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

Autor: Kinoshita-Kikuta, Emiko, Tanikawa, Ayane, Hosokawa, Takuro, Kiwado, Aya, Moriya, Koko, Kinoshita, Eiji, Koike, Tohru, Utsumi, Toshihiko
Předmět:
Zdroj: PLoS ONE; 11/21/2019, Vol. 14 Issue 11, p1-18, 18p
Abstrakt: 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. [ABSTRACT FROM AUTHOR]
Databáze: Complementary Index
Externí odkaz:
Nepřihlášeným uživatelům se plný text nezobrazuje