[14] Determination and location of phosphoserine in proteins and peptides by conversion to S-ethylcysteine

Meyer, Helmut E.; Hoffmann-Posorske, Edeltraut; Heilmeyer, Ludwig

doi:10.1016/0076-6879(91)01016-u

Cited by 110 publications

(66 citation statements)

References 11 publications

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“…2b) were separated by HPLC (Fig. 2c), and the phosphorylated residues were determined by gas phase sequencing (for methods see [29]). The sites phosphorylated at stage 3 are summarized in Table I.…”

Section: Resultsmentioning

confidence: 99%

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The Alzheimer‐like phosphorylation of tau protein reduces microtubule binding and involves Ser‐Pro and Thr‐Pro motifs

et al. 1992

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Tau protein can be transformed into an Alzheimer-like state by pho~phorylation with a kinase activity from brain EMBO J, 11, 1593EMBO J, 11, -1597], Here we show that the phosphorylation at Set-Pro motifs strongly decreases tau's affinity for mierotubul~s. The major reduction occurs during the first of the three main stages of phosphorylation, The data explain the lower stability of microtubules resulting from the pathological tau phosphorylation,

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Section: Resultsmentioning

confidence: 99%

“…Radioactive labeling was done with [y. '~P]ATP (NEN Du Pont) at 10 mCi/ml, radioactive t~ptic peptides were generated, isolated by HPLC, and sequenced as described [3,30], For details on sequencing methods see [8,29].…”

Section: Methodsmentioning

confidence: 99%

The Alzheimer‐like phosphorylation of tau protein reduces microtubule binding and involves Ser‐Pro and Thr‐Pro motifs

et al. 1992

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“…This analysis indicated that phosphoserine could be present in the peptide. To confirm the identification of phosphoserine at position 5 in the shark MGP sequence, the intact protein was treated with ethanethiol in order to convert the putative phosphoserine to S-ethylcysteine (Meyer et al, 1986(Meyer et al, , 1991 and then subjected to N-terminal sequence analysis. The HPLC separation of the PTH derivative released at the fifth sequencer cycle revealed the presence of a single peak that co-eluted with S-ethylcysteine and was present at the level predicted on the assumption that residue 5 was initially phosphoserine.…”

Section: Identification Of Phosphoserine In Mgp From Calcified Tissuesmentioning

confidence: 99%

Conserved phosphorylation of serines in the Ser‐X‐Glu/Ser(P) sequences of the vitamin K‐dependent matrix Gla protein from shark, lamb, rat, cow, and human

1994

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The present studies demonstrate that matrix Gla protein (MGP), a IO-kDa vitamin K-dependent protein, is phosphorylated at 3 serine residues near its N-terminus. Phosphoserine was identified at residues 3, 6 , and 9 of bovine, human, rat, and lamb MGP by N-terminal protein sequencing. All 3 modified serines are in tandemly repeated Ser-X-Glu sequences. Two of the serines phosphorylated in shark MGP, residues 2 and 5 , also have glutamate residues in the n + 2 position in tandemly repeated Ser-X-Glu sequences, whereas the third, shark residue 3, would acquire an acidic phosphoserine in the n + 2 position upon phosphorylation of serine 5 . The recognition motif found for MGP phosphorylation, Ser-X-Glu/Ser(P), has been seen previously in milk caseins, salivary proteins, and a number of regulatory peptides.A review of the literature has revealed an intriguing dichotomy in the extent of serine phosphorylation among secreted proteins that are phosphorylated at Ser-X-Glu/Ser(P) sequences. Those phosphoproteins secreted into milk or saliva are fully phosphorylated at each target serine, whereas phosphoproteins secreted into the extracellular environment of cells are partially phosphorylated at target serine residues, as we show here for MGP and others have shown for regulatory peptides and the insulin-like growth factor binding protein 1. We propose that the extent of serine phosphorylation regulates the activity of proteins secreted into the extracellular environment of cells, and that partial phosphorylation can therefore be explained by the need to ensure that the phosphoprotein be poised to gain or lose activity with regulated changes in phosphorylation status.The presence of 3 phosphoserine residues in all MGPs tested, which span shark to man, strongly supports the conclusion that phosphorylation of 3 serine residues is critical to the as yet unknown function of MGP. We have previously speculated that MGP is a locally acting regulator of cell growth and/or differentiation in the wide variety of cells that secrete the protein. Evidence for this hypothesis includes the strong induction of MGP expression by retinoic acid, a potent morphogen, and the independent discovery of MGP by differential cDNA screening as a gene that is overexpressed in transformed cells, in cells undergoing apoptosis, and in cells undergoing differentiation changes in culture. If MGP indeed acts on target cells near the point of its secretion to achieve changes in growth and/or differentiation, then regulated changes in the extent of MGP phosphorylation could provide a rapid and sensitive mechanism for controlling its activity.In the course of these studies we have made 2 improvements in the use of ethanethiol derivatization to identify phosphoserine residues during N-terminal protein sequencing, the demonstration that this reaction can be carried out on proteins bound to a polyvinylidene difluoride membrane and the observation that maximal conversion of phosphoserine to S-ethylcysteine requires 4 h at 60 "C rather than the previously pu...

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“…Alternatively, Michael addition is used to add a reactive thiol to dehydroaminobutyric acid or dehydroalanine to allow attachment of an affinity tag. Biotin is a widely used affinity tag and it permits purification of the chemically modified (previously phosphorylated) peptides (Meyer et al, 1991). This chemical modification is not applicable to phosphotyrosine residues and suffers from side reactions in which nonphosphorylated serine can be tagged.…”

Section: Biotin Tagging By β-Elimination and Michael Additionmentioning

confidence: 99%

Phosphoproteomics: Detection, Identification and Importance of Protein Phosphorylation

Jia¹,

Lin²,

Souchelnytskyi³

2012

Integrative Proteomics

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[14] Determination and location of phosphoserine in proteins and peptides by conversion to S-ethylcysteine

Cited by 110 publications

References 11 publications

The Alzheimer‐like phosphorylation of tau protein reduces microtubule binding and involves Ser‐Pro and Thr‐Pro motifs

The Alzheimer‐like phosphorylation of tau protein reduces microtubule binding and involves Ser‐Pro and Thr‐Pro motifs

Conserved phosphorylation of serines in the Ser‐X‐Glu/Ser(P) sequences of the vitamin K‐dependent matrix Gla protein from shark, lamb, rat, cow, and human

Phosphoproteomics: Detection, Identification and Importance of Protein Phosphorylation

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