Protein kinase A (PKA) is a key regulator of cellular functions by selectively phosphorylating
numerous substrates, including ion channels, enzymes, and transcription factors. It
has long served as a model system for understanding the eukaryotic kinases. Using
cryoelectron microscopy, we present complex structures of the PKA catalytic subunit
(PKA-C) bound to a full-length protein substrate, the cystic fibrosis transmembrane
conductance regulator (CFTR)-an ion channel vital to human health. CFTR gating requires
phosphorylation of its regulatory (R) domain. Unphosphorylated CFTR engages PKA-C
at two locations, establishing two "catalytic stations" near to, but not directly
involving, the R domain. This configuration, coupled with the conformational flexibility
of the R domain, permits transient interactions of the eleven spatially separated
phosphorylation sites. Furthermore, we determined two structures of the open-pore
CFTR stabilized by PKA-C, providing a molecular basis to understand how PKA-C stimulates
CFTR currents even in the absence of phosphorylation.