Protein phosphorylation is a post-translational modification widely used to regulate cellular responses. Recent studies showed that global phosphorylation analysis could be used to study signaling pathways and to identify targets of protein kinases in cells. A key objective of global phosphorylation analysis is to obtain an in-depth mapping of low abundance protein phosphorylation in cells; this necessitates the use of suitable separation techniques because of the complexity of the phosphoproteome. Here we developed a multidimensional chromatography technology, combining IMAC, hydrophilic interaction chromatography, and reverse phase LC, for phosphopeptide purification and fractionation.

Understanding the role of DNA damage checkpoint kinases in the cellular response to genotoxic stress requires the knowledge of their substrates. Here, we report the use of quantitative phosphoproteomics to identify in vivo kinase substrates of the yeast DNA damage checkpoint kinases Mec1, Tel1, and Rad53 (orthologs of human ATR, ATM, and CHK2, respectively). By analyzing 2,689 phosphorylation sites in wild-type and various kinase-null cells, 62 phosphorylation sites from 55 proteins were found to be controlled by the DNA damage checkpoint. Examination of the dependency of each phosphorylation on Mec1 and Tel1 or Rad53, combined with sequence and biochemical analysis, revealed that many of the identified targets are likely direct substrates of these kinases. In addition to several known targets, 50 previously undescribed targets of the DNA damage checkpoint were identified, suggesting that a wide range of cellular processes is likely regulated by Mec1, Tel1, and Rad53. mass spectrometry ͉ Mec1 ͉ N-isotag ͉ phosphorylation ͉ Rad53

Dominant mutations in two functionally related DNA/RNA-binding proteins, trans-activating response region (TAR) DNA-binding protein with a molecular mass of 43 KDa (TDP-43) and fused in sarcoma/ translocation in liposarcoma (FUS/TLS), cause an inherited form of ALS that is accompanied by nuclear and cytoplasmic aggregates containing TDP-43 or FUS/TLS. Using isogenic cell lines expressing wild-type or ALS-linked TDP-43 mutants and fibroblasts from a human patient, pulse-chase radiolabeling of newly synthesized proteins is used to determine, surprisingly, that ALS-linked TDP-43 mutant polypeptides are more stable than wild-type TDP-43. Tandem-affinity purification and quantitative mass spectrometry are used to identify TDP-43 complexes not only with heterogeneous nuclear ribonucleoproteins family proteins, as expected, but also with components of Drosha microprocessor complexes, consistent with roles for TDP-43 in both mRNA processing and microRNA biogenesis. A fraction of TDP-43 is shown to be complexed with FUS/TLS, an interaction substantially enhanced by TDP-43 mutants. Taken together, abnormal stability of mutant TDP-43 and its enhanced binding to normal FUS/TLS imply a convergence of pathogenic pathways from mutant TDP-43 and FUS/TLS in ALS.mass spectrometry | protein stability | amyotrophic lateral sclerosis | microRNA | ribonucleoproteins

The RNA-binding protein FUS/TLS, mutation in which is causative of the fatal motor neuron disease ALS, is demonstrated to directly bind to the U1-snRNP and SMN complexes. ALS-causative mutations in FUS/TLS are shown to abnormally enhance their interaction with SMN and dysregulate its function, including loss of Gems and altered levels of small nuclear RNAs (snRNAs). The same mutants are found to have reduced association with U1-snRNP. Correspondingly, global RNA analysis reveals a mutant-dependent loss of splicing activity, with ALS-linked mutants failing to reverse changes caused by loss of wild-type FUS/TLS. Furthermore, a common FUS/TLS mutant-associated RNA splicing signature is identified in ALS patient fibroblasts. Taken together, these studies establish potentially converging disease mechanisms in ALS and spinal muscular atrophy, with ALS-causative mutants acquiring properties representing both gain (dysregulation of SMN) and loss (reduced RNA processing mediated by U1-snRNP) of function.

Enhancers act to regulate cell type specific gene expression by facilitating the transcription of target genes. In mammalian cells active or primed enhancers are commonly marked by monomethylation of Histone H3 at lysine 4 (H3K4me1) in a cell-type specific manner. Whether and how this histone modification regulates enhancer-dependent transcription programs in mammals is unclear. In this study, we conducted SILAC Mass-spec experiments with mono-nucleosomes and identified multiple H3K4me1 associated proteins, including many involved in chromatin remodeling. We demonstrate that H3K4me1 augments the association of the chromatin remodeling complex BAF to enhancers in vivo and that in vitro, H3K4me1 nucleosomes are more efficiently remodeled by the BAF complex. Crystal structures of BAF component BAF45c reveal that monomethylation, but not trimethylation, is accommodated by BAF45c’s H3K4 binding site. Our results suggest that H3K4me1 plays an active role at enhancers by facilitating the binding of the BAF complex and possibly other chromatin regulators.

Suppression of duplication-mediated gross chromosomal rearrangements (GCRs) is essential to maintain genome integrity in eukaryotes. Here we report that SUMO ligase Mms21 has a strong role in suppressing GCRs in Saccharomyces cerevisiae, while Siz1 and Siz2 have weaker and partially redundant roles. Understanding the functions of these enzymes has been hampered by a paucity of knowledge of their substrate specificity in vivo. Using a new quantitative SUMO-proteomics technology, we found that Siz1 and Siz2 redundantly control the abundances of most sumoylated substrates, while Mms21 more specifically regulates sumoylation of RNA polymerase-I and the SMC-family proteins. Interestingly, Esc2, a SUMO-like domain-containing protein, specifically promotes the accumulation of sumoylated Mms21-specific substrates and functions with Mms21 to suppress GCRs. On the other hand, the Slx5-Slx8 complex, a SUMO-targeted ubiquitin ligase, suppresses the accumulation of sumoylated Mms21-specific substrates. Thus, distinct SUMO ligases work in concert with Esc2 and Slx5-Slx8 to control substrate specificity and sumoylation homeostasis to prevent GCRs.

SUMMARY Mutations in cancer reprogram amino acid metabolism to drive tumor growth, but the molecular mechanisms are not well understood. Using an unbiased proteomic screen, we identified mTORC2 as a critical regulator of amino acid metabolism in cancer via phosphorylation of the cystine-glutamate antiporter xCT. mTORC2 phosphorylates serine 26 at the cytosolic N-terminus of xCT, inhibiting its activity. Genetic inhibition of mTORC2, or pharmacologic mTOR kinase inhibition, promotes glutamate secretion, cystine uptake and incorporation into glutathione linking growth factor receptor signaling with amino acid uptake and utilization. These results identify an unanticipated mechanism regulating amino acid metabolism in cancer, enabling tumor cells to adapt to changing environmental conditions.

We present an approach for quantitative analysis of changes in the composition and phosphorylation of protein complexes by MS. It is based on a new class of stable isotope-labeling reagent, the amine-reactive isotope tag (N-isotag), for specific and quantitative labeling of peptides following proteolytic digestion of proteins. Application of the N-isotag method to the analysis of Rad53, a DNA damage checkpoint kinase in Saccharomyces cerevisiae, led to the identification of dynamic associations between Rad53 and the nuclear transport machinery, histones, and chromatin assembly proteins in response to DNA damage. Over 30 phosphorylation sites of Rad53 and its associated proteins were identified and quantified, and they showed different changes in phosphorylation in response to DNA damage. Interestingly, Ser 789 of Rad53 was found to be a major initial phosphorylation site, and its phosphorylation regulates the Rad53 abundance in response to DNA damage. Collectively, these results demonstrate that N-isotag-based quantitative MS is generally applicable to study dynamic changes in the composition of protein complexes and their phosphorylation patterns in a site-specific manner in response to different cell stimuli. Molecular & Cellular Proteomics 4:1358 -1369, 2005.MS is a central tool in proteomics and is widely used to study protein complexes and their post-translational modifications. For stably associated protein complexes, it is possible to carry out a multistep purification to obtain highly purified proteins for MS analysis (1, 2). However, numerous biologically important protein-protein interactions are dynamic and are regulated by reversible protein modifications such as phosphorylation. It is therefore important to develop new tools for quantitative analysis of dynamic changes in protein complexes under different cell stimuli. Given the nature of dynamic protein-protein interactions, rapid single step protein purification should help to preserve the interaction (3, 4). However, such purification procedures often result in higher levels of contaminating proteins, which may obscure the observation of less abundant yet specifically associated proteins by conventional in-gel digestion and MS approaches. Recently, the ICAT method was used to identify specific interacting proteins in Pol II general transcriptional complexes, thus demonstrating the potential of quantitative analysis of protein complexes by stable isotope labeling (5). However, only cysteine-containing peptides were labeled by ICAT; thus it is not applicable to quantify noncysteine-containing proteins and various protein modifications.MS has also been used to identify phosphorylation sites of proteins (6). Many signaling proteins are subjected to dynamic phosphorylation at many sites, thus understanding the biological function of phosphorylation requires analysis of phosphorylation in a site-specific and quantitative fashion (7). Existing approaches for quantitative analysis of protein phosphorylation include the use of heavy amino acids in cell cu...