History of Phos-tag technology for phosphoproteomics

Kinoshita, Eiji; Kinoshita–Kikuta, Emiko; Koike, Takao

doi:10.1016/j.jprot.2021.104432

Cited by 15 publications

(5 citation statements)

References 39 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Phos-tag TM , a binuclear metal complex, selectively binds phosphate monoesters in aqueous solutions. We employed the first application of Phos-tag TM , utilizing Phostag TM derivatives and hydrophilic chromatography to enrich phosphorylated Bt2 and Sh2 peptides [34]. Alkaline phosphatase, a phosphatase enzyme capable of dephosphorylating substrates, was used to remove phosphorylation from Sh2.…”

Section: Discussionmentioning

confidence: 99%

Serine 31 Phosphorylation-Driven Regulation of AGPase Activity: Potential Implications for Enhanced Starch Yields in Crops

Yu,

Mou,

Shoaib

et al. 2023

IJMS

View full text Add to dashboard Cite

ADP-Glc pyrophosphorylase (AGPase), which catalyzes the transformation of ATP and glucose-1-phosphate (Glc-1-P) into adenosine diphosphate glucose (ADP-Glc), acts as a rate-limiting enzyme in crop starch biosynthesis. Prior research has hinted at the regulation of AGPase by phosphorylation in maize. However, the identification and functional implications of these sites remain to be elucidated. In this study, we identified the phosphorylation site (serine at the 31st position of the linear amino acid sequence) of the AGPase large subunit (Sh2) using iTRAQTM. Subsequently, to ascertain the impact of Sh2 phosphorylation on AGPase, we carried out site-directed mutations creating Sh2-S31A (serine residue replaced with alanine) to mimic dephosphorylation and Sh2-S31D (serine residue replaced with aspartic acid) or Sh2-S31E (serine residue replaced with glutamic acid) to mimic phosphorylation. Preliminary investigations were performed to determine Sh2 subcellular localization, its interaction with Bt2, and the resultant AGPase enzymatic activity. Our findings indicate that phosphorylation exerts no impact on the stability or localization of Sh2. Furthermore, none of these mutations at the S31 site of Sh2 seem to affect its interaction with Bt2 (smaller subunit). Intriguingly, all S31 mutations in Sh2 appear to enhance AGPase activity when co-transfected with Bt2, with Sh2-S31E demonstrating a substantial five-fold increase in AGPase activity compared to Sh2. These novel insights lay a foundational groundwork for targeted improvements in AGPase activity, thus potentially accelerating the production of ADP-Glc (the primary substrate for starch synthesis), promising implications for improved starch biosynthesis, and holding the potential to significantly impact agricultural practices.

show abstract

Section: Discussionmentioning

confidence: 99%

Serine 31 Phosphorylation-Driven Regulation of AGPase Activity: Potential Implications for Enhanced Starch Yields in Crops

Yu,

Mou,

Shoaib

et al. 2023

IJMS

View full text Add to dashboard Cite

show abstract

“…Phos-tag gel electrophoresis is a versatile technique using a metal chelator, called “Phos-tag”, that forms transient complexes with phosphorylated amino acid residues. − When a Phos-tag acrylamide derivative is copolymerized into SDS-PAGE gels (with added Mn 2+ or Zn 2+ ), the migration of phosphorylated protein forms is slowed and proteins are separated based on their phosphorylation status as well as size (Figure B). Importantly, the extent to which migration is slowed depends on (i) the number of phosphorylation sites, (ii) the specific context of these sites within the protein, and (iii) the type of phosphorylation (e.g., pSer vs pThr) .…”

Section: Genetic Code Expansion As a Tool To Generate Phosphorylated ...mentioning

confidence: 99%

Genetic Encoding of Phosphorylated Amino Acids into Proteins

Allen,

Karplus,

Mehl

et al. 2024

Chem. Rev.

View full text Add to dashboard Cite

Reversible phosphorylation is a fundamental mechanism for controlling protein function. Despite the critical roles phosphorylated proteins play in physiology and disease, our ability to study individual phospho-proteoforms has been hindered by a lack of versatile methods to efficiently generate homogeneous proteins with site-specific phosphoamino acids or with functional mimics that are resistant to phosphatases. Genetic code expansion (GCE) is emerging as a transformative approach to tackle this challenge, allowing direct incorporation of phosphoamino acids into proteins during translation in response to amber stop codons. This genetic programming of phospho-protein synthesis eliminates the reliance on kinase-based or chemical semisynthesis approaches, making it broadly applicable to diverse phospho-proteoforms. In this comprehensive review, we provide a brief introduction to GCE and trace the development of existing GCE technologies for installing phosphoserine, phosphothreonine, phosphotyrosine, and their mimics, discussing both their advantages as well as their limitations. While some of the technologies are still early in their development, others are already robust enough to greatly expand the range of biologically relevant questions that can be addressed. We highlight new discoveries enabled by these GCE approaches, provide practical considerations for the application of technologies by non-GCE experts, and also identify avenues ripe for further development.

show abstract

“…(B) The dinuclear metal complex 1,3-bis[bis(pyridin-2-ylmethyl)-amino]propan-2olato has been found as selectively binding phosphate [114]. Based on this feature, various phosphate-binding tag derivatives including Phos-tag biotin and Phos-tag acrylamide have been developed for phosphoproteome research [115][116][117]. The Phos-tag biotin method is suitable for the detection of phosphorylation status of purified proteins obtained using, for example, GFP-trap and other methods.…”

Section: A Brief Guideline For Studying Phosphorylation In Plant Auto...mentioning

confidence: 99%

Shedding Light on the Role of Phosphorylation in Plant Autophagy

Liao

Zheng

et al. 2022

FEBS Letters

View full text Add to dashboard Cite

Autophagy is a highly conserved quality control process that maintains cellular health by eliminating deleterious cargoes. Compared with the extensive studies in yeast and mammalian models, the molecular details and significance of post-translational modifications (PTMs) in the autophagy process in plants remain less well defined. In this review, we discuss recent progress in our understanding of phosphorylation, one of the most extensively studied PTMs, in the regulation of autophagosome biogenesis and autophagic degradation in plants. Based on the plant mass spectrometric database, we summarize the experimentally verified phosphorylation sites of the core autophagy machinery in plants. Furthermore, we put forward several approaches to test the roles of phosphorylation in the regulation of plant autophagy.

show abstract

History of Phos-tag technology for phosphoproteomics

Cited by 15 publications

References 39 publications

Serine 31 Phosphorylation-Driven Regulation of AGPase Activity: Potential Implications for Enhanced Starch Yields in Crops

Serine 31 Phosphorylation-Driven Regulation of AGPase Activity: Potential Implications for Enhanced Starch Yields in Crops

Genetic Encoding of Phosphorylated Amino Acids into Proteins

Shedding Light on the Role of Phosphorylation in Plant Autophagy

Contact Info

Product

Resources

About