An Approach to Locate Phosphorylation Sites in a Phosphoprotein: Mass Mapping by Combining Specific Enzymatic Degradation with Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry

Liao, Pao Chi; Leykam, Joe; Andrews, Philip C.; Gage, Douglas A.; Allison, John

doi:10.1006/abio.1994.1224

Cited by 155 publications

(142 citation statements)

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“…Phosphopeptides were incubated for 15 min at 37°C with or without alkaline phosphatase (Calbiochem) (24). For phosphopeptide enrichment of native dephosphorylated and re-phosphorylated CAPS tryptic peptides, a Ga(III)-based phosphopeptide isolation kit (Pierce) was used as per the manufacturer's instructions.…”

Section: Methodsmentioning

confidence: 99%

CAPS Activity in Priming Vesicle Exocytosis Requires CK2 Phosphorylation

Nojiri

Loyet²,

Klenchin

et al. 2009

Journal of Biological Chemistry

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CAPS (Ca 2؉ -dependent activator protein for secretion) functions in priming Ca 2؉ -dependent vesicle exocytosis, but the regulation of CAPS activity has not been characterized. Here we show that phosphorylation by protein kinase CK2 is required for CAPS activity. Dephosphorylation eliminated CAPS activity in reconstituting Ca 2؉ -dependent vesicle exocytosis in permeable and intact PC12 cells. Ser-5, -6, and -7 and Ser-1281 were identified by mass spectrometry as the major phosphorylation sites in the 1289 residue protein. Ser-5, -6, and -7 but not Ser-1281 to Ala substitutions abolished CAPS activity. Protein kinase CK2 phosphorylated CAPS in vitro at these sites and restored the activity of dephosphorylated CAPS. CK2 is the likely in vivo CAPS protein kinase based on inhibition of phosphorylation by tetrabromo-2-benzotriazole in PC12 cells and by the identity of in vivo and in vitro phosphorylation sites. CAPS phosphorylation by CK2 was constitutive, but the elevation of Ca 2؉ in synaptosomes increased CAPS Ser-5 and -6 dephosphorylation, which terminates CAPS activity. These results identify a functionally important N-terminal phosphorylation site that regulates CAPS activity in priming vesicle exocytosis.Regulated neurotransmitter secretion is central to intercellular communication in the nervous system. Two types of secretory vesicles mediate neurotransmitter release; that is, synaptic vesicles that release transmitters such as glutamate at synapses and dense-core vesicles that release modulatory transmitters and neuropeptides at non-synaptic sites. Both types of secretory vesicles are recruited to docking sites on the plasma membrane where they are primed to a ready release state to undergo fusion in response to Ca 2ϩ elevations. Many of the proteins that mediate the targeting, docking, priming, and Ca 2ϩ -dependent fusion of vesicles with the plasma membrane function in both synaptic vesicle and dense-core vesicle pathways (1). CAPS-1 2 (also known as Cadps1) is a 1289-residue protein that reconstitutes Ca 2ϩ -triggered dense-core vesicle exocytosis in permeable neuroendocrine cells at a priming step (2-4). CAPS is required for secretion of a subset of transmitters in Caenorhabditis elegans (5) and Drosophila melanogaster (6) and for priming dense-core vesicle exocytosis in neuroendocrine cells (7) and synaptic vesicle exocytosis in neurons (8). Vesicle priming reactions are extensively modulated during physiological demand (9), but mechanisms that regulate CAPS function remain to be identified.Reversible protein phosphorylation is a major mechanism for the regulation of cellular processes including vesicle exocytosis. Many proteins that function in evoked vesicle exocytosis are phosphoproteins (10, 11). The neuronal SNARE proteins syntaxin 1A, VAMP-2, and SNAP-25 are phosphorylated by several protein kinases in vitro (12)(13)(14). Protein kinase C and protein kinase A sites on SNAP-25 affect refilling rates and size, respectively, of the primed pool of vesicles in chromaffin cells (15,16). Several SNARE...

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Section: Methodsmentioning

confidence: 99%

CAPS Activity in Priming Vesicle Exocytosis Requires CK2 Phosphorylation

Nojiri

Loyet²,

Klenchin

et al. 2009

Journal of Biological Chemistry

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“…In recent years, mass spectrometry has become a key technology for characterization of protein phosphorylation and phosphoproteome analysis. Two complementary ionization techniques, MALDI and ESI, in combination with a variety of mass analyzers, have been used to identify phosphopeptides and determine the phosphorylated amino acids on the peptides (3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16). In most cases, characterization of phosphopeptides by MS requires selection of phosphorylated peptides from complex peptide mixtures resulting from proteolysis of phosphorylated proteins followed by MS/MS to confirm the phosphorylation and to identify the phosphorylated amino acid residues on phosphopeptides containing more than a single serine, threonine, or tyrosine residue.…”

Section: Molecular and Cellular Proteomics 4:809 -818 2005mentioning

confidence: 99%

Identification of Phosphopeptides by MALDI Q-TOF MS in Positive and Negative Ion Modes after Methyl Esterification

Xu¹,

Lu²,

et al. 2005

Molecular & Cellular Proteomics

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. However, this type of experiment has been limited by low ionization efficiency of phosphopeptides in positive ion mode while selecting precursor ions of phosphopeptides. Our method entails neutralizing negative charges on acidic groups of nonphosphorylated peptides by methyl esterification before mass spectrometry in positive and negative ion modes. Methyl esterification significantly increases the relative signal intensity generated by phosphopeptides in negative ion mode compared with positive ion mode and greatly increases selectivity for phosphopeptides by suppressing the signal intensity generated by acidic peptides in negative ion mode. We used the method to identify 12 phosphopeptides containing 22 phosphorylation sites from low femtomolar amounts of a tryptic digest of ␤-casein and ␣-s-casein. We also identified 10 phosphopeptides containing five phosphorylation sites from an in-gel tryptic digest of 100 fmol of an in vitro autophosphorylated fibroblast growth factor receptor kinase domain and an additional phosphopeptide containing another phosphorylation site when 500 fmol of the digest was examined. The results demonstrate that the method is a fast, robust, and sensitive means of characterizing phosphopeptides present in low abundance mixtures of phosphorylated and nonphosphorylated peptides. Molecular & Cellular Proteomics 4:809 -818, 2005.Reversible phosphorylation of serine, threonine, and tyrosine residues is one of the most common and important regulatory modifications of proteins and often is a key event in cellular signal transduction (1, 2). In recent years, mass spectrometry has become a key technology for characterization of protein phosphorylation and phosphoproteome analysis. Two complementary ionization techniques, MALDI and ESI, in combination with a variety of mass analyzers, have been used to identify phosphopeptides and determine the phosphorylated amino acids on the peptides (3-16). In most cases, characterization of phosphopeptides by MS requires selection of phosphorylated peptides from complex peptide mixtures resulting from proteolysis of phosphorylated proteins followed by MS/MS to confirm the phosphorylation and to identify the phosphorylated amino acid residues on phosphopeptides containing more than a single serine, threonine, or tyrosine residue. Despite many recent advances in methodology, identification of phosphorylation sites on proteins remains a difficult challenge (17).High sensitivity, resolution, and mass accuracy make Q-TOF MS a powerful tool for the characterization of phosphopeptides. The MALDI Q-TOF allows for increased efficiency and sample throughput because identification of phosphopeptides by MS and characterization by CID MS/MS can be performed on a single sample spot (18). However, a limitation of this type of experiment is the low ionization efficiency of phosphopeptides in positive ion mode, resulting in low sensitivity of phosphopeptide detection and consumption of a large portion of the sample during the search for phosphorylated precursor peptides b...

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“…Nevertheless, these phosphorylation sites could have important functions. Mass spectrometry has made possible the identification of in vivo phosphorylation sites in live cells grown under physiological phosphate concentrations and in tissues (19,27,42,43). We have identified the tyrosine phosphorylation sites of ephrin-B1 and EphB2 in a tissue where these proteins are normally expressed and tyrosinephosphorylated.…”

Section: Ephrin-b1 and Ephb2 In Vivo Tyrosine Phosphorylation Sitesmentioning

confidence: 99%

In Vivo Tyrosine Phosphorylation Sites of Activated Ephrin-B1 and EphB2 from Neural Tissue

Kalo

Pasquale

2001

Journal of Biological Chemistry

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EphB2 is a receptor tyrosine kinase of the Eph family and ephrin-B1 is one of its transmembrane ligands. In the embryo, EphB2 and ephrin-B1 participate in neuronal axon guidance, neural crest cell migration, the formation of blood vessels, and the development of facial structures and the inner ear. Interestingly, EphB2 and ephrin-B1 can both signal through their cytoplasmic domains and become tyrosine-phosphorylated when bound to each other. Tyrosine phosphorylation regulates EphB2 signaling and likely also ephrin-B1 signaling. Embryonic retina is a tissue that highly expresses both ephrin-B1 and EphB2. Although the expression patterns of EphB2 and ephrin-B1 in the retina are different, they partially overlap, and both proteins are substantially tyrosine-phosphorylated. To understand the role of ephrin-B1 phosphorylation, we have identified three tyrosines of ephrin-B1 as in vivo phosphorylation sites in transfected 293 cells stimulated with soluble EphB2 by using mass spectrometry and site-directed mutagenesis. These tyrosines are also physiologically phosphorylated in the embryonic retina, although the extent of phosphorylation at each site may differ.

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An Approach to Locate Phosphorylation Sites in a Phosphoprotein: Mass Mapping by Combining Specific Enzymatic Degradation with Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry

Cited by 155 publications

References 0 publications

CAPS Activity in Priming Vesicle Exocytosis Requires CK2 Phosphorylation

CAPS Activity in Priming Vesicle Exocytosis Requires CK2 Phosphorylation

Identification of Phosphopeptides by MALDI Q-TOF MS in Positive and Negative Ion Modes after Methyl Esterification

In Vivo Tyrosine Phosphorylation Sites of Activated Ephrin-B1 and EphB2 from Neural Tissue

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