Figure 3: Y26 phosphorylation results in increased H11 phosphorylation of PGAM1 in vitro and in vivo. | Nature Communications

Figure 3: Y26 phosphorylation results in increased H11 phosphorylation of PGAM1 in vitro and in vivo.

From: Tyr26 phosphorylation of PGAM1 provides a metabolic advantage to tumours by stabilizing the active conformation

Figure 3

(a) Detection of H11 phosphorylation in PGAM1 using LC-MS/MS. Tandem mass spectrometry (MS/MS) spectrum of phosphorylated PGAM1 peptide pHGESAWNLENR (residue number 11–21) collected using collision-induced dissociation (CID) fragmentation. Underlined nominal masses above and below the sequence denote the b and y ions, respectively that were annotated from the spectrum. The expected and observed mass to charge ratio (m/z) for the [M+2H+]2+ precursor ion is provided. [M-H3PO4]+2 represents molecular ion with loss of the phosphate group. (b,c) Phosphorylation by rFGFR1 leads to increased H11 phosphorylation of Flag-PGAM1 WT but not Y26F in the presence of 10 μM 2,3-BPG (b). Panel (c) shows relative quantification of H11-phosphorylated PGAM1 using reverse phase liquid chromatography. MS data for HGESAWNLENR (upper three red spectra in each panel) and pHGESAWNLENR (lower three green spectra in each panel) were used to calculate the relative levels of phosphorylated and unphosphorylated peptide across all observable charge states in Flag-tagged PGAM1 WT and Y26F mutant incubated with active FGFR1 and 2,3-BPG for one hour (c, left and right, respectively). The relative abundance was normalised to the largest peak of each scan, which corresponds to 5.5E7 (c, left) and 2.26E7 (c, right). (d,e) Rescue cells expressing mouse PGAM1 (mPGAM1) Y26F demonstrate reduced H11-phosphorylated levels compared with cells with mPGAM1 WT (d). WT or Y26F: cells with stable knockdown of endogenous hPGAM1 and rescue expression of mPGAM1 WT or Y26F mutant, respectively. Panel (e) shows relative quantification of H11-phosporylated PGAM1 using reverse phase liquid chromatography with Flag-tagged PGAM1 WT and Y26F mutant proteins isolated from rescue H1299 cells (e, left and right, respectively). The relative abundance was normalised to the largest peak of each scan, which corresponds to 1.12E7 (e, left) and 1.96E7 (e, right). The error bars represent mean values±s.d. from two independent experiments (*0.01<P<0.05; **P<0.01).

Back to article page