Structures of the PKA RIα holoenzyme with the FLHCC driver J-PKAcα or wild type PKAcα

Abstract

1Fibrolamellar hepatocellular carcinoma (FLHCC) is driven by J-PKAcα, a kinase fusion chimera 2 2 of the J-domain of DnaJB1 with PKAcα, the catalytic subunit of Protein Kinase A (PKA). Here 2 3 we report the crystal structures of the chimeric fusion RIα 2 :J-PKAcα 2 holoenzyme formed by J-2 4 PKAcα and the PKA regulatory (R) subunit RIα, and the wild type (wt) RIα 2 :PKAcα 2 2 5 holoenzyme. The chimeric and wt RIα holoenzymes have quaternary structures different from 2 6 the previously solved wt RIβ and RIIβ holoenzymes. The chimeric holoenzyme shows an 2 7isoform-specific interface dominated by antiparallel interactions between the N3A-N3A' motifs 2 8of RIα that serves as an anchor for RIα structural rearrangements during cAMP activation. The 2 9wt RIα holoenzyme showed the same configuration as well as a distinct second conformation. In 3 0 the structure of the chimeric fusion RIα 2 :J-PKAcα 2 holoenzyme, the presence of the J-domain 3 1 does not prevent formation of the holoenzymes, and is positioned away from the symmetrical 3 2 interface between the two RIα:J-PKAcα heterodimers in the holoenzyme. The J-domains have 3 3 significantly higher temperature factors than the rest of the holoenzyme, implying a large degree 3 4 of conformational flexibility. Furthermore molecular dynamics simulations were applied to 3 5 analyze the conformational states of chimeric fusion and wt RIα holoenzymes, and showed an 3 6 ensemble of conformations in the majority of which the J-domain was dynamic and rotated away 3 7 from the R:J-PKAcα interface. Thus, rather than affecting the interactions with the regulatory 3 8 subunits, the fusion of the J-domain to the PKAcα alters the conformational landscape of the 3 9 chimeric fusion holoenzymes and potentially, as result, the interactions with other molecules.4 0The structural and dynamic features of these holoenzymes enhance our understanding of the 4 1 fusion chimera protein J-PKAcα that drives FLHCC as well as the isoform specificity of PKA. 4 2 4 3

Two PKA RIα holoenzyme states define ATP as an isoform-specific orthosteric inhibitor that competes with the allosteric activator, cAMP

“…3 H and I). Molecule A features an N3A-N3A′ interface similar to what was described earlier in the RIα homodimer (13), and this was also seen in a recent holoenzyme structure with a cancer-driving fusion protein of the C-subunit (45). Hydrophobic and hydrogen bonding interactions both contribute to the N3A-N3A′ interface.…”

Evaluation of PRKACA as a Therapeutic Target for Fibrolamellar Carcinoma

“…This study is the first to evaluate the therapeutic potential for PRKACA inhibition in FLC. We characterized and validated an FLC PDX mouse model ( 27 (40). In addition, primary and metastatic FLC were shown to be sensitive to clinically available inhibitors of topoisomerase 1 and histone deacetylases, and to napabucasin (41), suggesting these agents as potential therapeutic strategies for FLC.…”

“…PRKACA is expressed in virtually all tissues and plays a critical role in cardiac function, making a strategy to selectively inhibit DNAJB1-PRKACA while sparing WT PRKACA desirable. The alternative conformation of the holoenzyme containing the DNAJB1-PRKACA fusion protein and the PKA regulatory subunit may constitute a strategy for selectively targeting the activity of the DNAJB1-PRKACA fusion protein (40). In addition, primary and metastatic FLC were shown to be sensitive to clinically available inhibitors of topoisomerase 1 and histone deacetylases, and to napabucasin (41), suggesting these agents as potential therapeutic strategies for FLC.…”

Allosteric regulation of protein kinase a by nucleotides and metal ions

“…From the stability essays, ATP binding assays and the studies of the kinetics, the fusion tetramer did not differ significantly from the wild-type tetramer. The addition of the J domain might create more interaction surfaces with other proteins 75 .…”