A strategy to identify protein-N-myristoylation-dependent phosphorylation reactions of cellular proteins by using Phos-tag SDS-PAGE.

Emiko Kinoshita-Kikuta,Toshihiko Utsumi,Aya Kiwado,Ayane Tanikawa,Tohru Koike,Takuro Hosokawa,Koko Moriya,Eiji Kinoshita,Paul A Randazzo

doi:10.1371/journal.pone.0225510

Emiko Kinoshita-Kikuta, Toshihiko Utsumi + Show 7 more

Open Access

https://doi.org/10.1371/journal.pone.0225510

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Journal: PloS one	Publication Date: Nov 21, 2019
Citations: 6	License type: CC BY 4.0

Affiliation: Hiroshima University, Yamaguchi University

Abstract

To establish a strategy for identifying protein-N-myristoylation-dependent phosphorylation of cellular proteins, Phos-tag SDS-PAGE was performed on wild-type (WT) and nonmyristoylated mutant (G2A-mutant) FMNL2 and FMNL3, phosphorylated N-myristoylated model proteins expressed in HEK293 cells. The difference in the banding pattern in Phos-tag SDS-PAGE between the WT and G2A-mutant FMNL2 indicated the presence of N-myristoylation-dependent phosphorylation sites in FMNL2. Phos-tag SDS-PAGE of FMNL2 mutants in which the putative phosphorylation sites listed in PhosphoSitePlus (an online database of phosphorylation sites) were changed to Ala revealed that Ser-171 and Ser-1072 are N-myristoylation-dependent phosphorylation sites in FMNL2. Similar experiments with FMNL3 demonstrated that N-myristoylation-dependent phosphorylation occurs at a single Ser residue at position 174, which is a Ser residue conserved between FMNL2 and FMNL3, corresponding to Ser-171 in FMNL2. The facts that phosphorylation of Ser-1072 in FMNL2 has been shown to play a critical role in integrin β1 internalization mediated by FMNL2 and that Ser-171 in FMNL2 and Ser-174 in FMNL3 are novel putative phosphorylation sites conserved between FMNL2 and FMNL3 indicate that the strategy used in this study is a useful tool for identifying and characterizing physiologically important phosphorylation reactions occurring on N-myristoylated proteins.

Highlights

Protein N-myristoylation is a typical lipid modification that occurs on eukaryotic and viral proteins [1,2,3,4,5,6]
To confirm that protein N-myristoylation occurred on FMNL2 and FMNL3, cellular metabolic labeling experiments were performed by using cDNA encoding C-terminally FLAGtagged FMNL2 and FMNL3
Immunofluorescence staining of FMNL2-FLAG and FMNL3-FLAG expressed in COS-1 cells revealed that these proteins were largely localized to the plasma membrane and that they induced remarkable cellular morphological changes such as the formation of filopodia and membrane protrusions, whereas immunofluorescence staining of FMNL2-G2A-FLAG and FMNL3-G2A-FLAG showed a diffuse cytosolic distribution without the induction of cellular morphological changes (Fig 2)

Summary

Introduction

Protein N-myristoylation is a typical lipid modification that occurs on eukaryotic and viral proteins [1,2,3,4,5,6]. Protein N-myristoylation is an irreversible cotranslational protein modification. Analysis of protein-N-myristoylation-dependent phosphorylation study design, data collection and analysis, decision to publish, or preparation of the manuscript

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