Isoform-specific PKA holoenzymes differ in structure, function and allosteric regulation
- Author(s): Lu, Tsanwen
- Advisor(s): Taylor, Susan S
- et al.
Protein Kinase A (PKA) is the master switch for cAMP-mediated signaling. The inactive PKA holoenzymes (R2C2) are comprised of a cAMP-binding regulatory (R)-subunit dimer and 2 catalytic (C)-subunits, while cAMP binding to the R-subunits unleashes the activity of the C-subunits. Of the 4 functionally non-redundant R-subunits (RIα, RIβ, RIIα, RIIβ), RIα and RIβ contain PKA pseudo-substrate sites, in contrast to RIIα and RIIβ, which have substrate sites in their inhibitor sequences.
One of my main projects was to study the isoform-specific features of the PKA holoenzymes. I solved the crystal structure of the full-length RIα holoenzyme in two conformations and identified a novel isoform-specific function. The two conformations differ by their ATP-dependency. ATP also facilitates RIα holoenzyme formation by locking it into an inactive state that becomes more resistant to cAMP. ATP has no effect on the RIIβ holoenzyme conformation; instead it is a substrate that phosphorylates the inhibitor sequence in RIIβ. When the RIIβ holoenzyme is phosphorylated, it is easier to active with cAMP. The structures, combined with functional and biochemical data, reveal distinct isoform-specific quaternary structures and allosteric mechanisms. Both the in vitro and in vivo studies reveal that elevating ATP levels could turn on RIIα/RIIβ-related signaling pathways, while RIα/RIβ -related signaling would be triggered by depressing the levels of ATP.
Another part of my research focused on a fusion oncogene of the C-subunit, DnaJB1-PKAc (J-C) that drives fibrolamellar hepatocellular carcinoma (FL-HCC). DnaJB1-PKAc holoenzymes formed with RIα and RIIβ reveal different PKA dysfunctions. The crystal structure and biochemical properties of the RIα2J-C2 holoenzyme are similar to the wild-type holoenzyme. However, this mutant can disrupt RIα holoenzyme localization and cAMP signaling compartmentation. The RIIβ2J-C2 cryoEM structure is similar to the wild-type holoenzyme, whereas MD simulations and biochemical studies reveal that J-C can alter RIIb dynamics and make the RIIβ holoenzyme easier to be activated by cAMP.
My research highlights distinct PKA isoform-specific allostery in addition to the structural diversity of the isoforms. I also demonstrated that the J-C affects PKA signaling in isoform-specific ways. My studies not only shed light on our mechanistic understanding of PKA signaling, but also provides new insights for future therapeutic directions. How to target different isoforms of J-C holoenzymes and develop unique treatments for different holoenzyme isoforms will be future challenges and directions for curing FL-HCC.