Accurate and quantitative methods for measuring the dynamic fluctuations of
protein kinase activities are critically needed as diagnostic tools and for the evaluation of
kinase-targeted inhibitors, which represent a major therapeutic development area in the treatment of
cancer and other diseases. In particular, rapid and economical methods that utilize simple instrumentation and provide quantitative data in a high throughput format will have the most impact on basic research in systems biology and medicine. There are over 500
protein kinases in the human kinome. Among these, the
mitogen activated
protein (MAP)
kinases are recognized to be central players in key cellular signaling events and are associated with essential processes including growth, proliferation, differentiation, migration, and apoptosis. The major challenge with MAP
kinase sensor development is achieving high selectivity since these
kinases rely acutely on secondary interactions distal to the phosphorylation site to impart substrate specificity. Herein we describe the development and application of selective sensors for three MAP
kinase subfamilies, ERK1/2, p38α/β, and JNK1/2/3. The new sensors are based on a modular design, which includes a sensing
element that exploits a sulfonamido-
oxine (Sox) fluorophore for reporting phosphorylation, a recognition and specificity
element based on reported docking domain motifs and a variable linker, which can be engineered to optimize the intermodule distance and relative orientation. Following rigorous validation, the capabilities of the new sensors are exemplified through the quantitative analysis of the target MAP
kinases in
breast cancer progression in a cell culture model, which reveals a strong correlation between p38α/β activity and increased tumorgenicity.