Global, In Vivo, and Site-Specific Phosphorylation Dynamics in Signaling Networks

Olsen, Jesper V.; Blagoev, Blagoy; Gnad, Florian; Maček, Boris; Kumar, Chanchal; Mortensen, Poul Erik; Mann, Matthias

doi:10.1016/j.cell.2006.09.026

Cited by 3,236 publications

(3,651 citation statements)

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“…The small response at the phosphoproteome level detected here is quite different from global quantitative phosphoproteomics studies that use cellular stimuli to directly regulate a particular kinase [72][73][74][75] or those studying cellular processes known to be greatly dependent on phosphorylation. 18,76 Much greater effects on phosphorylation throughout the entire signaling cascade are also observed, as expected.…”

Section: View Article Onlinementioning

confidence: 65%

Quantitative global phosphoproteomics of human umbilical vein endothelial cells after activation of the Rap signaling pathway

et al. 2013

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The small GTPase Rap1 is required for proper cell-cell junction formation and also plays a key role in mediating cAMP-induced tightening of adherens junctions and subsequent increased barrier function of endothelial cells. To further study how Rap1 controls barrier function, we performed quantitative global phosphoproteomics in human umbilical vein endothelial cells (HUVECs) prior to and after Rap1 activation by the Epac-selective cAMP analog 8-pCPT-2 0 -O-Me-cAMP-AM (007-AM). Tryptic digests were labeled using stable isotope dimethyl labeling, enriched with phosphopeptides by strong cation exchange (SCX), followed by titanium(IV) immobilized metal affinity chromatography (Ti 4+ -IMAC) and analyzed by high resolution mass spectrometry. We identified 19 859 unique phosphopeptides containing 17 278 unique phosphosites on 4594 phosphoproteins, providing the largest HUVEC phosphoproteome to date. Of all identified phosphosites, 220 (B1%) were more than 1.5-fold up-or downregulated upon Rap activation, in two independent experiments. Compatible with the function of Rap1, these alterations were found predominantly in proteins regulating the actin cytoskeleton, cell-cell junctions and cell adhesion.

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Section: View Article Onlinementioning

confidence: 65%

Quantitative global phosphoproteomics of human umbilical vein endothelial cells after activation of the Rap signaling pathway

et al. 2013

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“…This argues that binding of PP1 to p68 functions to regulate phosphorylation states of Pol d itself or of its neighbors within the replication machinery. The immediate target may be p68; phosphoproteomic studies have shown that p68 is phosphorylated at a minimum of six sites in vivo [Olsen et al, 2006]. These sites are in sequence contexts that indicate that they are sites for CK2 phosphorylation, and it has been shown that p68 is phosphorylated at multiple sites in vitro by CK2 [Gao et al, 2008;Lemmens et al, 2008].…”

Section: Regulation Of Pol D Activity By Protein Phosphorylationmentioning

confidence: 99%

Regulation of human DNA polymerase delta in the cellular responses to DNA damage

Lee

Zhang

Lin

et al. 2012

Environ and Mol Mutagen

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The p12 subunit of polymerase delta (Pol d) is degraded in response to DNA damage induced by UV, alkylating agents, oxidative, and replication stresses. This leads to the conversion of the Pol d4 holoenzyme to the heterotrimer, Pol d3. We review studies that establish that Pol d3 formation is an event that could have a major impact on cellular processes in genomic surveillance, DNA replication, and DNA repair. p12 degradation is dependent on the apical ataxia telangiectasia and Rad3 related (ATR) kinase and is mediated by the ubiquitin-proteasome system. Pol d3 exhibits properties of an ''antimutator'' polymerase, suggesting that it could contribute to an increased surveillance against mutagenesis, for example, when Pol d carries out bypass synthesis past small base lesions that engage in spurious base pairing. Chromatin immunoprecipitation analysis and examination of the spatiotemporal recruitment of Pol d to sites of DNA damage show that Pol d3 is the primary form of Pol d associated with cyclobutane pyrimidine dimer lesions and therefore should be considered as the operative form of Pol d engaged in DNA repair. We propose a model for the switching of Pol d with translesion polymerases, incorporating the salient features of the recently determined structure of monoubiquitinated proliferating cell nuclear antigen and emphasizing the role of Pol d3. Because of the critical role of Pol d activity in DNA replication and repair, the formation of Pol d3 in response to DNA damage opens the prospect that pleiotropic effects may ensue. This opens the horizons for future exploration of how this novel response to DNA damage contributes to genomic stability. Environ. Mol. Mutagen. 53:683-698, 2012. V V C 2012 Wiley Periodicals, Inc.

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“…Already, investigators have begun using phosphoproteomics to address real biological and clinical problems, such as unraveling important signaling networks and discovering how these networks malfunction in various types of cancer. [9][10][11][12][13] Despite the success of some initial biological and clinical applications of phosphoproteomics, researchers want to see more progress in the methods. " [Phosphoproteomics] should become routine," says Figeys, "the same way as doing protein identification from a gel band 15 years ago was very difficult, and now any decent lab in the world can do it."…”

Section: To Mainstream Research and Beyondmentioning

confidence: 99%

Phosphoproteomics: Miles To Go Before It’s Routine

Piggee¹

2009

Anal. Chem.

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Researchers see major technological advances but still have significant challenges to overcome.Not so long ago, the process for determining protein phosphorylation was a labor-intensive and serial process. Typically, a researcher would use 32 P-labeling coupled with 2D gel electrophoresis phosphopeptide mapping, autoradiography, band excision, and Edman sequencing. In addition to expending much time and effort, researchers had to work with radioactivity, and the method could not be multiplexed. Now in 2009, two complementary techniques for identification of phosphorylation sites, MS and microarrays, have provided significant increases in throughput. In discovery mode, researchers can identify thousands of phosphorylation sites with LC/MS n , and in profiling mode, researchers can detect thousands of phosphorylation events and predict the substrates of various protein kinases with planar, suspension, and reverse microarrays. 1 As the technology has improved and researchers have been able to glean impressive amounts of information from phosphoproteomics experiments, more labs have shown interest. "I think the field of phosphoproteomics is poised to take a big leap forward," says Timothy Veenstra of the U.S. National Cancer Institute. And the widespread enthusiasm among researchers is tangible. "It's probably the most exciting time right now in phosphoproteomics," says Daniel Figeys of the University of Ottawa. " [Phosphoproteomics] used to be kind of stuck a few years ago, where the numbers of phosphorylation sites that could be mapped were dreadfully small," he says.But mapping so many sites can be a problem if researchers cannot weed through the information to find what is important in a reasonable amount of time, as Matthias Mann of the Max Planck Institute of Biochemistry (Germany) explains. "Now we're in the paradoxical situation that we almost have too much of a good thing. So it's now possible to quantify even 10,000 phosphorylation sites in a single experiment. But in my view ... without then knowing which ones of the 10,000 are actually relevant for [a particular] biological system, then it's too much," he says.With this flood of new information about protein phosphorylation, the intricacy of the cell signaling network is becoming more apparent. "In the past with proteomics, people thought, 'Okay, one gene, one functionsone gene, one protein,'" says Albert Heck of Utrecht University (The Netherlands). "It makes you wonder and makes you humble about what we know already." Researchers are learning that the cell signaling pathways are incredibly complicated. "We think we understand sometimes how proteins are interacting with each other and how signaling networks are really working," says Emanuel Petricoin of George Mason University. "We go into these clinical trials where we think we can turn off these pathways, and you have some compensatory feedback loop that was unknown take over and actually turn on another pathway that you didn't even know was connected." Elucidating the structure and function of these ...

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Global, In Vivo, and Site-Specific Phosphorylation Dynamics in Signaling Networks

Cited by 3,236 publications

References 61 publications

Quantitative global phosphoproteomics of human umbilical vein endothelial cells after activation of the Rap signaling pathway

Quantitative global phosphoproteomics of human umbilical vein endothelial cells after activation of the Rap signaling pathway

Regulation of human DNA polymerase delta in the cellular responses to DNA damage

Phosphoproteomics: Miles To Go Before It’s Routine

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