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Homologous to E6AP C-terminal (HECT) ubiquitin (Ub) ligases (E3s) are a large class of enzymes that bind to their substrates and catalyze ubiquitination through the formation of a Ub thioester intermediate. The mechanisms by which these E3s assemble polyubiquitin chains on their substrates remain poorly defined. We report here that the Nedd4 family HECT E3, WWP1, assembles substrate-linked Ub chains containing Lys-63, Lys-48, and Lys-11 linkages (Lys-63 > Lys-48 > Lys-11). Our results demonstrate that WWP1 catalyzes the formation of Ub chains through a sequential addition mechanism, in which Ub monomers are transferred in a successive fashion to the substrate, and that ubiquitination by WWP1 requires the presence of a low-affinity, noncovalent Ub-binding site within the HECT domain. Unexpectedly, we find that the formation of Ub chains by WWP1 occurs in two distinct phases. In the first phase, chains are synthesized in a unidirectional manner and are linked exclusively through Lys-63 of Ub. In the second phase, chains are elongated in a multidirectional fashion characterized by the formation of mixed Ub linkages and branched structures. Our results provide new insight into the mechanism of Ub chain formation employed by Nedd4 family HECT E3s and suggest a framework for understanding how this family of E3s generates Ub signals that function in proteasome-independent and proteasome-dependent pathways.

Shotgun proteomics generates valuable information from large-scale and target protein characterizations, including protein expression, protein quantification, protein post-translational modifications (PTMs), protein localization, and protein–protein interactions. Typically, peptides derived from proteolytic digestion, rather than intact proteins, are analyzed by mass spectrometers because peptides are more readily separated, ionized and fragmented. The amino acid sequences of peptides can be interpreted by matching the observed tandem mass spectra to theoretical spectra derived from a protein sequence database. Identified peptides serve as surrogates for their proteins and are often used to establish what proteins were present in the original mixture and to quantify protein abundance. Two major issues exist for assigning peptides to their originating protein. The first issue is maintaining a desired false discovery rate (FDR) when comparing or combining multiple large datasets generated by shotgun analysis and the second issue is properly assigning peptides to proteins when homologous proteins are present in the database. Herein we demonstrate a new computational tool, ProteinInferencer, which can be used for protein inference with both small- or large-scale data sets to produce a well-controlled protein FDR. In addition, ProteinInferencer introduces confidence scoring for individual proteins, which makes protein identifications evaluable.This article is part of a Special Issue entitled: Computational Proteomics.

SummaryHistidine 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.

Intact protein analysis via top-down mass spectrometry (MS) provides a bird’s eye view over the protein complexes and complex protein mixtures with the unique capability of characterizing protein variants, splice isoforms, and combinatorial post-translational modifications (PTMs). Here we applied capillary electrophoresis (CE) through a sheathless CE–electrospray ionization interface coupled to an LTQ Velos Orbitrap Elite mass spectrometer to analyze the Dam1 complex from Saccharomyces cerevisiae. We achieved a 100-fold increase in sensitivity compared to a reversed-phase liquid chromatography coupled MS analysis of recombinant Dam1 complex with a total loading of 2.5 ng (12 amol). N-terminal processing forms of individual subunits of the Dam1 complex were observed as well as their phosphorylation stoichiometry upon Mps1p kinase treatment.

Summary 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.

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.

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 novel 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 pro-senescent function of Tip60 and PRAK, respectively. These results have defined a novel signaling pathway that mediate oncogene-induced senescence, and identified novel posttranslational modifications that regulate the enzymatic activity and biological functions of Tip60 and PRAK.

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.

Central to the replication checkpoint are two protein kinases, ATR, and its downstream target kinase, Chk1. Signaling pathways leading to activation of ATR-Chk1 have been extensively investigated; however, events that mediate checkpoint termination and replication fork restart are less well understood. Here, we define a coupled activation-destruction mechanism of Chk1 that regulates checkpoint termination and cellular sensitivity to replicative stress. DNA damage-induced phosphorylation or mutation of a conserved motif of Chk1 both activates Chk1 and exposes a degron-like region at the carboxyl-terminus of Chk1 to a Fbx6-containing SCF (Skp1-Cul1-F-box) E3 ligase, which mediates the ubiquitination and degradation of Chk1, and, in turn, terminates the checkpoint. The expression levels of Chk1 and Fbx6 proteins showed an inverse correlation in both cultured cancer cell lines and in a small cohort of 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 killing by the chemotherapeutic agent, camptothecin (CPT). 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.

Mass spectrometry has been widely used to analyze biological samples and has evolved into an indispensable tool for proteomics research. Our desire to understand the proteome has led to new technologies that push the boundary of mass spectrometry capabilities, which in return has allowed mass spectrometry to address an ever-increasing array of biological questions. The recent development of a novel mass spectrometer (Orbitrap) and new dissociation methods such as electron-transfer dissociation has made possible the exciting new areas of proteomic application. Although bottom-up proteomics (analysis of proteolytic peptide mixtures) remains the workhorse for proteomic analysis, middle-down and top-down strategies (analysis of longer peptides and intact proteins, respectively) should allow more complete characterization of protein isoforms and post-translational modifications. Finally, stable isotope labeling strategies have transformed mass spectrometry from merely descriptive to a tool for measuring dynamic changes in protein expression, interaction, and modification.

Myc and E2f1 promote cell cycle progression, but overexpression of either can trigger p53-dependent apoptosis. Mice expressing an Eμ- Myc transgene in B lymphocytes develop lymphomas, the majority of which sustain mutations of either the Arf or p53 tumor suppressors. Eμ- 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 .

The development of mammalian tumors is dependent upon the disruption of two key biological activities, the control of cellular proliferation and the apoptotic response (Hanahan and Weinberg 2000). Remarkably, the Ink4a/Arf locus encodes two distinct tumor-suppressor proteins, p16Ink4a and p19Arf (p14Arf in humans), that influence one or both of these processes (Chin et al. 1998; Sherr 2001). p16Ink4a is a core component of the cell cycle control machinery (Sherr and Roberts 1999). It controls the activity of the G1 kinase, cyclinD · cdk4/6, and consequently, the phosphorylation status of the pocket protein family. This family includes the retinoblastoma protein (pRB) tumor suppressor and its relatives, p107 and p130. In the unphosphorylated state, the pocket proteins bind to the E2F family of transcription factors and prevent the expression of genes that are essential for entry into, and passage through the cell cycle (Trimarchi and Lees 2002). This inhibition occurs through two distinct mechanisms. pRB binds to the activating E2Fs, E2F1, E2F2, and E2F3a, and blocks their transcriptional activity. At the same time, the repressive E2Fs, E2F4, and E2F5 recruit p107 or p130 and their associated histone deactylases to E2F-responsive promoters. Under these conditions, the cell is blocked in G0/G1. Mitogenic signaling activates cell cycle re-entry by allowing cyclinD · cdk4/6 to overcome the repression by p16Ink4a. The consequent phosphorylation of the pocket proteins causes them to dissociate from E2F, enabling activation of E2F-responsive genes. In normal cells, the p16Ink4a–cyclinD · cdk4/6–pRB–E2F pathway responds to both positive and negative growth regulatory signals to determine whether or not a cell will divide (Sherr and Roberts 1999). This pathway is disrupted in most, if not all, mammalian tumors through loss of p16Ink4a, up-regulation of cyclinD · cdk4/6 or loss of pRB (Sherr 1996). The resulting deregulated proliferation is due, at least in part, to the inappropriate activation of E2F (Pan et al. 1998; Tsai et al. 1998; Yamasaki et al. 1998; McCaffrey et al. 1999; Ziebold et al. 2001, 2003). The second product of the Ink4a/Arf locus, p19Arf,isa key component of the p53 tumor-surveillance network (Sherr 2001). p19Arf exists at low or undetectable levels in most normal cell and tissue types (Zindy et al. 2003). However, its expression is specifically activated by abnormal proliferative signals. These include the continued in vitro culturing of mouse embryonic fibroblasts (MEFs; Kamijo et al. 1997) and the inappropriate expression of proliferative oncogenes including activated ras, c-myc, E2F, E1A, and v-Abl (Serrano et al. 1997; de Stanchina et al. 1998; Palmero et al. 1998; Radfar et al. 1998; Zindy et al. 1998; Dimri et al. 2000). Once it is expressed, p19Arf inhibits the p53 ubiquitin ligase, mdm2, allowing activation of the p53 tumor suppressor (Pomerantz et al. 1998; Stott et al. 1998; Zhang et al. 1998; Honda and Yasuda 1999; Weber et al. 1999; Llanos et al. 2001). Depending on the cellular context, p53 triggers either cell cycle arrest (via induction of the cdk inhibitor, p21Cip1) or apoptosis (through activation of various apoptosis inducers). In either case, this counteracts the effect of the abnormal proliferative signals. Essentially, p19Arf acts as a defense to oncogenic signals. The recent analysis of a mouse strain that expresses GFP in place of p19Arf confirms that Arf is induced by the oncogenic signals present in incipient tumors (Zindy et al. 2003). This explains why inactivation of the p19Arf–p53 network is essential for the survival and proliferation of tumor cells in vivo (Sherr 2001). The ability of Arf to specifically respond to inappropriate, but not normal proliferative signals must require a careful balance of transcriptional signals. Understanding how this is achieved remains a major challenge. Numerous studies have implicated E2F in this process (Phillips and Vousden 2001). The Arf promoter contains consensus E2F-binding sites and the overexpression of E2F1 is sufficient to trigger its transcriptional activation (DeGregori et al. 1997; Bates et al. 1998). However, it is unclear whether this regulation is direct because the identified E2F sites are not required for E2F-dependent activation (Parisi et al. 2002; Berkovich et al. 2003). There is also considerable debate as to which E2F family members might activate Arf (Trimarchi and Lees 2002). Some groups conclude that this is an E2F1-specific activity, whereas others propose that this is a shared property of the activating E2Fs. Certainly, E2F1 is not required for Arf induction in numerous settings (Palmero et al. 2002; Baudino et al. 2003) and p19Arf itself is dispensable for E2F-dependent apoptosis (Russell et al. 2002; Tolbert et al. 2002; Tsai et al. 2002). These findings could reflect redundancy; perhaps multiple E2Fs can activate a large panel of apoptotic inducers that includes p19Arf. Alternatively, E2F may not contribute to Arf activation in vivo. Others have suggested that Arf is regulated by repressive E2F · pocket protein complexes (Rowland et al. 2002). However, unlike classic E2F-responsive genes, Arf is not appreciably induced during cell cycle entry. Thus, if Arf is a genuine E2F target, it must be regulated in a distinct manner from classic E2F-responsive genes. In this study, we use E2f3-deficient MEFs to probe the role of E2F in Arf regulation. This analysis shows that a single member of the E2F family, E2f3, is required to maintain the transcriptional repression of Arf under normal proliferative conditions.

SummaryArginylation is an emerging posttranslational modification mediated by Arg-tRNA-protein-transferase (ATE1). It is believed that ATE1 links Arg solely to the N terminus of proteins, requiring prior proteolysis or action by Met-aminopeptidases to expose the arginylated site. Here, we tested the possibility of Arg linkage to midchain sites within intact protein targets and found that many proteins in vivo are modified on the side chains of Asp and Glu by unconventional chemistry that targets the carboxy rather than the amino groups at the target sites. Such arginylation appears to be functionally regulated, and it can be directly mediated by ATE1, in addition to the more conventional ATE1-mediated linkage of Arg to the N-terminal alpha amino group. This midchain arginylation implies an unconventional mechanism of ATE1 action that likely facilitates its major biological role.

SummaryLINE-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.