p190RhoGAP Filters Competing Signals to Resolve Axon Guidance Conflicts.

The rich functional diversity of the nervous system is founded in the specific connectivity of the underlying neural circuitry. Neurons are often preprogrammed to respond to multiple axon guidance signals because they use sequential guideposts along their pathways, but this necessitates a strict spatiotemporal regulation of intracellular signaling to ensure the cues are detected in the correct order. We performed a mouse mutagenesis screen and identified the Rho GTPase antagonist p190RhoGAP as a critical regulator of motor axon guidance. Rather than acting as a compulsory signal relay, p190RhoGAP uses a non-conventional GAP-independent mode to transiently suppress attraction to Netrin-1 while motor axons exit the spinal cord. Once in the periphery, a subset of axons requires p190RhoGAP-mediated inhibition of Rho signaling to target specific muscles. Thus, the multifunctional activity of p190RhoGAP emerges from its modular design. Our findings reveal a cell-intrinsic gate that filters conflicting signals, establishing temporal windows of signal detection.

An internal ribosome entry site in the coding region of tyrosyl-DNA phosphodiesterase 2 drives alternative translation start.

Tyrosyl-DNA phosphodiesterase 2 (TDP2) is a multifunctional protein that has been implicated in a myriad of cellular pathways. Although most well-known for its phosphodiesterase activity removing stalled topoisomerase 2 from DNA, TDP2 has also been shown to interact with both survival and apoptotic mitogen-activated protein kinase (MAPK) signaling cascades. Moreover, it facilitates enterovirus replication and has been genetically linked to neurological disorders ranging from Parkinson's disease to dyslexia. To accurately evaluate TDP2 as a therapeutic target, we need to understand how TDP2 performs such a wide diversity of functions. Here, we use cancer cell lines modified with CRISPR/Cas9 or stably-expressed TDP2-targeted shRNA and transfection of various TDP2 mutants to show that its expression is regulated at the translational level via an internal ribosome entry site (IRES) that initiates translation at codon 54, the second in-frame methionine of the TDP2 coding sequence. We observed that this IRES drives expression of a shorter, N-terminally truncated isoform of TDP2, ΔN-TDP2, which omits a nuclear localization sequence. Additionally, we noted that ΔN-TDP2 retains phosphodiesterase activity and is protective against etoposide-induced cell death, but co-immunoprecipitates with fewer high-molecular-weight ubiquitinated peptide species, suggesting partial loss-of-function of TDP2's ubiquitin-association domain. In summary, our findings suggest the existence of an IRES in the 5' coding sequence of TDP2 that translationally regulates expression of an N-terminally truncated, cytoplasmic isoform of TDP2. These results shed light on the regulation of this multifunctional protein and may inform the design of therapies targeting TDP2 and associated pathways.

Heterochromatin-Encoded Satellite RNAs Induce Breast Cancer.

Heterochromatic repetitive satellite RNAs are extensively transcribed in a variety of human cancers, including BRCA1 mutant breast cancer. Aberrant expression of satellite RNAs in cultured cells induces the DNA damage response, activates cell cycle checkpoints, and causes defects in chromosome segregation. However, the mechanism by which satellite RNA expression leads to genomic instability is not well understood. Here we provide evidence that increased levels of satellite RNAs in mammary glands induce tumor formation in mice. Using mass spectrometry, we further show that genomic instability induced by satellite RNAs occurs through interactions with BRCA1-associated protein networks required for the stabilization of DNA replication forks. Additionally, de-stabilized replication forks likely promote the formation of RNA-DNA hybrids in cells expressing satellite RNAs. These studies lay the foundation for developing novel therapeutic strategies that block the effects of non-coding satellite RNAs in cancer cells.

Secreted Glioblastoma Nanovesicles Contain Intracellular Signaling Proteins and Active Ras Incorporated in a Farnesylation-dependent Manner.

Glioblastomas (GBMs) are malignant brain tumors with a median survival of less than 18 months. Redundancy of signaling pathways represented within GBMs contributes to their therapeutic resistance. Exosomes are extracellular nanovesicles released from cells and present in human biofluids that represent a possible biomarker of tumor signaling state that could aid in personalized treatment. Herein, we demonstrate that mouse GBM cell-derived extracellular nanovesicles resembling exosomes from an H-RasV12 myr-Akt mouse model for GBM are enriched for intracellular signaling cascade proteins (GO: 0007242) and Ras protein signal transduction (GO: 0007265), and contain active Ras. Active Ras isolated from human and mouse GBM extracellular nanovesicles lysates using the Ras-binding domain of Raf also coprecipitates with ESCRT (endosomal sorting complex required for transport)-associated exosome proteins Vps4a and Alix. Although we initially hypothesized a role for active Ras protein signaling in exosome biogenesis, we found that GTP binding of K-Ras was dispensable for its packaging within extracellular nanovesicles and for the release of Alix. By contrast, farnesylation of K-Ras was required for its packaging within extracellular nanovesicles, yet expressing a K-Ras farnesylation mutant did not decrease the number of nanovesicles or the amount of Alix protein released per cell. Overall, these results emphasize the primary importance of membrane association in packaging of extracellular nanovesicle factors and indicate that screening nanovesicles within human fluids could provide insight into tissue origin and the wiring of signaling proteins at membranes to predict onset and behavior of cancer and other diseases linked to deregulated membrane signaling states.

Primate-specific ORF0 contributes to retrotransposon-mediated diversity.

LINE-1 retrotransposons are fast-evolving mobile genetic entities that play roles in gene regulation, pathological conditions, and evolution. Here, we show that the primate LINE-1 5'UTR contains a primate-specific open reading frame (ORF) in the antisense orientation that we named ORF0. The gene product of this ORF localizes to promyelocytic leukemia-adjacent nuclear bodies. ORF0 is present in more than 3,000 loci across human and chimpanzee genomes and has a promoter and a conserved strong Kozak sequence that supports translation. By virtue of containing two splice donor sites, ORF0 can also form fusion proteins with proximal exons. ORF0 transcripts are readily detected in induced pluripotent stem (iPS) cells from both primate species. Capped and polyadenylated ORF0 mRNAs are present in the cytoplasm, and endogenous ORF0 peptides are identified upon proteomic analysis. Finally, ORF0 enhances LINE-1 mobility. Taken together, these results suggest a role for ORF0 in retrotransposon-mediated diversity.

Monoclonal 1- and 3-Phosphohistidine Antibodies: New Tools to Study Histidine Phosphorylation.

Histidine phosphorylation (pHis) is well studied in bacteria; however, its role in mammalian signaling remains largely unexplored due to the lack of pHis-specific antibodies and the lability of the phosphoramidate (P-N) bond. Both imidazole nitrogens can be phosphorylated, forming 1-phosphohistidine (1-pHis) or 3-phosphohistidine (3-pHis). We have developed monoclonal antibodies (mAbs) that specifically recognize 1-pHis or 3-pHis; they do not cross-react with phosphotyrosine or the other pHis isomer. Assays based on the isomer-specific autophosphorylation of NME1 and phosphoglycerate mutase were used with immunoblotting and sequencing IgG variable domains to screen, select, and characterize anti-1-pHis and anti-3-pHis mAbs. Their sequence independence was determined by blotting synthetic peptide arrays, and they have been tested for immunofluorescence staining and immunoaffinity purification, leading to putative identification of pHis-containing proteins. These reagents should be broadly useful for identification of pHis substrates and functional study of pHis using a variety of immunological, proteomic, and biological assays.

Identification of small ubiquitin-like modifier substrates with diverse functions using the Xenopus egg extract system.

Post-translational modification by SUMO is a highly conserved pathway in eukaryotes that plays very important regulatory roles in many cellular processes. Deregulation of the SUMO pathway contributes to the development and progression of many diseases including cancer. Therefore, identifying additional SUMO substrates and studying how their cellular and biological functions are regulated by sumoylation should provide new insights. Our studies showed that sumoylation activity was significant in Xenopus egg extracts, and that a high level of sumoylation was associated with sperm chromatin when SUMO was incubated with Xenopus egg extracts. By isolating SUMO-conjugated substrates using His-tagged SUMO1 or SUMO2 proteins under denaturing conditions, we identified 346 proteins by mass spectrometry analysis that were not present in control pull-downs. Among them, 167 proteins were identified from interphase egg extracts, 86 proteins from mitotic phase egg extracts, and 93 proteins from both. Thirty-three proteins were pulled down by SUMO1, 85 proteins by SUMO2, and 228 proteins by both. We validated the sumoylation of five candidates, CKB, ATXN10, BTF3, HABP4, and BZW1, by co-transfecting them along with SUMO in HEK293T cells. Gene ontology analysis showed that SUMO substrates identified in this study were involved in diverse biological processes. Additionally, SUMO substrates identified from different cell cycle stages or pulled down by different SUMO homologs were enriched for distinct cellular components and functional categories. Our results comprehensively profile the sumoylation occurring in the Xenopus egg extract system.

Escargot restricts niche cell to stem cell conversion in the Drosophila testis.

Stem cells reside within specialized microenvironments, or niches, that control many aspects of stem cell behavior. Somatic hub cells in the Drosophila testis regulate the behavior of cyst stem cells (CySCs) and germline stem cells (GSCs) and are a primary component of the testis stem cell niche. The shutoff (shof) mutation, characterized by premature loss of GSCs and CySCs, was mapped to a locus encoding the evolutionarily conserved transcription factor Escargot (Esg). Hub cells depleted of Esg acquire CySC characteristics and differentiate as cyst cells, resulting in complete loss of hub cells and eventually CySCs and GSCs, similar to the shof mutant phenotype. We identified Esg-interacting proteins and demonstrate an interaction between Esg and the corepressor C-terminal binding protein (CtBP), which was also required for maintenance of hub cell fate. Our results indicate that niche cells can acquire stem cell properties upon removal of a single transcription factor in vivo.

Census 2: isobaric labeling data analysis.

MOTIVATION: We introduce Census 2, an update of a mass spectrometry data analysis tool for peptide/protein quantification. New features for analysis of isobaric labeling, such as Tandem Mass Tag (TMT) or Isobaric Tags for Relative and Absolute Quantification (iTRAQ), have been added in this version, including a reporter ion impurity correction, a reporter ion intensity threshold filter and an option for weighted normalization to correct mixing errors. TMT/iTRAQ analysis can be performed on experiments using HCD (High Energy Collision Dissociation) only, CID (Collision Induced Dissociation)/HCD (High Energy Collision Dissociation) dual scans or HCD triple-stage mass spectrometry data. To improve measurement accuracy, we implemented weighted normalization, multiple tandem spectral approach, impurity correction and dynamic intensity threshold features. AVAILABILITY AND IMPLEMENTATION: Census 2 supports multiple input file formats including MS1/MS2, DTASelect, mzXML and pepXML. It requires JAVA version 6 or later to run. Free download of Census 2 for academic users is available at http://fields.scripps.edu/census/index.php. CONTACT: jyates@scripps.edu SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Mass spectrometry-based quantification of the cellular response to methyl methanesulfonate treatment in human cells.

Faithful transmission of genetic material is essential for cell viability and organism health. The occurrence of DNA damage, due to either spontaneous events or environmental agents, threatens the integrity of the genome. The consequences of these insults, if allowed to perpetuate and accumulate over time, are mutations that can lead to the development of diseases such as cancer. Alkylation is a relevant DNA lesion produced endogenously as well as by exogenous agents including certain chemotherapeutics. We sought to better understand the cellular response to this form of DNA damage using mass spectrometry-based proteomics. For this purpose, we performed sub-cellular fractionation to monitor the effect of methyl methanesulfonate (MMS) treatment on protein localization to chromatin. The levels of over 500 proteins were increased in the chromatin-enriched nuclear lysate including histone chaperones. Levels of ubiquitin and subunits of the proteasome were also increased within this fraction, suggesting that ubiquitin-mediated degradation by the proteasome has an important role in the chromatin response to MMS treatment. Finally, the levels of some proteins were decreased within the chromatin-enriched lysate including components of the nuclear pore complex. Our spatial proteomics data demonstrate that many proteins that influence chromatin organization are regulated in response to MMS treatment, presumably to open the DNA to allow access by other DNA damage response proteins. To gain further insight into the cellular response to MMS-induced DNA damage, we also performed phosphorylation enrichment on total cell lysates to identify proteins regulated via post-translational modification. Phosphoproteomic analysis demonstrated that many nuclear phosphorylation events were decreased in response to MMS treatment. This reflected changes in protein kinase and/or phosphatase activity in response to DNA damage rather than changes in total protein abundance. Using these two mass spectrometry-based approaches, we have identified a novel set of MMS-responsive proteins that will expand our understanding of DNA damage signaling.

Psy2 targets the PP4 family phosphatase Pph3 to dephosphorylate Mth1 and repress glucose transporter gene expression.

The reversible nature of protein phosphorylation dictates that any protein kinase activity must be counteracted by protein phosphatase activity. How phosphatases target specific phosphoprotein substrates and reverse the action of kinases, however, is poorly understood in a biological context. We address this question by elucidating a novel function of the conserved PP4 family phosphatase Pph3-Psy2, the yeast counterpart of the mammalian PP4c-R3 complex, in the glucose-signaling pathway. Our studies show that Pph3-Psy2 specifically targets the glucose signal transducer protein Mth1 via direct binding of the EVH1 domain of the Psy2 regulatory subunit to the polyproline motif of Mth1. This activity is required for the timely dephosphorylation of the downstream transcriptional repressor Rgt1 upon glucose withdrawal, a critical event in the repression of HXT genes, which encode glucose transporters. Pph3-Psy2 dephosphorylates Mth1, an Rgt1 associated corepressor, but does not dephosphorylate Rgt1 at sites associated with inactivation, in vitro. We show that Pph3-Psy2 phosphatase antagonizes Mth1 phosphorylation by protein kinase A (PKA), the major protein kinase activated in response to glucose, in vitro and regulates Mth1 function via putative PKA phosphorylation sites in vivo. We conclude that the Pph3-Psy2 phosphatase modulates Mth1 activity to facilitate precise regulation of HXT gene expression by glucose.

A posttranslational modification cascade involving p38, Tip60, and PRAK mediates oncogene-induced senescence.

Oncogene-induced senescence is an important tumor-suppressing defense mechanism. However, relatively little is known about the signaling pathway mediating the senescence response. Here, we demonstrate that a multifunctional acetyltransferase, Tip60, plays an essential role in oncogenic ras-induced senescence. Further investigation reveals a cascade of posttranslational modifications involving p38, Tip60, and PRAK, three proteins that are essential for ras-induced senescence. Upon activation by ras, p38 induces the acetyltransferase activity of Tip60 through phosphorylation of Thr158; activated Tip60 in turn directly interacts with and induces the protein kinase activity of PRAK through acetylation of K364 in a manner that depends on phosphorylation of both Tip60 and PRAK by p38. These posttranslational modifications are critical for the prosenescent function of Tip60 and PRAK, respectively. These results have defined a signaling pathway that mediates oncogene-induced senescence, and identified posttranslational modifications that regulate the enzymatic activity and biological functions of Tip60 and PRAK.

The RING finger protein RNF8 ubiquitinates Nbs1 to promote DNA double-strand break repair by homologous recombination.

Ubiquitination plays an important role in the DNA damage response. We identified a novel interaction of the E3 ubiquitin ligase RNF8 with Nbs1, a key regulator of DNA double-strand break (DSB) repair. We found that Nbs1 is ubiquitinated both before and after DNA damage and is a direct ubiquitination substrate of RNF8. We also identified key residues on Nbs1 that are ubiquitinated by RNF8. By using laser microirradiation and live-cell imaging, we observed that RNF8 and its ubiquitination activity are important for promoting optimal binding of Nbs1 to DSB-containing chromatin. We also demonstrated that RNF8-mediated ubiquitination of Nbs1 contributes to the efficient and stable binding of Nbs1 to DSBs and is important for HR-mediated DSB repair. Taken together, these studies suggest that Nbs1 is one important target of RNF8 to regulate DNA DSB repair.

Dual recruitment of Cdc48 (p97)-Ufd1-Npl4 ubiquitin-selective segregase by small ubiquitin-like modifier protein (SUMO) and ubiquitin in SUMO-targeted ubiquitin ligase-mediated genome stability functions.

Protein modification by SUMO and ubiquitin critically impacts genome stability via effectors that "read" their signals using SUMO interaction motifs or ubiquitin binding domains, respectively. A novel mixed SUMO and ubiquitin signal is generated by the SUMO-targeted ubiquitin ligase (STUbL), which ubiquitylates SUMO conjugates. Herein, we determine that the "ubiquitin-selective" segregase Cdc48-Ufd1-Npl4 also binds SUMO via a SUMO interaction motif in Ufd1 and can thus act as a selective receptor for STUbL targets. Indeed, we define key cooperative DNA repair functions for Cdc48-Ufd1-Npl4 and STUbL, thereby revealing a new signaling mechanism involving dual recruitment by SUMO and ubiquitin for Cdc48-Ufd1-Npl4 functions in maintaining genome stability.

Methylation of the retinoblastoma tumor suppressor by SMYD2.

The retinoblastoma tumor suppressor (RB) is a central cell cycle regulator and tumor suppressor. RB cellular functions are known to be regulated by a diversity of post-translational modifications such as phosphorylation and acetylation, raising the possibility that RB may also be methylated in cells. Here we demonstrate that RB can be methylated by SMYD2 at lysine 860, a highly conserved and novel site of modification. This methylation event occurs in vitro and in cells, and it is regulated during cell cycle progression, cellular differentiation, and in response to DNA damage. Furthermore, we show that RB monomethylation at lysine 860 provides a direct binding site for the methyl-binding domain of the transcriptional repressor L3MBTL1. These results support the idea that a code of post-translational modifications exists for RB and helps guide its functions in mammalian cells.

The F box protein Fbx6 regulates Chk1 stability and cellular sensitivity to replication stress.

ATR and Chk1 are two key protein kinases in the replication checkpoint. Activation of ATR-Chk1 has been extensively investigated, but checkpoint termination and replication fork restart are less well understood. Here, we report that DNA damage not only activates Chk1, but also exposes a degron-like region at the carboxyl terminus of Chk1 to an Fbx6-containing SCF (Skp1-Cul1-F box) E3 ligase, which mediates the ubiquitination and degradation of Chk1 and, in turn, terminates the checkpoint. The protein levels of Chk1 and Fbx6 showed an inverse correlation in both cultured cancer cells and in human breast tumor tissues. Further, we show that low levels of Fbx6 and consequent impairment of replication stress-induced Chk1 degradation are associated with cancer cell resistance to the chemotherapeutic agent, camptothecin. We propose that Fbx6-dependent Chk1 degradation contributes to S phase checkpoint termination and that a defect in this mechanism might increase tumor cell resistance to certain anticancer drugs.

p53-deficient cells rely on ATM- and ATR-mediated checkpoint signaling through the p38MAPK/MK2 pathway for survival after DNA damage.

In response to DNA damage, eukaryotic cells activate ATM-Chk2 and/or ATR-Chk1 to arrest the cell cycle and initiate DNA repair. We show that, in the absence of p53, cells depend on a third cell-cycle checkpoint pathway involving p38MAPK/MK2 for cell-cycle arrest and survival after DNA damage. MK2 depletion in p53-deficient cells, but not in p53 wild-type cells, caused abrogation of the Cdc25A-mediated S phase checkpoint after cisplatin exposure and loss of the Cdc25B-mediated G2/M checkpoint following doxorubicin treatment, resulting in mitotic catastrophe and pronounced regression of murine tumors in vivo. We show that the Chk1 inhibitor UCN-01 also potently inhibits MK2, suggesting that its clinical efficacy results from the simultaneous disruption of two critical checkpoint pathways in p53-defective cells.

Repression of the Arf tumor suppressor by E2F3 is required for normal cell cycle kinetics.

Tumor development is dependent upon the inactivation of two key tumor-suppressor networks, p16(Ink4a)-cycD/cdk4-pRB-E2F and p19(Arf)-mdm2-p53, that regulate cellular proliferation and the tumor surveillance response. These networks are known to intersect with one another, but the mechanisms are poorly understood. Here, we show that E2F directly participates in the transcriptional control of Arf in both normal and transformed cells. This occurs in a manner that is significantly different from the regulation of classic E2F-responsive targets. In wild-type mouse embryonic fibroblasts (MEFs), the Arf promoter is occupied by E2F3 and not other E2F family members. In quiescent cells, this role is largely fulfilled by E2F3b, an E2F3 isoform whose function was previously undetermined. E2f3 loss is sufficient to derepress Arf, triggering activation of p53 and expression of p21(Cip1). Thus, E2F3 is a key repressor of the p19(Arf)-p53 pathway in normal cells. Consistent with this notion, Arf mutation suppresses the activation of p53 and p21(Cip1) in E2f3-deficient MEFs. Arf loss also rescues the known cell cycle re-entry defect of E2f3(-/-) cells, and this correlates with restoration of appropriate activation of classic E2F-responsive genes. Our data also demonstrate a direct role for E2F in the oncogenic activation of Arf. Specifically, we observe recruitment of the endogenous activating E2Fs, E2F1, and E2F3a, to the Arf promoter. Thus, distinct E2F complexes directly contribute to the normal repression and oncogenic activation of Arf. We propose that monitoring of E2F levels and/or activity is a key component of Arf's ability to respond to inappropriate, but not normal cellular proliferation.

Myc-mediated proliferation and lymphomagenesis, but not apoptosis, are compromised by E2f1 loss.

Myc and E2f1 promote cell cycle progression, but overexpression of either can trigger p53-dependent apoptosis. Mice expressing an Emu-Myc transgene in B lymphocytes develop lymphomas, the majority of which sustain mutations of either the Arf or p53 tumor suppressors. Emu-Myc transgenic mice lacking one or both E2f1 alleles exhibited a slower onset of lymphoma development associated with increased expression of the cyclin-dependent kinase inhibitor p27(Kip1) and a reduced S phase fraction in precancerous B cells. In contrast, Myc-induced apoptosis and the frequency of Arf and p53 mutations in lymphomas were unaffected by E2f1 loss. Therefore, Myc does not require E2f1 to induce Arf, p53, or apoptosis in B cells, but depends upon E2f1 to accelerate cell cycle progression and downregulate p27(Kip1).