Although cancer is a diverse set of diseases, cancer cells share characteristics distinct from normal cells, including sustained proliferative signaling, resistance to cell death, and active invasion and metastasis. While less recognized, dysregulated pH is also a distinct feature of most cancers, regardless of tissue origin or genetic background. Compared with normal adult cells, cancer cells have a higher intracellular pH (pHi) of ~ 7.6 and a lower extracellular pH of ~ 7.1. Constitutively increased pHi promotes cell proliferation, limits apoptosis, and is obligatory for efficient directed cell migration. However, how a higher pHi enables disease progression and the molecular mechanisms mediating pHi-dependent cancer cell behaviors are understudied and mostly not understood. One challenge is that increased pHi enables cancer progression through multiple mechanisms. A second challenge is that the molecular basis for pHi-dependent cell behaviors is difficult to determine because it is dependent on protonation/deprotonation of proteins, which cannot be resolved by mass spectrometry or antibodies. We are addressing both of these challenges by bridging protein structure dynamics and cell biology to determine the design principles and functions of pH sensors, defined as proteins with activities or ligand-binding affinities regulated by cellular changes in pH1. To characterize pH sensors we use biochemistry, cell biology, molecular dynamics simulations and NMR. Relevant to cancer, we determined the molecular basis for increased pHi promoting cell migration by revealing the structural dynamics and functional properties of the cytoskeleton-associated pH sensors cofilin2, talin3 and focal adhesion kinase4. We are currently identifying pH sensors for additional cancer cell behaviors, including tumorigenesis, metabolic reprogramming and retention of recurring charge-changing mutations. For tumorigenesis, we found that beta-catenin is a pH sensor, with higher pHi increasing beta-catenin degradation, which is mediated by deprotonation of a conserved histidine (His 36 in human beta-catenin) for increased binding of the E3 ligase beta-TrCP1. For metabolic reprogramming we are determining pH sensing by the glycolytic enzyme phosphofructokinase-1 (PFK1). Based on our recently resolved crystal structure of PFK15 we found that in the human muscle isoform, His242 is necessary for increased enzyme activity at higher pH. For charge-changing mutations, we found that Arg>His substitutions, which are enriched in cancers, can confer a gain in pH sensing that enables cancer behaviors that we confirmed for p53-R273H and EGFR-R776H6. These findings provide new insights on molecular mechanisms enabling cancer progression that can inform therapeutic approaches. Because constitutively increased pHi is a distinct, conserved feature of cancer cells, pH sensors hold promise as therapeutic targets to achieve efficacy with high specificity.