Back-Pyrophosphorylation Assay to Detect In Vivo InsP7-Dependent Protein Pyrophosphorylation in Mammalian Cells.
Methods Mol Biol
; 2091: 93-105, 2020.
Article
em En
| MEDLINE
| ID: mdl-31773573
ABSTRACT
Protein pyrophosphorylation involves the transfer of a high-energy ß-phosphate from inositol pyrophosphates, such as diphosphoinositol pentakisphosphate (InsP7) to phosphorylated serine residues. Over a decade of research has established several proteins, involved in diverse physiological processes, as substrates of InsP7-mediated pyrophosphorylation. However, the need for detection of this posttranslational modification on endogenous proteins is paramount. "Back-pyrophosphorylation" is a simple technique to test whether a native protein undergoes InsP7-mediated pyrophosphorylation inside cells. The basis of this technique relies on the fact that a target protein isolated from cells with lower InsP7 levels exists in a hypo-pyrophosphorylated form as compared to the same protein isolated from cells with normal InsP7 levels. Hence, when radiolabeled InsP7 is added to a target protein immunoprecipitated from both these cell types, the hypopyrophosphorylated protein accepts a higher amount of radiolabeled phosphate when compared to the protein isolated from wild-type cells. This chapter provides detailed methods to identify an InsP7 target protein and conduct a back-pyrophosphorylation assay on a target protein immunoprecipitated from cells with normal versus reduced InsP7 levels, to confirm its endogenous pyrophosphorylation status.
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Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Fosfoproteínas
/
Fosfatos de Inositol
Limite:
Humans
Idioma:
En
Ano de publicação:
2020
Tipo de documento:
Article