Intracellular pH regulates β-catenin with low pHi increasing adhesion and signaling functions.

Intracellular pH (pHi) dynamics are linked to cell proliferation, migration, and differentiation. The adherens junction (AJ) and signaling protein β-catenin has decreased abundance at high pHi due to increased proteasomal-mediated degradation. However, the effects of low pHi on β-catenin abundance and function have not been characterized. Here, we use population-level and single-cell assays to show that low pHi stabilizes β-catenin, increasing junctional, cytoplasmic, and nuclear abundance. We assayed single-cell protein degradation rates to show that β-catenin half-life is longer at low pHi and shorter at high pHi compared to control. Importantly, a constitutively stabilized and pHi-insensitive β-catenin mutant (β-catenin-H36R), has a longer and pHi-independent half-life. We also determined that the pH-dependent stability of β-catenin affects both its adhesion and signaling functions. We show that the composition of AJs changes with pHi; at low pHi, E-cadherin-containing AJs are enriched in β-catenin while plakoglobin abundance is reduced. Conversely, when β-catenin is lost from E-cadherin-containing AJs at high pHi, plakoglobin is increased. We also found that cell area was reduced at low pHi and increased at high pHi compared to control while cell volume was unaffected, suggesting pHi alters cell-cell adhesion. Finally, we show that low pHi increases β-catenin transcriptional activity in single cells and is indistinguishable from a Wnt-on state, while high pHi reduces β-catenin transcriptional activity compared to control cells. This work characterizes pHi as a true rheostat regulating β-catenin abundance, stability, and function, solidifying β-catenin as a molecular mediator of pHi-dependent cell processes via pH-dependent adhesion and signaling functions.