of this chemoresistance, we compared the ability of immature CD34 þ versus mature CD34À AML cell lines (KG1a and U937, respectively) to recover from a DNA damage-induced cell cycle checkpoint in G2. Here, we report that KG1a cells have a more stringent G2/M checkpoint response than U937 cells. We show that in both cell types, the CDC25B phosphatase participates in the G2/M checkpoint recovery and that its expression is upregulated. Furthermore, we show that CHK1 inhibition by UCN-01 in immature KG1a cells allows checkpoint exit and induces sensitivity to genotoxic agents. Similarly, UCN-01 treatment potentializes genotoxic-induced inhibition of colony formation efficiency of primary leukemic cells from AML patients. Altogether, our results demonstrate that checkpoint stringency varies during the maturation process and indicate that targeting checkpoint mechanisms might represent an attractive therapeutic opportunity for chemoresistant immature AML cells.