Cellular decision-making, especially metabolism, is precisely regulated by protein kinases such as protein kinase A (PKA). PKA is a holoenzyme composed of two regulatory subunits (PKA-R) and two catalytic subunits (PKA-C). Classical biochemical studies have established that cAMP binding promotes release of PKA- C from PKA-R, but these approaches lacked the spatiotemporal resolution necessary to resolve molecular heterogeneity across the subcellular landscape. Emerging evidence suggests that dissociation may be incomplete, with a weak post-activation association persisting between PKA-R and PKA-C. However, the existence of partially dissociated states and their role in regulating signaling dynamics is unknown. Here, we present our progress on studying local nanoscale PKA signaling events using emerging quantitative super resolution imaging strategies, allowing us to directly observe individual molecular signaling events in well-defined biochemical and native cellular contexts. We accomplish this by incorporating fluorescently tagged PKA subunits into an in vitro reconstitution system and endogenous knock-in cell lines. These tools enable direct visualization of PKA dynamics at physiological concentrations. This work will provide the first direct visualization of single PKA molecules during activation, advancing our mechanistic understanding of PKA biology and ultimately defining how nanoscale dynamics and heterogeneous dissociation regulate signaling.
Liu et al. (Sun,) studied this question.