Structures of the PKA RIα Holoenzyme with the FLHCC Driver J-PKAcα or Wild-Type PKAcα

Abstract

Fibrolamellar hepatocellular carcinoma (FLHCC) is driven by J-PKAca, a kinase fusion chimera of the J domain of DnaJB1 with PKAca, the catalytic subunit of protein kinase A (PKA). Here we report the crystal structures of the chimeric fusion RIa 2 :J-PKAca 2 holoenzyme formed by J-PKAca and the PKA regulatory (R) subunit RIa, and the wild-type (WT) RIa 2 : PKAca 2 holoenzyme. The chimeric and WT RIa holoenzymes have quaternary structures different from the previously solved WT RIb and RIIb holoenzymes. The WT RIa holoenzyme showed the same configuration as the chimeric RIa 2 :J-PKAca 2 holoenzyme and a distinct second conformation. The J domains are positioned away from the symmetrical interface between the two RIa:J-PKAca heterodimers in the chimeric fusion holoenzyme and are highly dynamic. The structural and dynamic features of these holoenzymes enhance our understanding of the fusion chimera protein J-PKAca that drives FLHCC as well as the isoform specificity of PKA. ACKNOWLEDGMENTSWe thank Drs. Alexandr Kornev, Di Xia, and Kylie Walters for critical reading of the manuscript and helpful discussions.

Structural analyses of the PKA RIIβ holoenzyme containing the oncogenic DnaJB1-PKAc fusion protein reveal protomer asymmetry and fusion-induced allosteric perturbations in fibrolamellar hepatocellular carcinoma

“…Second is the cross communication between the CNB-B domain with the J-Domain, which is revealed in the cryo-EM structure. As seen in previous structures, the J-domain is very flexible [11,2]; however, in the RIIβ holoenzyme, we see that the flexibility of the J-domain also appear to influence the dynamic features of its adjacent CNB-B domain as well as the A-helix that it is fused to. The J-domain can also further influence the dynamic features of the CNB-B domains in the RIIβ holoenzyme, which we did not see in the RIα holoenzyme.…”

“…Biochemically, we show that the deletion of residues 1 to 14 (exon 1) in WT C subunit can introduce instability and can also affect holoenzyme function [49]. Our previous study also indicated that the J-C subunit is less thermostable than WT C subunit [11,12]. Several posttranslational modification sites in the WT C subunit are localized at the other end of the A-helix in exon 1, and these are missing in the fusion protein.…”

“…The activity of PKA can be stimulated by the elevated concentration of cAMP, through binding of cAMP to R subunits which unleashes the activity. Each of 4 functionally nonredundant R subunit isoforms (RIα, RIβ, RIIα, and RIIβ) has distinct quaternary holoenzyme structures, cAMP sensitivity, and cellular localization [7][8][9][10][11]. All 4 R subunit isoforms share a similar domain organization which consists of an N-terminal Dimerization/Docking (D/D) domain followed by an intrinsically disordered linker that connects to 2 tandem cyclic nucleotide binding (CNB) domains.…”

An acquired scaffolding function of the DNAJ-PKAc fusion contributes to oncogenic signaling in fibrolamellar carcinoma

“…Since aberrant kinase activity is known to drive many cancers, we further reasoned that augmented PKA activity could also contribute to the pathobiology of FLC (Druker et al, 2001; Turnham and Scott, 2016). However, one confounding factor is that the pathological DNAJ-PKAc fusion and its native kinase counterpart share similar sensitivities to the inhibitor PKI and efficiently bind R subunits to form type I and type II PKA holoenzymes (Cao et al, 2019; Cheung et al, 2015; Riggle et al, 2016a; Scott et al, 1985). Although physiochemically similar, notable differences between the PKA holoenzyme subtypes include lack of an autoregulatory phosphorylation site in RI isoforms, different in vitro binding affinities for cAMP and dispersal to distinct subcellular sites via interaction with distinct AKAPs (Aggarwal et al, 2019; Burgers et al, 2012; Feramisco et al, 1980; Means et al, 2011; Smith et al, 2018; Taylor et al, 2012).…”

Activation of PKA via asymmetric allosteric coupling of structurally conserved cyclic nucleotide binding domains

“…The allosteric networks we describe here may be amplified in the PKA hetero-tetramer composed of two regulatory and two catalytic subunits, where the potential cross-talk between PKA subunits is expanded 12,34,40 . Structural studies of the PKA hetero-tetramer formed with RI isoforms showed the N3A motif of one regulatory subunit stacked against the N3A motif of the neighboring one, forming a helical bundle with several hydrophobic interactions 41,42 . Therefore, it is possible that the dynamic switching response of the N3A motif described here may play additional allosteric regulatory roles by communicating the two regulatory subunits in the PKA hetero-tetramer via quaternary interactions.…”