Back-Pyrophosphorylation Assay to Detect In Vivo InsP₇-Dependent Protein Pyrophosphorylation in Mammalian Cells

Abstract

Protein pyrophosphorylation involves the transfer of a high-energy β-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.