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Lessons Learned from PKA: from Motifs to the Dynamic Assembly of Isoform‐specific Macromolecular Switches

Twenty‐five years after the first protein kinase structure, we are still learning about the fundamental properties that define this large enzyme family, and cAMP‐dependent protein kinase (PKA) continues to serve as a prototype for the superfamily. Each protein kinase has evolved to be a highly regulated and dynamic molecular switch. For most kinases the assembly of the active kinase is dynamically and transiently regulated by the alignment of a hydrophobic regulatory spine. In the case of PKA, however, the active catalytic (C) subunit is then packaged as an inactive R2C2 holoenzyme with dimeric regulatory (R) subunits that are receptors for cAMP. PKA thus combines two of the major regulatory mechanisms in biology, protein phosphorylation and cAMP signaling. Specificity in PKA signaling is achieved in large part by four functionally non‐redundant R‐subunits (RIa, RIb, RIIa, and RIIb), and targeting of these holoenzymes to specific sites in the cell creates yet another layer of specificity. The functional non‐redundancy of the R‐subunits is reflected in the structural diversity of the holoenzymes. Many diseases result from dysfunctional PKA signaling, and these diseases also reflect the diversity and unique functions of the R‐subunit isoforms. While we have learned much from the individual subunits, it was not until we solved the structure of the full length R2C2 holoenzyme that we could appreciate allostery and also why the four holoenzymes were functionally so different. Although each R‐subunit has the same domain organization that includes an N‐terminal dimerization/docking (D/D) domain and two tandem cAMP binding (CNB) domains, the quaternary organization of each holoenzyme is distinct due in large part to the intrinsically disordered linker that joins the D/D and CNB domains. Embedded in the linker is an inhibitor site that docks into the active site of the C‐subunit in the holoenzyme rendering it inactive, and these linkers in large part drive the conformational state of each holoenzyme. Our major challenge now is to understand how the flexible linkers drive the assembly and regulation of the holoenzyme. Capturing this dynamic space is a major challenge for PKA signaling and indeed a fundamental challenge for all protein kinases. Recognizing this critical roadblock, we are now using cryo Electron Microscopy (cryoEM) in parallel with crystallography and SAXS/SANS to explore the domain dynamics that contribute to PKA signaling.