Phosphorylation stoichiometries of human eukaryotic initiation factors

Abstract

Eukaryotic translation initiation factors are the principal molecular effectors regulating the process converting nucleic acid to functional protein. Commonly referred to as eIFs (eukaryotic initiation factors), this suite of proteins is comprised of at least 25 individual subunits that function in a coordinated, regulated, manner during mRNA translation. Multiple facets of eIF regulation have yet to be elucidated; however, many of the necessary protein factors are phosphorylated. Herein, we have isolated, identified and quantified phosphosites from eIF2, eIF3, and eIF4G generated from log phase grown HeLa cell lysates. Our investigation is the first study to globally quantify eIF phosphosites and illustrates differences in abundance of phosphorylation between the residues of each factor. Thus, identification of those phosphosites that exhibit either high or low levels of phosphorylation under log phase growing conditions may aid researchers to concentrate their investigative efforts to specific phosphosites that potentially harbor important regulatory mechanisms germane to mRNA translation.

Figure 1

Quantification of phosphorylation on Thr-66 on eIF2γ. (A) Precursor ion mass scan of the [M + 2H]²⁺ ion is shown; (B) MS/MS spectra of m/z ion 582.9 illustrating indicative b- and y-ions for peptide spanning residues 60-68 of eIF2γ; and (C) Zoomed in view of the m/z region containing the TMT (tandem mass tags) reporter ions. Calculation of the reporter ion ratio reveals a phosphorylation level of 70.5% for Thr-66.

Figure 2

Quantification of phosphorylation on eIF3. (A) MS/MS spectra of m/z ion 406.6, z = 3. Peptide spans residues 1329–1337 for eIF3a; (B) Zoomed in view of the m/z region containing the TMT reporter ions. Calculation of the reporter ion ratio reveals a phosphorylation level of 18% for Ser-1336; (C) MS/MS spectra of m/z ion 405.5, z = 3. Peptide spans residues 1194–1201 for eIF3a; and (D) Zoomed in view of the m/z region containing the TMT reporter ions. Calculation of the reporter ion ratio reveals a phosphorylation level of 92% for Ser-1198.

Figure 3

Quantification of phosphorylation on eIF4G. (A) MS/MS spectra of m/z ion 992.6, z = 2. Peptide spans residues 1072–1085; (B) Zoomed in view of the m/z region containing the TMT reporter ions. Calculation of the reporter ion ratio reveals a phosphorylation level of 15% for Ser-1077; (C) MS/MS spectra of m/z ion 520.8, z = 2. Peptide spans residues 1091–1095; and (D) Zoomed in view of the m/z region containing the TMT reporter ions. Calculation of the reporter ion ratio reveals a phosphorylation level of 40% for Ser-1092.