Integrated Phosphoproteomics for Identifying Substrates of Human Protein Kinase A (PRKACA) and Its Oncogenic Mutant...

The DNAJB1-PRKACA fusion is the signature genetic event of fibrolamellar hepatocellular carcinoma (FL-HCC), a rare but lethal liver cancer that primarily affects adolescents and young adults. A deletion fuses the first exon of the HSP40 gene (DNAJB1), with exons 2-10 of protein kinase A (PRKACA), producing the chimeric kinase DNAJB1-PKA_ca (J-PKA_ca). The HSP40 portion's scaffolding/chaperone function has been implicated in redirecting substrate recognition to upregulate oncogenic pathways, but the direct substrates of this fusion are not fully known. We integrated cell-based and in vitro phosphoproteomics to identify substrates targeted directly by PKA and J-PKA_ca, comparing phosphoproteome profiles from cells with in vitro rephosphorylation of peptides and proteins from lysates using recombinant enzymes. We identified a subset of phosphorylation sites in both cell-based and in vitro experiments, as well as altered pathways and proteins consistent with observations from related studies. We also treated cells with PKA inhibitors that function by two different mechanisms (rpcAMPs and PKI) and examined phosphoproteome profiles, finding some substrates that persisted in the presence of inhibitors and revealing differences between WT and chimera. Overall, these results provide potential insights into J-PKA_ca's oncogenic activity in a complex cellular system and may provide candidate targets for therapeutic follow-up.