Lessons Learned from PKA: Assembly of Dynamic Macromolecular Switches

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ABSTRACT. My entire career, funded under the umbrella of NIGMS, has been guided by the principle that structure will provide an understanding of function with the ultimate goal being to elucidate how protein phos- phorylation regulates biology. My specific focus has been to solve structures of molecules that are associated with PKA signaling beginning with the crystal structure of the catalytic (C) subunit, which was the first protein kinase structure to be solved. While many functional insights have come from structures of the regulatory (R) and C-subunits and from R:C heterodimers, PKA signaling in cells is mediated by full-length R2C2 holoenzy- mes that are targeted, typically through A Kinase Anchoring Proteins (AKAPs), to discreet sites in the cell near dedicated substrates. It is not possible to comprehensively understand PKA signaling in cells without having a detailed portrait of the targeted holoenzymes, and this includes not only the R:C domains which reveal so much about symmetry, catalysis and allostery but also the dynamic linkers and domains that evade classic crystallography. So much important biology is embedded in these linkers that drive the assembly, targeting and regulation of all kinases. Our recent work in solving structures and elucidating features of the full-length holo- enzymes shows how higher levels of complexity and specificity are achieved. It also revealed the remarkable structural and functional non-redundancy of the four PKA holoenzymes, which is so essential for achieving specificity. The major challenge now is to understand how flexible linkers drive the assembly and regulation of each holoenzyme. To meet this challenge we are building cryo electron microscopy (cryoEM) and eventually cryo electron tomography (cryoET) into our portfolio of techniques that we need as well as high-resolution mosaic imaging (HRMI) in tissues. With these tools in hand we expect to create a dynamic portrait of the RIIb and RIa holoenzymes as they toggle between their active and inactive states. To simultaneously enhance our understanding of disease we will focus on three diseases that are caused directly by mutant PKA subunits. FL- HCC is a rare childhood liver cancer that is driven by the fusion of the J domain of DNA-JB1 to the N-terminus of the PKA Ca subunit. Carney Complex Disease (CNC) and Acrodysostosis (ACRDYS) are endocrine dis- orders caused by mutations in RIa. We believe that holoenzymes formed with these mutants will drive our understanding of the wt proteins. In parallel we will do an HRMI profile of the liver and compare normal liver to tissues where FL-HCC is expressed. The ACRDYS and CNC mutants in RIa highlight the allosteric network that controls activation. For targeted PKA we will focus on two systems: the RIIb holoenzyme and calcineurin bound to AKAP79 and RIa bound to the newly discovered AKAP motif in the C-terminal tail of the cilia-specific GPCR, GPR161. With our exceptional team of collaborators we are poised to make rapid progress. Our long- term goal is to establish PKA as the prototypical kinase for demonstrating how polyvalent macromolecular signaling complexes are assembled and regulated and become dysfunctional as a consequence of disease.