Scott M. Carlson

Mcl-1 integrates the opposing actions of signaling pathways that mediate survival and apoptosis.

ABSTRACT Mcl-1 is a member of the Bcl2-related protein family that is a critical mediator of cell survival. Exposure of cells to stress causes inhibition of Mcl-1 mRNA translation and rapid destruction of Mcl-1 protein by proteasomal degradation mediated by a phosphodegron created by glycogen synthase kinase 3 (GSK3) phosphorylation of Mcl-1. Here we demonstrate that prior phosphorylation of Mcl-1 by the c-Jun N-terminal protein kinase (JNK) is essential for Mcl-1 phosphorylation by GSK3. Stress-induced Mcl-1 degradation therefore requires the coordinated activity of JNK and GSK3. Together, these data establish that Mcl-1 functions as a site of signal integration between the proapoptotic activity of JNK and the prosurvival activity of the AKT pathway that inhibits GSK3.

Large-Scale Discovery of ERK2 Substrates Identifies ERK-Mediated Transcriptional Regulation by ETV3

A chemical genetics strategy identified 67 previously unknown substrates of the kinase ERK2, including the transcriptional repressor ETV3. Discovering ERK2 Substrates Extracellular signal–regulated kinase 2 (ERK2) is one of two closely related mitogen-activated protein kinases (MAPKs) that function downstream of many receptors. Because ERK2 and ERK1 are usually activated under the same conditions and are recognized by the same antibodies, it can be difficult to assign specific substrates and functions to these related proteins. Carlson et al. combined mass spectrometry analysis with chemical genetics to identify ERK2-specific substrates. Their work identified targets in diverse pathways suggesting mechanisms for crosstalk with MAPK signaling. Additionally, detailed analysis of the transcriptional repressor ETV3 suggested that phosphorylation by ERK2 on sites in both canonical and noncanonical motifs inhibited its DNA binding capacity, which may poise cells for a transient transcriptional burst in response to stimuli that activate ERK signaling or those that arrive concurrently. The mitogen-activated protein kinase (MAPK) extracellular signal–regulated kinase 2 (ERK2) is ubiquitously expressed in mammalian tissues and is involved in a wide range of biological processes. Although MAPKs have been intensely studied, identification of their substrates remains challenging. We have optimized a chemical genetic system using analog-sensitive ERK2, a form of ERK2 engineered to use an analog of adenosine 5′-triphosphate (ATP), to tag and isolate ERK2 substrates in vitro. This approach identified 80 proteins phosphorylated by ERK2, 13 of which are known ERK2 substrates. The 80 substrates are associated with diverse cellular processes, including regulation of transcription and translation, mRNA processing, and regulation of the activity of the Rho family guanosine triphosphatases. We found that one of the newly identified substrates, ETV3 (a member of the E twenty-six family of transcriptional regulators), was extensively phosphorylated on sites within canonical and noncanonical ERK motifs. Phosphorylation of ETV3 regulated transcription by preventing its binding to DNA at promoters for several thousand genes, including some involved in negative feedback regulation of itself and of upstream signals.

Proteome-wide enrichment of proteins modified by lysine methylation

We present a protocol for using the triple malignant brain tumor domains of L3MBTL1 (3xMBT), which bind to mono- and di-methylated lysine with minimal sequence specificity, in order to enrich for such methylated lysine from cell lysates. Cells in culture are grown with amino acids containing light or heavy stable isotopic labels. Methylated proteins are enriched by incubating cell lysates with 3xMBT, or with the binding-null D355N mutant as a negative control. Quantitative liquid chromatography and tandem mass spectrometry (LC-MS/MS) are then used to identify proteins that are specifically enriched by 3xMBT pull-down. The addition of a third isotopic label allows the comparison of protein lysine methylation between different biological conditions. Unlike most approaches, our strategy does not require a prior hypothesis of candidate methylated proteins, and it recognizes a wider range of methylated proteins than any available method using antibodies. Cells are prepared by growing in isotopic labeling medium for about 7 d; the process of enriching methylated proteins takes 3 d and analysis by LC-MS/MS takes another 1–2 d.

Using small molecules and chemical genetics to interrogate signaling networks.

The limited clinical success of therapeutics targeting cellular signaling processes is due to multiple factors, including off-target effects and complex feedback regulation encoded within the signaling network. To understand these effects, chemical proteomics and chemical genetics tools have been developed to map the direct targets of kinase inhibitors, determine the network-level response to inhibitor treatment, and to infer network topology. Here we provide an overview of chemical phosphoproteomic and chemical genetic methods, including specific examples where these methods have been applied to yield biological insight regarding network structure and the system-wide effects of targeted therapeutics. The challenges and caveats associated with each method are described, along with approaches being used to resolve some of these issues. With the broad array of available techniques the next decade should see a rapid improvement in our understanding of signaling networks regulation and response to targeted perturbations, leading to more efficacious therapeutic strategies.

A Potential Biomarker in the Cord Blood of Preterm Infants Who Develop Retinopathy of Prematurity

Preterm infants are at risk of developing sepsis, necrotizing enterocolitis (NEC), chronic lung disease (CLD), and retinopathy of prematurity (ROP). We used high-throughput mass spectrometry to investigate whether cord blood proteins can be used to predict development of these morbidities. Cord blood plasma from 44 infants with a birth weight of <1500 g was analyzed by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF). Six infants developed ROP ≥stage II, 10 CLD, three sepsis, and one NEC. We detected 814 protein signals representing 330 distinct protein species. Nineteen biomarkers were associated with development of ≥stage II ROP [false-discovery rate (FDR) <5%] and none with CLD. Several proteins with molecular weight (Mr) 15–16 kD and pI 4–5 were detected with increased abundance in infants with ROP, while similar Mr proteins with pI 7–9 were less abundant in these patients. Sodium dodecylsulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and sequence analysis identified these proteins as α-, β-, and γ-globin chains. Partial deamidation of Asn139 in β-globin chains was observed only in the pI 4–5 proteins. We conclude that there are several promising biomarkers for the risk of ROP. Deamidation of globin chains is especially promising and may indicate underlying prenatal pathologic mechanisms in ROP. Validation studies will be undertaken to determine their clinical utility.

Coordination of stress signals by the lysine methyltransferase SMYD2 promotes pancreatic cancer

Pancreatic ductal adenocarcinoma (PDAC) is a lethal form of cancer with few therapeutic options. We found that levels of the lysine methyltransferase SMYD2 (SET and MYND domain 2) are elevated in PDAC and that genetic and pharmacological inhibition of SMYD2 restricts PDAC growth. We further identified the stress response kinase MAPKAPK3 (MK3) as a new physiologic substrate of SMYD2 in PDAC cells. Inhibition of MAPKAPK3 impedes PDAC growth, identifying a potential new kinase target in PDAC. Finally, we show that inhibition of SMYD2 cooperates with standard chemotherapy to treat PDAC cells and tumors. These findings uncover a pivotal role for SMYD2 in promoting pancreatic cancer.

Nonhistone Lysine Methylation in the Regulation of Cancer Pathways.

Proteins are regulated by an incredible array of posttranslational modifications (PTMs). Methylation of lysine residues on histone proteins is a PTM with well-established roles in regulating chromatin and epigenetic processes. The recent discovery that hundreds and likely thousands of nonhistone proteins are also methylated at lysine has opened a tremendous new area of research. Major cellular pathways involved in cancer, such as growth signaling and the DNA damage response, are regulated by lysine methylation. Although the field has developed quickly in recent years many fundamental questions remain to be addressed. We review the history and molecular functions of lysine methylation. We then discuss the enzymes that catalyze methylation of lysine residues, the enzymes that remove lysine methylation, and the cancer pathways known to be regulated by lysine methylation. The rest of the article focuses on two open questions that we suggest as a roadmap for future research. First is understanding the large number of candidate methyltransferase and demethylation enzymes whose enzymatic activity is not yet defined and which are potentially associated with cancer through genetic studies. Second is investigating the biological processes and cancer mechanisms potentially regulated by the multitude of lysine methylation sites that have been recently discovered.

A Proteomic Strategy Identifies Lysine Methylation of Splicing Factor snRNP70 by the SETMAR Enzyme.

Background: SETMAR is a lysine methyltransferase (KMT) that contributes to DNA repair, but its biochemical function is not well understood. Results: A novel proteomic strategy identifies splicing factor snRNP70 as a SETMAR substrate. Conclusion: SETMAR is the first KMT identified to target splicing factors. Significance: Proteomics can be harnessed to discover methyltransferase substrates. Lysine methylation may be a new mode of regulation for mRNA splicing. The lysine methyltransferase (KMT) SETMAR is implicated in the response to and repair of DNA damage, but its molecular function is not clear. SETMAR has been associated with dimethylation of histone H3 lysine 36 (H3K36) at sites of DNA damage. However, SETMAR does not methylate H3K36 in vitro. This and the observation that SETMAR is not active on nucleosomes suggest that H3K36 methylation is not a physiologically relevant activity. To identify potential non-histone substrates, we utilized a strategy on the basis of quantitative proteomic analysis of methylated lysine. Our approach identified lysine 130 of the mRNA splicing factor snRNP70 as a SETMAR substrate in vitro, and we show that the enzyme primarily generates monomethylation at this position. Furthermore, we show that SETMAR methylates snRNP70 Lys-130 in cells. Because snRNP70 is a key early regulator of 5′ splice site selection, our results suggest a model in which methylation of snRNP70 by SETMAR regulates constitutive and/or alternative splicing. In addition, the proteomic strategy described here is broadly applicable and is a promising route for large-scale mapping of KMT substrates.

Integrated data management and validation platform for phosphorylated tandem mass spectrometry data

MS/MS is a widely used method for proteome‐wide analysis of protein expression and PTMs. The thousands of MS/MS spectra produced from a single experiment pose a major challenge for downstream analysis. Standard programs, such as MASCOT, provide peptide assignments for many of the spectra, including identification of PTM sites, but these results are plagued by false‐positive identifications. In phosphoproteomic experiments, only a single peptide assignment is typically available to support identification of each phosphorylation site, and hence minimizing false positives is critical. Thus, tedious manual validation is often required to increase confidence in the spectral assignments. We have developed phoMSVal, an open‐source platform for managing MS/MS data and automatically validating identified phosphopeptides. We tested five classification algorithms with 17 extracted features to separate correct peptide assignments from incorrect ones using over 2600 manually curated spectra. The naïve Bayes algorithm was among the best classifiers with an AUC value of 97% and PPV of 97% for phosphotyrosine data. This classifier required only three features to achieve a 76% decrease in false positives as compared with MASCOT while retaining 97% of true positives. This algorithm was able to classify an independent phosphoserine/threonine data set with AUC value of 93% and PPV of 91%, demonstrating the applicability of this method for all types of phospho‐MS/MS data. PhoMSVal is available at http://csbi.ltdk.helsinki.fi/phomsval.

The epigenetic regulator SIRT7 guards against mammalian cellular senescence induced by ribosomal DNA instability

In the yeast Saccharomyces cerevisiae, genomic instability in rDNA repeat sequences is an underlying cause of cell aging and is suppressed by the chromatin-silencing factor Sir2. In humans, rDNA instability is observed in cancers and premature aging syndromes, but its underlying mechanisms and functional consequences remain unclear. Here, we uncovered a pivotal role of sirtuin 7 (SIRT7), a mammalian Sir2 homolog, in guarding against rDNA instability and show that this function of SIRT7 protects against senescence in primary human cells. We found that, mechanistically, SIRT7 is required for association of SNF2H (also called SMARCA5, SWI/SNF-related matrix-associated actin-dependent regulator of chromatin, subfamily A, member 5), a component of the nucleolar heterochromatin-silencing complex NoRC, with rDNA sequences. Defective rDNA–heterochromatin silencing in SIRT7-deficient cells unleashed rDNA instability, with excision and loss of rDNA gene copies, which in turn induced acute senescence. Mounting evidence indicates that accumulation of senescent cells significantly contributes to tissue dysfunction in aging-related pathologies. Our findings identify rDNA instability as a driver of mammalian cellular senescence and implicate SIRT7-dependent heterochromatin silencing in protecting against this process.