An efficient chemical method for dephosphorylation of phosphopeptides

Kuyama, Hiroki; Toda, Chikako; Watanabe, Mamoru; Tanaka, Kōichi; Nishimura, Osamu

doi:10.1002/rcm.1078

Cited by 36 publications

(40 citation statements)

References 28 publications

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“…These results indicated that the stimulation might be caused by the binding of the BAF in SC to ⌬TM, which is known to mediate the binding of emerin to chromatin, because (i) the stimulation of the binding was not suppressed by kinase inhibitors, (ii) the stimula- (upper, 11-13, 17-25). Slashed boxes indicate the hydrophobic amino acid-rich region, which was demonstrated by Wolff et al (31). Two GST-fused emerin fragments were constructed and used in this study (middle and lower).…”

Section: Resultsmentioning

confidence: 90%

Dissociation of Emerin from Barrier-to-autointegration Factor Is Regulated through Mitotic Phosphorylation of Emerin in a Xenopus Egg Cell-free System

Hirano

Segawa

Ouchi

et al. 2005

Journal of Biological Chemistry

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Emerin is the gene product of STA whose mutations cause Emery-Dreifuss muscular dystrophy. It is an inner nuclear membrane protein and phosphorylated in a cell cycle-dependent manner. However, the means of phosphorylation of emerin are poorly understood. We investigated the regulation mechanism for the binding of emerin to chromatin, focusing on its cell cycle-dependent phosphorylation in a Xenopus egg cell-free system. It was shown that emerin dissociates from chromatin depending on mitotic phosphorylation of the former, and this plays a critical role in the dissociation of emerin from barrier-to-autointegration factor (BAF). Then, we analyzed the mitotic phosphorylation sites of emerin. Emerin was strongly phosphorylated in an M-phase Xenopus egg cell-free system, and five phosphorylated sites, Ser 49 , Ser 66 , Thr 67 , Ser 120 , and Ser 175 , were identified on analysis of chymotryptic and tryptic emerin peptides using a phosphopeptide-concentrating system coupled with a Titansphere column, which specifically binds phosphopeptides, and tandem mass spectrometry sequencing. An in vitro binding assay involving an emerin S175A point mutant protein suggested that phosphorylation at Ser 175 regulates the dissociation of emerin from BAF. The nuclear envelope (NE)2 is a highly dynamic structure that disassembles at the onset of mitosis and reassembles on the surface of chromatin during telophase in vertebrates. These changes of NE are crucial for cell cycle progression. The NE consists of an outer nuclear membrane, inner nuclear membrane, nuclear pore complex, and nuclear lamina. The inner nuclear membrane contains integral membrane proteins, i.e. lamin B receptor (LBR), lamina-associated polypeptide-2␤ (LAP2␤), emerin, MAN1, and others, which interact with DNA and/or chromatin, and these proteins are proposed to participate in nuclear membrane targeting to chromatin at an early stage of nuclear assembly (1). The interactions between some of the inner nuclear membrane proteins and chromatin are regulated through phosphorylation of these inner nuclear membrane proteins. The phosphorylation mechanisms for LBR and LAP2␣ and 2␤ are well understood (2-7). LBR directly binds to DNA in vitro and dissociates on phosphorylation by cdc2 kinase and other kinase(s) in a mitotic egg extract (3). LAP2␤ binds to lamin B1 and chromatin, and cell cycle-dependent phosphorylation of LAP2␤ cancels this binding (4). Phosphorylation of these inner nuclear proteins, therefore, is likely to be one of the key mechanisms that control the interactions between the inner nuclear proteins and components of the nuclear lamina as well as chromatin. In this study, we focused on the mitotic phosphorylation of emerin, one of the inner nuclear membrane proteins.Human emerin is a serine-rich protein exhibiting an apparent mass of 34 kDa on SDS-PAGE (8) and is phosphorylated in a cell cycle-dependent manner (9). Emerin belongs to the LEM (LAP2␤, emerin, MAN1) protein family, whose members have approximately a 40-residue domain named the LEM (10). These ...

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Section: Resultsmentioning

confidence: 90%

Dissociation of Emerin from Barrier-to-autointegration Factor Is Regulated through Mitotic Phosphorylation of Emerin in a Xenopus Egg Cell-free System

Hirano

Segawa

Ouchi

et al. 2005

Journal of Biological Chemistry

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“…The chemical dephosphorylation of patatin was performed with hydrogen fluoride-pyridine (HF-P) as previously described [28,29], with some modification [10]. An amount of 1 mg of total protein extract from tuber of cv.…”

Section: Chemical Dephosphorylation Of Patatinmentioning

confidence: 99%

“…Second, direct and rapid in-gel multiplex identification and mapping of phosphorylated isoforms of the patatin were achieved using the Pro-Q Diamond phosphoprotein stain (Pro-Q DPS), which specifically binds to the phosphate moieties of phosphoproteins [27]. Third, quantitative profiling of phosphorylated patatin isoforms was assessed by chemical dephosphorylation of phosphoproteins with hydrogen fluoride-pyridine (HF-P) [28][29][30]. For this purpose, the volume difference between phosphorylated and dephosphorylated 2-DE patatin spots was used to quantify protein phosphorylation levels.…”

Section: Introductionmentioning

confidence: 99%

Identification and Mapping of Phosphorylated Isoforms of the Major Storage Protein of Potato Based on Two- Dimensional Electrophoresis

Bernal¹,

López‐Pedrouso²,

Daprà³

et al. 2017

Advances in Seed Biology

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Protein phosphorylation plays a key role in the synthesis and degradation of dry seed storage proteins. In contrast, no evidence for phosphorylation has been reported to date in vegetative storage proteins (VSPs). The patatin multigene family encodes the major VSP of the potato, Solanum tuberosum L. This study addresses for the first time the identification and mapping of phosphorylated patatin forms based on high-resolution two-dimensional electrophoresis (2-DE) profiles. Patatin isoforms from mature tubers of cultivar Kennebec were separated by 2-DE and subsequently identified by tandem mass spectrometry. In-gel identification and mapping of phosphorylated isoforms were performed using the multiplex phosphoprotein-specific staining Pro-Q DPS. We found that phosphorylation is a ubiquitous post-translational protein modification associated with isoforms of patatin. In addition, protein dephosphorylation with hydrogen fluoride-pyridine coupled to 2-DE was used for quantitative profiling of phosphorylated patatin. This experimental approach showed that patatin comprises multiple isoforms with very different phosphorylation level. Thus, phosphorylation rates over isoforms ranged from 4.6 to 52.3%. Overall, the identification and mapping of differentially phosphorylated patatin opens up new exploratory ways to unravel the molecular mechanisms underlying its mobilization along the tuber life cycle.

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“…Achieving this relies on complete de phosphorylation of proteins in a sample by hydrogen fluoride treatment. Chemical de phosphorylation by hydrogen fluoride has been shown to non-specifically remove post-translational modifications from proteins without affecting its integrity [23]. To determine in vivo phosphorylation status of a specific protein using RIKA, the protein is purified from the cell lysate using a specific tag or antibody raised against that protein.…”

Section: Reverse In-gel Kinase Assay (Rika)mentioning

confidence: 99%

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2016

MOJPB

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Protein kinases and protein phosphorylation play an important role in almost every process within a cell. Identifying specific substrates downstream of individual protein kinases is critical to understanding their function and role in human health and diseases. Given its importance, several techniques have been developed to determine direct substrates of protein kinases in cells. Here, I have summarized the advantages and limitations of some of the most widely used current techniques available to profile substrates of protein kinases.

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An efficient chemical method for dephosphorylation of phosphopeptides

Abstract: Phosphate moieties found on serine, threonine or tyrosine residues in peptides, e.g., in proteolytic digests of proteins, were cleaved using hydrofluoric acid or hydrogen fluoride-pyridine without side reactions.

Cited by 36 publications

References 28 publications

Dissociation of Emerin from Barrier-to-autointegration Factor Is Regulated through Mitotic Phosphorylation of Emerin in a Xenopus Egg Cell-free System

Dissociation of Emerin from Barrier-to-autointegration Factor Is Regulated through Mitotic Phosphorylation of Emerin in a Xenopus Egg Cell-free System

Identification and Mapping of Phosphorylated Isoforms of the Major Storage Protein of Potato Based on Two- Dimensional Electrophoresis

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