The limitations of revolutionary new mutation-specific inhibitors of BRAFV600E include the universal recurrence seen in melanoma patients treated with this novel class of drugs. Recently, our lab showed that simultaneous activation of the Wnt/β-catenin signaling pathway and targeted inhibition of BRAFV600E by PLX4720 synergistically induces apoptosis across a spectrum of BRAFV600E melanoma cell lines. As a follow-up to that study, treatment of BRAF-mutant and NRAS-mutant melanoma lines with WNT3A and the MEK inhibitor AZD6244 also induces apoptosis. The susceptibility of BRAF-mutant lines and NRAS-mutant lines to apoptosis correlates with negative regulation of Wnt/β-catenin signaling by ERK/MAPK signaling and dynamic decreases in abundance of the downstream scaffolding protein, AXIN1. Apoptosis-resistant NRAS-mutant lines can sensitize to AZD6244 by pretreatment with AXIN1 siRNA, similar to what we previously reported in BRAF-mutant cell lines. Taken together, these findings indicate that NRAS-mutant melanoma share with BRAF-mutant melanoma the potential to regulate apoptosis upon MEK inhibition through WNT3A and dynamic regulation of cellular AXIN1. Understanding the cellular context that makes melanoma cells susceptible to this combination treatment will contribute to the study and development of novel therapeutic combinations that may lead to more durable responses.
BRAF; MEK; apoptosis; melanoma; wnt
The E2F family of transcription factors contributes to oncogenesis through activation of multiple genes involved in cellular proliferation, a process that is opposed by the Retinoblastoma tumor suppressor protein (RB). RB also increases E2F1 stability by inhibiting its proteasome-mediated degradation, but the consequences of this post-translational regulation of E2F1 remain unknown. To better understand the mechanism of E2F stabilization and its physiological relevance, we examined the streamlined Rbf1-dE2F1 network in Drosophila. During embryonic development, Rbf1 is insulated from ubiquitin-mediated turnover by the COP9 signalosome, a multi-protein complex that modulates E3 ubiquitin ligase activity. Here, we report that the COP9 signalosome also protects the Cullin4-E3 ligase that is responsible for dE2F1 proteasome-mediated destruction. This dual role of the COP9 signalosome may serve to buffer E2F levels, enhancing its turnover via Cul4 protection and its stabilization through protection of Rbf1. We further show that Rbf1-mediated stabilization of dE2F1 and repression of dE2F1 cell cycle-target genes are distinct properties. Removal of an evolutionarily conserved Rbf1 C terminal degron disabled Rbf1 repression without affecting dE2F1 stabilization. This mutant form of Rbf1 also enhanced G1-to-S phase progression when expressed in Rbf1-containing S2 embryonic cells, suggesting that such mutations may generate gain-of-function properties relevant to cellular transformation. Consistent with this idea, several studies have identified mutations in the homologous C terminal domains of RB and p130 in human cancer.
retinoblastoma; tumor suppressor; E2F1; repression; proteasome; ubiquitin; degron
DNA interstrand cross-links (ICLs) present a major challenge to cells, preventing separation of the two strands of duplex DNA and blocking major chromosome transactions, including transcription and replication. Due to the complexity of removing this form of DNA damage, no single DNA repair pathway has been shown to be capable of eradicating ICLs. In eukaryotes, ICL repair is a complex process, principally because several repair pathways compete for ICL repair intermediates in a strictly cell cycle-dependent manner. Yeast cells require a combination of nucleotide excision repair, homologous recombination repair and postreplication repair/translesion DNA synthesis to remove ICLs. There are also a number of additional ICL repair factors originally identified in the budding yeast Saccharomyces cerevisiae, called Pso1 though 10, of which Pso2 has an apparently dedicated role in ICL repair. Mammalian cells respond to ICLs by a complex network guided by factors mutated in the inherited cancer-prone disorder Fanconi anemia (FA). Although enormous progress has been made over recent years in identifying and characterizing FA factors as well as in elucidating certain aspects of the biology of FA, the mechanistic details of the ICL repair defects in FA patients remain unknown. Dissection of the FA DNA damage response pathway has, in part, been limited by the absence of FA-like pathways in highly tractable model organisms, such as yeast. Although S. cerevisiae possesses putative homologs of the FA factors FANCM, FANCJ and FANCP (Mph1, Chl1 and Slx4, respectively) as well as of the FANCM-associated proteins MHF1 and MHF2 (Mhf1 and Mhf2), the corresponding mutants display no significant increase in sensitivity to ICLs. Nevertheless, we and others have recently shown that these FA homologs, along with several other factors, control an ICL repair pathway, which has an overlapping or redundant role with a Pso2-controlled pathway. This pathway acts in S-phase and serves to prevent ICL-stalled replication forks from collapsing into DNA double-strand breaks.
Fanconi anemia; DNA interstrand cross-link repair; S-phase; Saccharomyces cerevisiae
During important cellular processes such as centrosome and spindle positioning, dynein at the cortex interacts with dynamic microtubules in an apparent “end-on” fashion. It is well-established that dynein can generate forces by moving laterally along the microtubule lattice, but much less is known about dynein’s interaction with dynamic microtubule ends. In this paper, we review recent in vitro experiments that show that dynein, attached to an artificial cortex, is able to capture microtubule ends, regulate microtubule dynamics and mediate the generation of pulling forces on shrinking microtubules. We further review existing ideas on the involvement of dynein-mediated cortical pulling forces in the positioning of microtubule organizing centers such as centrosomes. Recent in vitro experiments have demonstrated that cortical pulling forces in combination with pushing forces can lead to reliable centering of microtubule asters in quasi two-dimensional microfabricated chambers. In these experiments, pushing leads to slipping of microtubule ends along the chamber boundaries, resulting in an anisotropic distribution of cortical microtubule contacts that favors centering, once pulling force generators become engaged. This effect is predicted to be strongly geometry-dependent, and we therefore finally discuss ongoing efforts to repeat these experiments in three-dimensional, spherical and deformable geometries.
microtubules; centrosome; dynein; positioning; centering; aster; pulling; pushing; slipping; microfabricated chambers; emulsion droplets; GUVs; cytoskeleton; molecular motors
PCTAIRE kinases (PCTK) are a highly conserved, but poorly characterized, subgroup of cyclin-dependent kinases (CDK). They are characterized by a conserved catalytic domain flanked by N- and C-terminal extensions that are involved in cyclin binding. Vertebrate genomes contain three highly similar PCTAIRE kinases (PCTK1,2,3, a.k.a., CDK16,17,18), which are most abundant in post-mitotic cells in brain and testis. Consistent with this restricted expression pattern, PCTK1 (CDK16) has recently been shown to be essential for spermatogenesis. PCTAIREs are activated by cyclin Y (CCNY), a highly conserved single cyclin fold protein. By binding to N-myristoylated CCNY, CDK16 is targeted to the plasma membrane. Unlike conventional cyclin-CDK interactions, binding of CCNY to CDK16 not only requires the catalytic domain, but also domains within the N-terminal extension. Interestingly, phosphorylation within this domain blocks CCNY binding, providing a novel means of cyclin-CDK regulation. By using these functional characteristics, we analyzed “PCTAIRE” sequence containing protein kinase genes in genomes of various organisms and found that CCNY and CCNY-dependent kinases are restricted to eumetazoa and possibly evolved along with development of a central nervous system. Here, we focus on the structure and regulation of PCTAIREs and discuss their established functions.
CDK; CDK16; PCTAIRE; PCTK; cyclin; development; evolution; spermatogenesis
Cilia are found on most human cells and exist as motile cilia or non-motile primary cilia. Primary cilia play sensory roles in transducing various extracellular signals, and defective ciliary functions are involved in a wide range of human diseases. Centrosomes are the principal microtubule-organizing centers of animal cells and contain two centrioles. We observed that DNA damage causes centriole splitting in non-transformed human cells, with isolated centrioles carrying the mother centriole markers CEP170 and ninein but not kizuna or cenexin. Loss of centriole cohesion through siRNA depletion of C-NAP1 or rootletin increased radiation-induced centriole splitting, with C-NAP1-depleted isolated centrioles losing mother markers. As the mother centriole forms the basal body in primary cilia, we tested whether centriole splitting affected ciliogenesis. While irradiated cells formed apparently normal primary cilia, most cilia arose from centriolar clusters, not from isolated centrioles. Furthermore, C-NAP1 or rootletin knockdown reduced primary cilium formation. Therefore, the centriole cohesion apparatus at the proximal end of centrioles may provide a target that can affect primary cilium formation as part of the DNA damage response.
centrosome; DNA damage response; kizuna; C-NAP1; NEK2
Centrosome duplication is controlled both negatively and positively by a number of proteins. The activities and stabilities of those regulatory proteins are in many cases controlled by posttranslational modifications. Although acetylation and deacetylation are highly common posttranslational modifications, their roles in the regulation of centrosome duplication had not been closely examined. Here, through focusing on the deacetylases, we investigated the role of acetylation/deacetylation in the regulation of centrosome duplication and induction of abnormal amplification of centrosomes. We found that the deacetylation event negatively controls centrosome duplication and amplification. Of the 18 total known deacetylases (HDAC1–11, SIRT1–7), ten deacetylases possess the activity to suppress centrosome amplification, and their centrosome amplification suppressing activities are strongly associated with their abilities to localize to centrosomes. Among them, HDAC1, HDAC5 and SIRT1 show the highest suppressing activities, but each of them suppresses centrosome duplication and/or amplification with its unique mechanism.
centrosome; deacetylase; histone deacetylase (HDAC); acetylation; deacetylation; centrosome duplication; centrosome amplification
The DNA damage checkpoint controls cell cycle arrest in response to DNA damage, and activation of this checkpoint is in turn cell cycle-regulated. Rad9, the ortholog of mammalian 53BP1, is essential for this checkpoint response and is phosphorylated by the cyclin-dependent kinase (CDK) in the yeast Saccharomyces cerevisiae. Previous studies suggested that the CDK consensus sites of Rad9 are important for its checkpoint activity. However, the precise CDK sites of Rad9 involved have not been determined. Here we show that CDK consensus sites of Rad9 function in parallel to its BRCT domain toward checkpoint activation, analogous to its fission yeast ortholog Crb2. Unlike Crb2, however, mutation of multiple rather than any individual CDK site of Rad9 is required to completely eliminate its checkpoint activity in vivo. Although Dpb11 interacts with CDK-phosphorylated Rad9, we provide evidence showing that elimination of this interaction does not affect DNA damage checkpoint activation in vivo, suggesting that additional pathway(s) exist. Taken together, these findings suggest that the regulation of Rad9 by CDK and the role of Dpb11 in DNA damage checkpoint activation are more complex than previously suggested. We propose that multiple phosphorylation of Rad9 by CDK may provide a more robust system to allow Rad9 to control cell cycle-dependent DNA damage checkpoint activation.
BRCT; CDK; Dpb11; Mec1; Rad53; Rad9
Tumor protein (TP)-p53 family members (TP63, TP63 and TP73) are guardians of the genome and key players in orchestrating the cellular response to cisplatin treatment. Cisplatin-induced phosphorylation of ΔNp63α was shown to have a role in regulating intracellular ΔNp63α protein levels. We previously found that squamous cell carcinoma (SCC) cells exposed to cisplatin displayed the ATM-dependent phosphorylation of ΔNp63α (p-ΔNp63α), which is critical for the transcriptional regulation of specific downstream mRNAs and microRNAs and is likely to underlie the chemoresistance of SCC cells. However, SCC cells expressing non-p-ΔNp63α became more cisplatin-resistant. We also found that p-ΔNp63α forms complexes with a number of proteins involved in cell death response through regulation of cell cycle arrest, apoptosis, autophagy, RNA splicing and chromatin modifications. Here, we showed that p-ΔNp63α induced ARG1, GAPDH, and CPT2 gene transcription in cisplatin-sensitive SCC cells, while non-p-ΔNp63α increased a transcription of CAD, G6PD and FASN genes in cisplatin-resistant SCC cells. We report that the p-ΔNp63α-dependent regulatory mechanisms implicated in the modulation of plethora of pathways, including amino acid, carbohydrate, lipid and nucleotide metabolisms, thereby affect tumor cell response to cisplatin-induced cell death, suggesting that the ATM-dependent ΔNp63α pathway plays a role in the resistance of tumor cells to platinum therapy.
P53; p63; cisplatin; squamous cell carcinomas; protein interactions; metabolomics
Rhabdomyosarcoma (RMS) is a pediatric tumor that arises from muscle precursor cells. RMS cells express several markers of early myogenic differentiation, but they fail to complete both differentiation program and cell cycle arrest, resulting in uncontrolled proliferation and incomplete myogenesis. Previous studies showed that EZH2, which is involved in both differentiation and cancer progression, is overexpressed in RMS, but a functional binding between its expression and its functional role in tumor formation or progression has not yet been demonstrated. We hypothesized that EZH2 is a key regulator of muscular differentiation program in RMS cells.
In this study, we demonstrated that EZH2 directly binds muscle specific genes in RD cells. Silencing of EZH2 promotes the recruitment of a multiprotein complex at muscle-specific promoters, their transcriptional activation and protein expression. Moreover, we demonstrated that EZH2 is directly involved in transcriptional repression of MyoD, the main factor promoting myogenesis. EZH2 ablation induces MyoD activation the recovery of its binding on muscle-specific genes.
PRC complex; EZH2; MyoD; rhabdomyosarcoma; myogenesis
Impaired DNA damage response pathways may create vulnerabilities of cancer cells that can be exploited therapeutically. One such selective vulnerability is the sensitivity of BRCA1- or BRCA2-defective tumors (hence defective in DNA repair by homologous recombination, HR) to inhibitors of the poly(ADP-ribose) polymerase-1 (PARP-1), an enzyme critical for repair pathways alternative to HR. While promising, treatment with PARP-1 inhibitors (PARP-1i) faces some hurdles, including (1) acquired resistance, (2) search for other sensitizing, non-BRCA1/2 cancer defects and (3) lack of biomarkers to predict response to PARP-1i. Here we addressed these issues using PARP-1i on 20 human cell lines from carcinomas of the breast, prostate, colon, pancreas and ovary. Aberrations of the Mre11-Rad50-Nbs1 (MRN) complex sensitized cancer cells to PARP-1i, while p53 status was less predictive, even in response to PARP-1i combinations with camptothecin or ionizing radiation. Furthermore, monitoring PARsylation and Rad51 foci formation as surrogate markers for PARP activity and HR, respectively, supported their candidacy for biomarkers of PARP-1i responses. As to resistance mechanisms, we confirmed the role of the multidrug resistance efflux transporters and its reversibility. More importantly, we demonstrated that shRNA lentivirus-mediated depletion of 53BP1 in human BRCA1-mutant breast cancer cells increased their resistance to PARP-1i. Given the preferential loss of 53BP1 in BRCA-defective and triple-negative breast carcinomas, our findings warrant assessment of 53BP1 among candidate predictive biomarkers of response to PARPi. Overall, this study helps characterize genetic and functional determinants of cellular responses to PARP-1i and contributes to the search for biomarkers to exploit PARP inhibitors in cancer therapy.
53BP1; BRCA1; DNA damage response; MRN complex; PARP-1 inhibitor; cancer treatment; p53; parsylation; predictive biomarkers; synthetic lethality or viability
Resveratrol is a polyphenol contained in red wine that has been amply investigated for its beneficial effects on organismal metabolism, in particular in the context of the so-called “French paradox,” i.e., the relatively low incidence of coronary heart disease exhibited by a population with a high dietary intake of cholesterol and saturated fats. At least part of the beneficial effect of resveratrol on human health stems from its capacity to promote autophagy by activating the NAD-dependent deacetylase sirtuin 1. However, the concentration of resveratrol found in red wine is excessively low to account alone for the French paradox. Here, we investigated the possibility that other mono- and polyphenols contained in red wine might induce autophagy while affecting the acetylation levels of cellular proteins. Phenolic compounds found in red wine, including anthocyanins (oenin), stilbenoids (piceatannol), monophenols (caffeic acid, gallic acid) glucosides (delphinidin, kuronamin, peonidin) and flavonoids (catechin, epicatechin, quercetin, myricetin), were all capable of stimulating autophagy, although with dissimilar potencies. Importantly, a robust negative correlation could be established between autophagy induction and the acetylation levels of cytoplasmic proteins, as determined by a novel immunofluorescence staining protocol that allows for the exclusion of nuclear components from the analysis. Inhibition of sirtuin 1 by both pharmacological and genetic means abolished protein deacetylation and autophagy as stimulated by resveratrol, but not by piceatannol, indicating that these compounds act through distinct molecular pathways. In support of this notion, resveratrol and piceatannol synergized in inducing autophagy as well as in promoting cytoplasmic protein deacetylation. Our results highlight a cause-effect relationship between the deacetylation of cytoplasmic proteins and autophagy induction by red wine components.
Beclin 1; LC3; longevity; p62/SQSTM1; trichostatin A; U2OS
DJ-1 (or PARK-7) is a multifunctional protein implicated in numerous pathologies including cancer, sterility and Parkinson disease (PD). The popular genetic model Drosophila melanogaster has two orthologs, dj-1: α and β. Dysfunction of dj-1β strongly impairs fly mobility in an age-dependent manner. In this study, we analyze in detail the molecular mechanism underlying the dj-1β mutant phenotype. Mitochondrial hydrogen peroxide production, but not superoxide production, was increased in mutant flies. An increase in peroxide leak from mitochondria causes oxidative damage elsewhere and explains the strong reduction in mobility caused by dj-1β mutation. However, at the same time, increased levels of hydrogen peroxide activated a pro-survival program characterized by (1) an alteration in insulin-like signaling, (2) an increase in mitochondrial biogenesis and (3) an increase in the de-acetylase activity of sirtuins. The activation of this pro-survival program was associated with increased longevity under conditions of moderate oxidative stress. Additionally, the dj-1β mutation unexpectedly accelerated development, a phenotype not previously associated with this mutation. Our results reveal an important role of dj-1β in oxidative stress handling, insulin-like signaling and development in Drosophila melanogaster.
Drosophila; dj-1β; lifespan; mitochondria; oxidative stress
CREBZF is a member of the mammalian ATF/CREB family of transcription factors. Here, we describe a novel functional interaction between CREBZF and the tumor suppressor p53. CREBZF was identified in a yeast two-hybrid screen using HEY1, recently characterized as an indirect p53 activator, as bait. CREBZF interacts in vitro with both HEY1 and p53, and CREBZF expression stabilizes and activates p53. Moreover, CREBZF cooperates synergistically with HEY1 to enhance p53 transcriptional activity. On the other hand, partial depletion of endogenous CREBZF diminishes p53 protein levels and inhibits HEY1-mediated activation of p53. CREBZF-positive effects on p53 signaling may reflect, at least in part, an observed induction of posttranslational modifications in p53 known to prevent its degradation. CREBZF expression protects HCT116 cells from UV radiation-induced cell death. In addition, CREBZF expression confers sensitivity to 5-fluorouracil, a p53-activating chemotherapeutic drug. Our study suggests that CREBZF may participate in the modulation of p53 tumor suppressor function.
CREBZF; p53; ultraviolet radiation; 5-fluorouracil; DNA damage; HEY1
Multiple myeloma (MM) is a serious, mostly incurable human cancer of malignant plasma cells. Chromosomal translocations affecting MAFB are present in a significant percentage of multiple myeloma patients. Genetically engineered Sca1-MafB mice, in which MafB expression is limited to hematopoietic stem/progenitor cells (HS/P-Cs), display the phenotypic features of MM. Contrary to many other types of cancer, it is not yet known if the p53 gene plays any essential role in the pathogenesis of this disease. Here, we show, taking advantage of the Sca1-MafB MM mouse model, that loss of p53 does not rescue the multiple myeloma disease, but instead accelerates its development and exacerbates the MM phenotype. Therefore, the efficiency of the MafB-induced MM reprogramming of normal HS/P-Cs to terminally differentiated malignant plasma cells is enhanced by p53 deficiency, in analogy to what happens in reprogramming to pluripotency. These results raise caution about interfering with p53 function when treating multiple myeloma.
stem cells; cancer stem cell; cell reprogramming; mouse models; oncogenes; cancer therapy; Mafb; multiple myeloma
Glutamine (Gln) and glucose (Glc) represent two important nutrients for proliferating cells, consistent with the observations that oncogenic processes are associated with enhanced glycolysis and glutaminolysis. Gln depletion and Glc depletion have been shown to trigger growth arrest and eventually cell death. Solid tumors often outgrow the blood supply, resulting in ischemia, which is associated with hypoxia and nutrient insufficiency. Whereas oxygen-sensing and adaptive mechanisms to hypoxia have been well-studied, how cells directly sense and respond to Gln and Glc insufficiency remains unclear. Using mRNA profiling techniques, we compared the gene expression profiles of acute Gln-depleted cells, Glc-depleted cells and cells adapted to Gln depletion. Here we report the global changes of the gene expression in those cells cultured under the defined nutrient conditions. Analysis of mRNA profiling data revealed that Gln and Glc depletion triggered dramatic gene expression reprogramming. Either Gln or Glc deletion leads to changes of the expression of cell cycle genes, but these conditions have distinctive effects on transcription regulators and gene expression profiles. Moreover, Gln and Glc depletion triggered distinguishable ER-stress responses. The gene expression patterns support that Gln and Glc have distinctive metabolic roles in supporting cell survival and proliferation, and cells use different mechanisms to sense and respond to Gln and Glc insufficiency. Our mRNA profiling database provides a resource for further investigating the nutrient-sensing mechanisms and potential effects of Glc and Gln abundance on the biological behaviors of cells.
ATF4; ATF6; ER stress; XBP1; glucose depletion; glutamine depletion; metabolism
It has long been argued that cell cycle regulators such as cyclins, cyclin-dependent kinases and their inhibitors affect the fate of neuronal progenitor cells. Recently, we identified that cyclin D2, which localizes at the basal tip of the radial glial cell (i.e., the neural progenitor in the developing neocortex), functions to give differential cell fates to its daughter cells just after cell division. This basally biased localization is due to transportation of cyclin D2 mRNA via its unique cis-regulatory sequence and local translation into cyclin D2 protein at the basal endfoot. During division of the neural progenitor cells, cyclin D2 protein is inherited by the daughter cell that retain the basal process, resulting in asymmetric distribution of cyclin D2 protein between the two daughter cells. Cyclin D2 is similarly localized in the human fetal cortical primordium, suggesting a common mechanism for the maintenance of neural progenitors and a possible scenario in evolution of primate brains. Here we introduce our recent findings and discuss how cyclin D2 functions in mammalian brain development and evolution.
asymmetric cell division; cyclin D2; localization; mRNA; post-transcriptional regulation; radial glia
CEP192 is a centrosome protein that plays a critical role in centrosome biogenesis and function in mammals, Drosophila and C. elegans.1-6 Moreover, CEP192-depleted cells arrest in mitosis with disorganized microtubules, suggesting that CEP192’s function in spindle assembly goes beyond its role in centrosome activity and pointing to a potentially more direct role in the regulation of the mitotic microtubule landscape.7 To better understand CEP192 function in mitosis, we used mass spectrometry to identify CEP192-interacting proteins. We previously reported that CEP192 interacts with NEDD1, a protein that associates with the γ-tubulin ring complex (γ-TuRC) and regulates its phosphorylation status during mitosis.8 Additionally, within the array of proteins that interact with CEP192, we identified the microtubule binding K63-deubiquitinase CYLD. Further analyses show that co-depletion of CYLD alleviates the bipolar spindle assembly defects observed in CEP192-depleted cells. This functional relationship exposes an intriguing role for CYLD in spindle formation and raises the tantalizing possibility that CEP192 promotes robust mitotic spindle assembly by regulating K63-polyubiquitin-mediated signaling through CYLD.
CEP192; CYLD; mitosis; spindle; microtubules; ubiquitination; K63; centrosome; proteomics
TLRs are a family of pattern recognition receptors that recognize conserved molecular structures/products from a wide variety of microbes. Following recognition of ligands, TLRs recruit signaling adapters to initiate a pro-inflammatory signaling cascade culminating in the activation of several transcription factor families. Additionally, TLR signals lead to activation of PI3K, affecting many aspects of the cellular response, including cell survival, proliferation and regulation of the pro-inflammatory response. The recent discovery of BCAP as a TLR signaling adaptor, crucial for linking TLRs to PI3K activation, allows new questions of the importance of PI3K activation downstream of TLRs. Here, we summarize the current understanding of signaling pathways activated by TLRs and provide our perspective on TLR mediated activation of PI3K and its impact on regulating cellular processes.
BCAP; PI3K; inflammation; macrophage; toll-like receptor
Origin recognition complex (ORC) is highly dynamic, with several ORC subunits getting posttranslationally modified by phosphorylation or ubiquitination in a cell cycle-dependent manner. We have previously demonstrated that a WD repeat containing protein ORC-associated (ORCA/LRWD1) stabilizes the ORC on chromatin and facilitates pre-RC assembly. Further, ORCA levels are cell cycle-regulated, with highest levels during G1, and progressively decreasing during S phase, but the mechanism remains to be elucidated. We now demonstrate that ORCA is polyubiquitinated in vivo, with elevated ubiquitination observed at the G1/S boundary. ORCA utilizes lysine-48 (K48) ubiquitin linkage, suggesting that ORCA ubiquitination mediates its regulated degradation. Ubiquitinated ORCA is re-localized in the form of nuclear aggregates and is predominantly associated with chromatin. We demonstrate that ORCA associates with the E3 ubiquitin ligase Cul4A-Ddb1. ORCA is ubiquitinated at the WD40 repeat domain, a region that is also recognized by Orc2. Furthermore, Orc2 associates only with the non-ubiquitinated form of ORCA, and Orc2 depletion results in the proteasome-mediated destabilization of ORCA. Based on the results, we suggest that Orc2 protects ORCA from ubiquitin-mediated degradation in vivo.
ORCA/LRWD1; replication; pre-RC; ORC; ubiquitination; Cul4A-Ddb1
Lin28 plays important roles in development, stem cell maintenance, oncogenesis and metabolism. As an RNA-binding protein, it blocks the biogenesis primarily of let-7 family miRNAs and also promotes translation of a cohort of mRNAs involved in cell growth, metabolism and pluripotency, likely through recognition of distinct sequence and structural motifs within mRNAs. Here, we show that one such motif, shared by multiple Lin28-responsive elements (LREs) present in Lin28 mRNA targets also participates in a Drosha-dependent regulation and may contribute to destabilization of its cognate mRNAs. We further show that the same mutations in the LREs known to abolish Lin28 binding and stimulation of translation also abrogate Drosha-dependent mRNA destabilization, and that this effect is independent of miRNAs, uncovering a previously unsuspected coupling between Drosha-dependent destabilization and Lin28-mediated regulation. Thus, Lin28-dependent stimulation of translation of target mRNAs may, in part, serve to compensate for their intrinsic instability, thereby ensuring optimal levels of expression of genes critical for cell viability, metabolism and pluripotency.
Lin28; Drosha; stem cell; oncogene; RNA stability; metabolism
It is well known that ligand binding to the high-affinity GM-CSF receptor (GMR) activates JAK2. However, how and where this event occurs in a cellular environment remains unclear. Here, we demonstrate that clathrin- but not lipid raft-mediated endocytosis is crucial for GMR signaling. Knockdown expression of clathrin heavy chain or intersectin 2 (ITSN2) attenuated GMR-mediated activation of JAK2, whereas inhibiting clathrin-coated pits or plagues to bud off the membrane by the dominant-negative mutant of dynamin enhanced such event. Moreover, unlike the wild-type receptor, an ITSN2-non-binding mutant of GMR defective in targeting to clathrin-coated pits or plagues [collectively referred to as clathrin-coated structures (CCSs) here] failed to activate JAK2 at such locations. Additional experiments demonstrate that ligand treatment not only enhanced JAK2/GMR association at CCSs, but also induced a conformational change of JAK2 which is required for JAK2 to be activated by CCS-localized CK2. Interestingly, ligand-independent activation of the oncogenic mutant of JAK2 (JAK2V617F) also requires the targeting of this mutant to CCSs. But JAK2V617F seems to be constitutively in an open conformation for CK2 activation. Together, this study reveals a novel functional role of CCSs in GMR signaling and the oncogenesis of JAK2V617F.
GM-CSF receptor; Jak2; casein kinase 2; clathrin-coated pit; myeloproliferative disorders
Accurate DNA replication is crucial for the maintenance of genome integrity. To this aim, cells have evolved complex surveillance mechanisms to prevent mitotic entry in the presence of partially replicated DNA. ATR and Chk1 are key elements in the signal transduction pathways of DNA replication checkpoint; however, other kinases also make significant contributions. We show here that the stress kinases p38 and JNK are activated when DNA replication is blocked, and that their activity allows S/M, but not G₂/M, checkpoint maintenance when Chk1 is inhibited. Activation of both kinases by DNA replication inhibition is not mediated by the caffeine-sensitive kinases ATR or ATM. Phosphorylation of MKK3/6 and MKK4, p38 and JNK upstream kinases was also observed upon DNA replication inhibition. Using a genetic approach, we dissected the p38 pathway and showed that both p38α and p38β isoforms collaborate to inhibit mitotic entry. We further defined MKK3/6 and MK2/3 as the key upstream and downstream elements in the p38 signaling cascade after replication arrest. Accordingly, we found that the stress signaling pathways collaborate with Chk1 to keep cyclin B1/Cdk1 complexes inactive when DNA replication is inhibited, thereby preventing cell cycle progression when DNA replication is stalled. Our results show a complex response to replication stress, where multiple pathways are activated and fulfill overlapping roles to prevent mitotic entry with unreplicated DNA.
Chk1; JNK; S/M checkpoint; SAPK; hydroxyurea; p38
The HECT-containing E3 ubiquitin ligase Itch mediates the degradation of several proteins, including p63 and p73, involved in cell specification and fate. Itch contains four WW domains, which are essential for recognition on the target substrate, which contains a short proline-rich sequence. Several signaling complexes containing these domains have been associated with human diseases such as muscular dystrophy, Alzheimer’s or Huntington’s diseases. To gain further insight into the structural determinants of the Itch-WW2 domain, we investigated its interaction with p63. We assigned, by 3D heteronuclear NMR experiments, the backbone and side chains of the uniformly ¹³C-¹⁵N-labeled Itch-WW2. In vitro interaction of Itch-WW2 domain with p63 was studied using its interactive p63 peptide, pep63. Pep63 is an 18-mer peptide corresponding to the region from 534–551 residue of p63, encompassing the PPxY motif that interacts with the Itch-WW domains, and we identified the residues involved in this molecular recognition. Moreover, here, a strategy of stabilization of the conformation of the PPxY peptide has been adopted, increasing the WW-ligand binding. We demonstrated that cyclization of pep63 leads to an increase of both the biological stability of the peptide and of the WW-ligand complex. Stable metal-binding complexes of the pep63 have been also obtained, and localized oxidative damage on Itch-WW2 domain has been induced, demonstrating the possibility of use of metal-pep63 complexes as models for the design of metal drugs to inhibit the Itch-WW-p63 recognition in vivo. Thus, our data suggest a novel strategy to study and inhibit the recognition mechanism of Itch E3-ligase.
p63; p53 family; E3 ubiquitin ligases; HECT; ubiquitilation; cyclization; metal-peptide
Heat shock protein 90 (Hsp90) is an essential, evolutionarily conserved molecular chaperone. Cancer cells rely on Hsp90 to chaperone mutated and/or activated oncoproteins, and its involvement in numerous signaling pathways makes it an attractive target for drug development. Surprisingly, however, the impact of Hsp90 inhibitors on cancer cells is frequently cytostatic in nature, and efforts to enhance the antitumor activity of Hsp90 inhibitors in the clinic remain a significant challenge. In agreement with previous data obtained using Wee1 siRNA, we show that dual pharmacologic inhibition of Wee1 tyrosine kinase and Hsp90 causes cancer cells to undergo apoptosis in vitro and in vivo. Gene expression profiling revealed that induction of the intrinsic apoptotic pathway by this drug combination coincided with transcriptional downregulation of Survivin and Wee1, an outcome not seen in cells treated separately with either agent. At the translational level, expression of these two proteins, as well as activated Akt, was completely abrogated. These data support the hypothesis that Wee1 inhibition sensitizes cancer cells to Hsp90 inhibitors; they establish combined Wee1/Hsp90 inhibition as a novel therapeutic strategy; and they provide a mechanistic rationale for enhancing the pro-apoptotic activity of Hsp90 inhibitors.
Wee1; apoptosis; cancer; heat shock protein 90; molecular targeted anticancer drugs