@article {49737,
	title = {Stable isotope labeling of phosphoproteins for large-scale phosphorylation rate determination.},
	journal = {Mol Cell Proteomics},
	volume = {13},
	year = {2014},
	month = {2014 Apr},
	pages = {1106-18},
	abstract = {<p>Signals that control responses to stimuli and cellular function are transmitted through the dynamic phosphorylation of thousands of proteins by protein kinases. Many techniques have been developed to study phosphorylation dynamics, including several mass spectrometry (MS)-based methods. Over the past few decades, substantial developments have been made in MS techniques for the large-scale identification of proteins and their post-translational modifications. Nevertheless, all of the current MS-based techniques for quantifying protein phosphorylation dynamics rely on the measurement of changes in peptide abundance levels, and many methods suffer from low confidence in phosphopeptide identification due to poor fragmentation. Here we have optimized an approach for the stable isotope labeling of amino acids by phosphate using [γ-{\textonesuperior}$^{8}$O$_{4}$]ATP in nucleo to determine global site-specific phosphorylation rates. The advantages of this metabolic labeling technique are increased confidence in phosphorylated peptide identification, direct labeling of phosphorylation sites, measurement phosphorylation rates, and the identification of actively phosphorylated sites in a cell-like environment. In this study we calculated approximate rate constants for over 1,000 phosphorylation sites based on labeling progress curves. We measured a wide range of phosphorylation rate constants from 0.34 min$^{-}${\textonesuperior} to 0.001 min$^{-}${\textonesuperior}. Finally, we applied stable isotope labeling of amino acids by phosphate to identify sites that have different phosphorylation kinetics during G1/S and M phase. We found that most sites had very similar phosphorylation rates under both conditions; however, a small subset of sites on proteins involved in the mitotic spindle were more actively phosphorylated during M phase, whereas proteins involved in DNA replication and transcription were more actively phosphorylated during G1/S phase. The data have been deposited to the ProteomeXchange with the identifier PXD000680.</p>},
	keywords = {cell cycle, HEK293 Cells, HeLa Cells, HUMANS, Isotope Labeling, Kinetics, Peptide Mapping, Phosphoproteins, Phosphorylation, proteomics, Tandem Mass Spectrometry},
	issn = {1535-9484},
	doi = {10.1074/mcp.O113.036145},
	author = {Molden, Rosalynn C and Goya, Jonathan and Khan, Zia and Garcia, Benjamin A}
}
@article {38536,
	title = {The transcription factor Eyes absent is a protein tyrosine phosphatase},
	journal = {NatureNature},
	volume = {426},
	year = {2003},
	note = {http://www.ncbi.nlm.nih.gov/pubmed/14628053?dopt=Abstract},
	type = {10.1038/nature02097},
	abstract = {Post-translational modifications provide sensitive and flexible mechanisms to dynamically modulate protein function in response to specific signalling inputs. In the case of transcription factors, changes in phosphorylation state can influence protein stability, conformation, subcellular localization, cofactor interactions, transactivation potential and transcriptional output. Here we show that the evolutionarily conserved transcription factor Eyes absent (Eya) belongs to the phosphatase subgroup of the haloacid dehalogenase (HAD) superfamily, and propose a function for it as a non-thiol-based protein tyrosine phosphatase. Experiments performed in cultured Drosophila cells and in vitro indicate that Eyes absent has intrinsic protein tyrosine phosphatase activity and can autocatalytically dephosphorylate itself. Confirming the biological significance of this function, mutations that disrupt the phosphatase active site severely compromise the ability of Eyes absent to promote eye specification and development in Drosophila. Given the functional importance of phosphorylation-dependent modulation of transcription factor activity, this evidence for a nuclear transcriptional coactivator with intrinsic phosphatase activity suggests an unanticipated method of fine-tuning transcriptional regulation.},
	keywords = {Amino Acid Motifs, Amino Acid Sequence, Animals, Antibodies, Phospho-Specific, Drosophila melanogaster, Drosophila Proteins, Embryonic Induction, eye, Eye Proteins, Gene Expression Regulation, Kinetics, Mice, Models, Molecular, Molecular Sequence Data, Mutation, Phosphorylation, Protein Conformation, Protein Tyrosine Phosphatases, Substrate Specificity, Transcription Factors},
	author = {Tootle, Tina L. and Silver, Serena J. and Davies, Erin L. and Newman, Victoria and Latek, Robert R. and Mills, Ishara A. and J. Selengut and Parlikar, Beth E. W. and Rebay, Ilaria}
}
@article {38143,
	title = {Carbonic anhydrase III: the phosphatase activity is extrinsic},
	journal = {Archives of biochemistry and biophysicsArchives of biochemistry and biophysics},
	volume = {377},
	year = {2000},
	note = {http://www.ncbi.nlm.nih.gov/pubmed/10845711?dopt=Abstract},
	type = {10.1006/abbi.2000.1793},
	abstract = {The carbonic anhydrases reversibly hydrate carbon dioxide to yield bicarbonate and hydrogen ion. They have a variety of physiological functions, although the specific roles of each of the 10 known isozymes are unclear. Carbonic anhydrase isozyme III is particularly rich in skeletal muscle and adipocytes, and it is unique among the isozymes in also exhibiting phosphatase activity. Previously published studies provided evidence that the phosphatase activity was intrinsic to carbonic anhydrase III, that it had specificity for tyrosine phosphate, and that activity was regulated by reversible glutathionylation of cysteine186. To study the mechanism of this phosphatase, we cloned and expressed the rat liver carbonic anhydrase III. The purified recombinant had the same specific activity as the carbonic anhydrase purified from rat liver, but it had virtually no phosphatase activity. We attempted to identify an activator of the phosphatase in rat liver and found a protein of approximately 14 kDa, the amount of which correlated with the phosphatase activity of the carbonic anhydrase III fractions. It was identified as liver fatty acid binding protein, which was then purified to test for activity as an activator of the phosphatase and for protein-protein interaction, but neither binding nor activation could be demonstrated. Immunoprecipitation experiments established that carbonic anhydrase III could be separated from the phosphatase activity. Finally, adding additional purification steps completely separated the phosphatase activity from the carbonic anhydrase activity. We conclude that the phosphatase activity previously considered to be intrinsic to carbonic anhydrase III is actually extrinsic. Thus, this isozyme exhibits only the carbon dioxide hydratase and esterase activities characteristic of the other mammalian isozymes, and the phosphatase previously shown to be activated by glutathionylation is not carbonic anhydrase III.},
	keywords = {Animals, Carbonic Anhydrases, Chromatography, High Pressure Liquid, Cloning, Molecular, Enzyme Activation, Glutathione, Kinetics, Liver, Male, Muscles, Phosphoric Monoester Hydrolases, Precipitin Tests, Rabbits, Rats, Rats, Inbred F344, Recombinant Proteins, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Time factors},
	author = {Kim, G. and J. Selengut and Levine, R. L.}
}
