SG00014292

1. This study describes comprehensive polling of transcription start and termination sites and analysis of previously unidentified full-length complementary DNAs derived from the mouse genome. We identify the 5' and 3' boundaries of 181,047 transcripts with extensive variation in transcripts arising from alternative promoter usage, splicing, and polyadenylation. There are 16,247 new mouse protein-coding transcripts, including 5154 encoding previously unidentified proteins. Genomic mapping of the transcriptome reveals transcriptional forests, with overlapping transcription on both strands, separated by deserts in which few transcripts are observed. The data provide a comprehensive platform for the comparative analysis of mammalian transcriptional regulation in differentiation and development. PMID: [16141072]

2. The National Institutes of Health's Mammalian Gene Collection (MGC) project was designed to generate and sequence a publicly accessible cDNA resource containing a complete open reading frame (ORF) for every human and mouse gene. The project initially used a random strategy to select clones from a large number of cDNA libraries from diverse tissues. Candidate clones were chosen based on 5'-EST sequences, and then fully sequenced to high accuracy and analyzed by algorithms developed for this project. Currently, more than 11,000 human and 10,000 mouse genes are represented in MGC by at least one clone with a full ORF. The random selection approach is now reaching a saturation point, and a transition to protocols targeted at the missing transcripts is now required to complete the mouse and human collections. Comparison of the sequence of the MGC clones to reference genome sequences reveals that most cDNA clones are of very high sequence quality, although it is likely that some cDNAs may carry missense variants as a consequence of experimental artifact, such as PCR, cloning, or reverse transcriptase errors. Recently, a rat cDNA component was added to the project, and ongoing frog (Xenopus) and zebrafish (Danio) cDNA projects were expanded to take advantage of the high-throughput MGC pipeline. PMID: [15489334]

3. Proper development of the mammalian brain requires the precise integration of numerous temporally and spatially regulated stimuli. Many of these signals transduce their cues via the reversible phosphorylation of downstream effector molecules. Neuronal stimuli acting in concert have the potential of generating enormous arrays of regulatory phosphoproteins. Toward the global profiling of phosphoproteins in the developing brain, we report here the use of a mass spectrometry-based methodology permitting the first proteomic-scale phosphorylation site analysis of primary animal tissue, identifying over 500 protein phosphorylation sites in the developing mouse brain. PMID: [15345747]

4. External stimuli trigger internal signaling events within a cell that may represent either a temporary or permanent shift in the phosphorylation state of its proteome. Numerous reports have elucidated phosphorylation sites from a variety of biological samples and more recent studies have monitored the temporal dynamics of protein phosphorylation as a given system is perturbed. Understanding which proteins are phosphorylated as well as when they are phosphorylated may indicate novel functional roles within a system and allow new therapeutic avenues to be explored. To elucidate the dynamics of protein phosphorylation within differentiating murine P19 embryonal carcinoma cells, we induced P19 cells to differentiate using all-trans-retinoic acid and developed a strategy that combines isotopically labeled methyl esterification, immobilized metal affinity chromatography, mass spectrometric analysis, and a rigorous and unique data evaluation approach. We present the largest differential phosphoproteomic analysis using isotopically labeled methyl esterification to date, identifying a total of 472 phosphorylation sites on 151 proteins; 56 of these proteins had altered abundances following treatment with retinoic acid and approximately one-third of these have been previously associated with cellular differentiation. A series of bioinformatic tools were used to extract information from the data and explore the implications of our findings. This study represents the first global gel-free analysis that elucidates protein phosphorylation dynamics during cellular differentiation. PMID: [17622165]

5. Protein phosphorylation is a complex network of signaling and regulatory events that affects virtually every cellular process. Our understanding of the nature of this network as a whole remains limited, largely because of an array of technical challenges in the isolation and high-throughput sequencing of phosphorylated species. In the present work, we demonstrate that a combination of tandem phosphopeptide enrichment methods, high performance MS, and optimized database search/data filtering strategies is a powerful tool for surveying the phosphoproteome. Using our integrated analytical platform, we report the identification of 5,635 nonredundant phosphorylation sites from 2,328 proteins from mouse liver. From this list of sites, we extracted both novel and known motifs for specific Ser/Thr kinases including a "dipolar" motif. We also found that C-terminal phosphorylation was more frequent than at any other location and that the distribution of potential kinases for these sites was unique. Finally, we identified double phosphorylation motifs that may be involved in ordered phosphorylation. PMID: [17242355]

6. The ability of macrophages to clear pathogens and elicit a sustained immune response is regulated by various cytokines, including interferon-gamma (IFN-gamma). To investigate the molecular mechanisms by which IFN-gamma modulates phagosome functions, we profiled the changes in composition, abundance, and phosphorylation of phagosome proteins in resting and activated macrophages by using quantitative proteomics and bioinformatics approaches. We identified 2415 phagosome proteins together with 2975 unique phosphorylation sites with a high level of sensitivity. Using network analyses, we determined that IFN-gamma delays phagosomal acquisition of lysosomal hydrolases and peptidases for the gain of antigen presentation. Furthermore, this gain in antigen presentation is dependent on phagosomal networks of the actin cytoskeleton and vesicle-trafficking proteins, as well as Src kinases and calpain proteases. Major histocompatibility complex class I antigen-presentation assays validated the molecular participation of these networks in the enhanced capacity of IFN-gamma-activated macrophages to crosspresent exogenous antigens to CD8(+) T cells. PMID: [19144319]