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

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 ?1 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.

SUBMITTER: Kinoshita-Kikuta E

PROVIDER: S-EPMC6872159 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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

Kinoshita-Kikuta Emiko E Tanikawa Ayane A Hosokawa Takuro T Kiwado Aya A Moriya Koko K Kinoshita Eiji E Koike Tohru T Utsumi Toshihiko T

PloS one 20191121 11

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 ...[more]

PMID: 31751425

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Project description:Recombinant protein technology provides an invaluable source of proteins for use in structure-function studies, as immunogens, and in the development of therapeutics. Recombinant proteins are typically engineered with "tags" that allow the protein to be purified from crude host cell extracts using affinity based chromatography techniques. Amelogenin is the principal component of the developing enamel matrix and a frequent focus for biomineralization researchers. Several groups have reported the successful production of recombinant amelogenins but the production of recombinant amelogenin free of any tags, and at single band purity on silver stained SDS PAGE is technically challenging. This is important, as rigorous structure-function research frequently demands a high degree of protein purity and fidelity of protein sequence. Our aim was to generate His-tagged recombinant amelogenin at single band purity on silver stained SDS PAGE for use in functionality studies after His-tag cleavage. An acetic acid extraction technique (previously reported to produce recombinant amelogenin at 95% purity directly from E. coli) followed by repeated rounds of nickel column affinity chromatography, failed to generate recombinant amelogenin at single band purity. This was because following an initial round of nickel column affinity chromatography, subsequent cleavage of the His-tag was not 100% efficient. A second round of nickel column affinity chromatography, used in attempts to separate the cleaved His-tag free recombinant from uncleaved His-tagged contaminants, was still unsatisfactory as cleaved recombinant amelogenin exhibited significant affinity for the nickel column. To solve this problem, we used preparative SDS PAGE to successfully purify cleaved recombinant amelogenins to single band purity on silver stained SDS PAGE. The resolving power of preparative SDS PAGE was such that His-tag based purification of recombinant amelogenin becomes redundant. We suggest that acetic acid extraction of recombinant amelogenin and subsequent purification using preparative SDS PAGE provides a simple route to highly purified His-tag free amelogenin for use in structure-function experiments and beyond.

Project description:Phosphorylation of viral proteins serves as a regulatory mechanism during the intracellular life cycle of infected viruses. There is therefore a pressing need to develop a method to efficiently purify and enrich phosphopeptides derived from viral particles in biological samples. In this study, we utilized Phos-tag technology to analyze the functional phosphorylation of the nucleocapsid protein (N protein; NP) of severe respiratory syndrome coronavirus 2 (SARS-CoV-2). Viral particles were collected from culture supernatants of SARS-CoV-2-infected VeroE6/TMPRSS2 cells by ultracentrifugation, and phosphopeptides were purified by Phos-tag magnetic beads for LC-MS/MS analysis. Analysis revealed that NP was reproducibly phosphorylated at serine 79 (Ser79). Multiple sequence alignment and phylogenetic analysis showed that the Ser79 was a distinct phospho-acceptor site in SARS-CoV-2 but not in other beta-coronaviruses. We also found that the prolyl-isomerase Pin1 bound to the phosphorylated Ser79 in NP and positively regulated the production of viral particles. These results suggest that SARS-CoV-2 may have acquired the potent virus-host interaction during its evolution mediated by viral protein phosphorylation. Moreover, Phos-tag technology can provide a useful means for analyzing the functional phosphorylation of viral proteins. SIGNIFICANCE: In this study, we aimed to investigate the functional phosphorylation of SARS-CoV-2 NP. For this purpose, we used Phos-tag technology to purify and enrich virus-derived phosphopeptides with high selectivity and reproducibility. This method can be particularly useful in analyzing viral phosphopeptides from cell culture supernatants that often contain high concentrations of fetal bovine serum and supplements. We newly identified an NP phosphorylation site at Ser79, which is important for Pin1 binding. Furthermore, we showed that the interaction between Pin1 and phosphorylated NP could enhance viral replication in a cell culture model.

Dataset Information

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

Publications

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

Similar Datasets

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