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

PLoS One. 2019 Nov 21;14(11):e0225510. doi: 10.1371/journal.pone.0225510. eCollection 2019.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • COS Cells
  • Chlorocebus aethiops
  • Electrophoresis, Polyacrylamide Gel
  • Formins / chemistry
  • Formins / genetics
  • Formins / metabolism*
  • HEK293 Cells
  • Humans
  • Mutation
  • Phosphorylation
  • Pyridines / chemistry*
  • Serine / chemistry*

Substances

  • 1,3-bis(bis(pyridin-2-ylmethyl)amino)propan-2-ol
  • FMNL2 protein, human
  • FMNL3 protein, human
  • Formins
  • Pyridines
  • Serine

Grants and funding

This work was supported in part by KAKENHI Grants (No.18K065960 to EK-K, No.19K071470 to EK, No.17K08237 to TK, and No.17K07758 to TU, https://www.jsps.go.jp/j-grantsinaid/index.html) from the Ministry of Education, Culture, Sports, Science and Technology of Japan. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.