Research ArticleCancer

Cancer-associated arginine-to-histidine mutations confer a gain in pH sensing to mutant proteins

See allHide authors and affiliations

Science Signaling  05 Sep 2017:
Vol. 10, Issue 495, eaam9931
DOI: 10.1126/scisignal.aam9931
  • Fig. 1 EGFR-R776H has pH-sensitive activity and downstream signaling, with increased activity at higher pHi.

    (A) Structures of inactive (left, 3GT8) and active (right, 2GS6) EGFR-WT. Arg776 is shown in stick. Red, αC helix; yellow, heteroatom distances (in Ångstrom). (B) In vitro kinase activity of WT and EGFR-R776H (R776H) was determined by [γ-32P]–adenosine 5′-triphosphate (ATP) incorporation into enzyme (autophosphorylation) or substrate peptide (substrate) at two pH values. Data were normalized to autophosphorylation of WT at pH 6.8. Data are from four independent experiments (means ± SEM). Representative gel and autoradiograph are shown in fig. S1A. (C) Representative immunoblots of lysates from quiescent MDA-MB-453 cells transfected with EGFR-WT, EGFR-R776H, or EGFR-R776G with (+) or without (−) EGF (50 ng/ml; 5 min) at indicated pHi values. Blots of total EGFR (EGFR), EGFR autophosphorylation (pTyr1173), AKT-pSer473 (pAKT), ERK1/2 (ERK), and ERK1/2-pThr202/Tyr204 (pERK). (D to F) Quantification of pTyr1173 (D), pAKT (E), and pERK (F), with data normalized to amount of total EGFR present. Data are from four independent experiments (Tukey box plots). For (B) and (D) to (F), Student’s t tests (unpaired, two-tailed) with Holm-Sidak multiple comparisons correction were used. *P < 0.05, **P < 0.01, ***P < 0.001.

  • Fig. 2 Protonation state of His776 alters EGFR αC helix conformations sampled in MD simulations.

    (A) JS divergence of protonated versus neutral R776H highlights major nonlocal differences localized around the N-terminal end of the αC helix. (B) αC helix conformational distribution for 300-ns MD simulations defined by two angle measures (see also fig. S5 for comparisons with R776G) that describe tilting and displacement of the helix relative to the active crystal structure. Circles denote the tilt and displacement of active (yellow) and inactive (black) crystal structures. c.o.m., center of mass; Norm. frac. obs., normalized fraction observed. (C to E) Final snapshots of simulations for WT (C), neutral His776 (D), and protonated His776 (E).

  • Fig. 3 EGFR-R776H has pH-sensitive proliferation and soft agar transformation.

    (A) Immunoblot of lysates from parental NIH3T3 (3T3) cells and cells stably expressing vectors encoding WT or mutant EGFR. (B) pHi in 3T3 cells when cultured with ammonium chloride (NH4Cl) or EIPA. Data are means ± SEM from four independent experiments. (C) Fold increase in cell number at given pHi for stable WT or mutant EGFR cell lines. Data are means ± SEM from three or more independent experiments, each normalized to parental 3T3 at pHi 7.4. (D) Soft agar transformation assay in NIH3T3 cells stably expressing WT or mutant EGFR or BRAF-V600E. Cells were maintained for 15 days in complete medium or medium supplemented with 5 mM NH4Cl and then fixed and stained with crystal violet. (E) Colonies >8 pixel2 (px2) were counted using ImageJ particle analyzer. Data are from four independent experiments (Tukey box plots). Comparison of 3T3 at pHi 7.2 and 7.4 in (C) used a one-sample t test (two-tailed). All other comparisons in (B), (C), and (E) used Student’s t tests (unpaired, two-tailed) with Holm-Sidak multiple comparisons correction. *P < 0.05, **P < 0.01, ***P < 0.001.

  • Fig. 4 p53-R273H has decreased DNA binding at higher pHi.

    (A) Structure of p53 (4HJE) with Arg273 and Arg175 in magenta stick. (B) Luciferase assay in PS120 cells transfected with WT or mutant p53 at indicated pHi (see fig. S7A for pHi measurements). Luciferase was normalized to β-galactosidase control and then within each experiment to WT at pHi 7.1. Data are means ± SEM from four independent experiments for WT, R273H, and R175H and three independent experiments for R273L. (C) RT-PCR from etoposide-treated MDA-MB-157 cells stably expressing WT or p53-R273H at two pHi values (see fig. S7B for pHi measurements). Scatter plots of 2−ΔCt on log10 scale for WT and R273H show transcripts that are more (red) or less (blue) abundant at pHi 7.2 relative to 7.6 (see table S1 for the list of altered transcripts). Data from three independent experiments [mean, identity (solid) and threefold boundary (dotted) lines]. (D) Cells were treated as in (C), and percentage of dead cells was determined by trypan blue exclusion. Data are means ± SEM from four independent experiments. For (B) and (D), Student’s t tests (unpaired, two-tailed) with Holm-Sidak multiple comparisons correction were used. *P < 0.05, ***P < 0.001. For (C), a threefold boundary significance cutoff was used.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/10/495/eaam9931/DC1

    Fig. S1. EGFR-R776H activity is pH-sensitive in vitro and titrates with increasing pH.

    Fig. S2. pHi control and pH-dependent EGFR-R776H activity in EGFR-null cells.

    Fig. S3. EGFR-R776H activity is pH-sensitive at distinct autophosphorylation sites.

    Fig. S4. Quantification of C helix flexibility and trajectory RMSD.

    Fig. S5. EGFR MD simulations metrics for analysis.

    Fig. S6. Full-well images of transformation assays.

    Fig. S7. pHi control for p53 assays.

    Table S1. pH-dependent transcriptional profiles.

  • Supplementary Materials for:

    Cancer-associated arginine-to-histidine mutations confer a gain in pH sensing to mutant proteins

    Katharine A. White, Diego Garrido Ruiz, Zachary A. Szpiech, Nicolas B. Strauli, Ryan D. Hernandez, Matthew P. Jacobson, Diane L. Barber*

    *Corresponding author. Email: diane.barber{at}ucsf.edu

    This PDF file includes:

    • Fig. S1. EGFR-R776H activity is pH-sensitive in vitro and titrates with increasing pH.
    • Fig. S2. pHi control and pH-dependent EGFR-R776H activity in EGFR-null cells.
    • Fig. S3. EGFR-R776H activity is pH-sensitive at distinct autophosphorylation sites.
    • Fig. S4. Quantification of C helix flexibility and trajectory RMSD.
    • Fig. S5. EGFR MD simulations metrics for analysis.
    • Fig. S6. Full-well images of transformation assays.
    • Fig. S7. pHi control for p53 assays.
    • Table S1. pH-dependent transcriptional profiles.

    [Download PDF]

    Technical Details

    Format: Adobe Acrobat PDF

    Size: 891 KB


    Citation: K. A. White, D. G. Ruiz, Z. A. Szpiech, N. B. Strauli, R. D. Hernandez, M. P. Jacobson, D. L. Barber, Cancer-associated arginine-to-histidine mutations confer a gain in pH sensing to mutant proteins. Sci. Signal. 10, eaam9931 (2017).

    © 2017 American Association for the Advancement of Science

Stay Connected to Science Signaling

Navigate This Article