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. 2014 Jul 24;289(37):25418–25430. doi: 10.1074/jbc.M114.547844

FIGURE 5.

FIGURE 5.

Effects of CAi-mediated CO2-pH coupling on kinase phosphorylation. A, Phosphorylation state of ERK1 (Thr-202/Tyr-204) and ERK2 (Thr-185/Tyr-187) was unaffected by changing pCO2 either tonically (5% or 20% for 30 min) or dynamically (six cycles of 5–20% fluctuations; cycle length of 4 min); densitometric analysis (n = 3). Time-averaged intracellular [H+] was measured in separate experiments. B, JNK1/2 were dephosphorylated (Thr-183/Tyr-185) by stably raised pCO2 but unaffected by oscillating pCO2 (n = 3). C, panel i, Western blot for S6 kinase phosphorylation (P) (readout of mTORC1 signaling) under 5% CO2, 20% CO2, and fluctuating pCO2 (six cycles; 5–20%) in the presence or absence of ATZ (100 μm). Panel ii, pCO2 fluctuations reduced S6 kinase phosphorylation but only under full CAi activity. S6K activity did not correlate with time-averaged [H+] (n = 3). D, panels i and ii, analysis for one cycle of pCO2 oscillation in HCT116 cells. E, at 5% CO2, mTOR activity was unaffected by ATZ but strongly inhibited by rapamycin (10 μm). F, six cycles of pCO2 fluctuations in CAII knockdown (KD) HCT116 cells (construct 695). G, six cycles of pCO2 fluctuations in MDA-MB-468 cells (low CAi activity). Error bars represent S.E. *, p > 0.05.