A strategy to identify protein-N-myristoylation-dependent phosphorylation reactions of cellular proteins by using Phos-tag SDS-PAGE
Autoři:
Emiko Kinoshita-Kikuta aff001; Ayane Tanikawa aff002; Takuro Hosokawa aff002; Aya Kiwado aff002; Koko Moriya aff002; Eiji Kinoshita aff001; Tohru Koike aff001; Toshihiko Utsumi aff002
Působiště autorů:
Department of Functional Molecular Science, Institute of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan
aff001; Graduate School of Sciences and Technology for Innovation, Yamaguchi University, Yamaguchi, Japan
aff002; Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University, Yamaguchi, Japan
aff003
Vyšlo v časopise:
PLoS ONE 14(11)
Kategorie:
Research Article
doi:
https://doi.org/10.1371/journal.pone.0225510
Souhrn
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.
Klíčová slova:
Cell membranes – Membrane proteins – Phosphorylation – Protein kinases – Protein sequencing – Sequence motif analysis – Polyacrylamides – Metabolic labeling
Zdroje
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