Defective internal allosteric network imparts dysfunctional ATP/substrate-binding cooperativity in oncogenic chimera of protein kinase A

Olivieri, Cristina; Walker, Caitlin; Karamafrooz, Adak; Wang, Yingjie; Manu, V.S.; Porcelli, Fernando; Blumenthal, Donald K.; Thomas, David D.; Bernlohr, David A.; Simon, Sanford M.; Taylor, Susan S.; Veglia, Gianluigi

doi:10.1038/s42003-021-01819-6

Cited by 26 publications

(38 citation statements)

References 65 publications

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“…These differences suggest some intrinsic distinctions in substrate recognition of J-PKA ca vs WT PKA ca for example, this may indicate that J-PKA ca substrate recognition is less reliant on the N-terminal basic amino acid motif, with the C-terminal motif being sufficient for phosphorylation by the mutant. It could also mean that the J-domain alters catalytic dynamics in a way that depends more on the C-terminal motif and leads to increased turnover for more substrates, which may not be reflected in the K m or V max values previously characterized using a limited set of substrates that all contained the N-terminal basic motif. , …”

Section: Resultsmentioning

confidence: 99%

Integrated Phosphoproteomics for Identifying Substrates of Human Protein Kinase A (PRKACA) and Its Oncogenic Mutant DNAJB1–PRKACA

Karamafrooz

Brennan²,

Thomas

et al. 2021

J. Proteome Res.

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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.

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Section: Resultsmentioning

confidence: 99%

Integrated Phosphoproteomics for Identifying Substrates of Human Protein Kinase A (PRKACA) and Its Oncogenic Mutant DNAJB1–PRKACA

Karamafrooz

Brennan²,

Thomas

et al. 2021

J. Proteome Res.

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“…The CSPs was calculated using the following equation 94 : Δ = %&' − )*+,-= . (Δ ) 2 + (0.154 × Δ ) 2 The PKIa(5-24) CSPs were originally published earlier 90 and are included here for reference. Purification of SMO pCT for PKA-C activity assays.…”

Section: Methodsmentioning

confidence: 99%

“…NMR spectroscopy. Recombinant human PKA-Ca (PRKACA, uniprot P17612) was expressed and purified as reported 89,90 . Briefly, transformed E.coli BL21 (DE3) pLysS (Agilent) cells were cultured in deuterated ( 2 H) M9 minimal medium supplemented with 15 NH4Cl.…”

Section: Methodsmentioning

confidence: 99%

A PKA Inhibitor Motif within Smoothened Controls Hedgehog Signal Transduction

Happ

Arveseth

Bruystens³

et al. 2021

Preprint

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The Hedgehog (Hh) cascade is central to development, tissue homeostasis, and cancer. A pivotal step in Hh signal transduction is the activation of GLI transcription factors by the atypical G protein-coupled receptor (GPCR) Smoothened (SMO). How SMO activates GLI has remained unclear for decades. Here we show that SMO employs a decoy substrate sequence to physically block the active site of the PKA catalytic subunit (PKA-C) and extinguish its enzymatic activity. As a result, GLI is released from phosphorylation-induced inhibition. Using a combination of in vitro, cellular, and organismal models, we demonstrate that interfering with SMO / PKA pseudosubstrate interactions prevents Hh signal transduction. The mechanism we uncovered echoes one utilized by the Wnt cascade, revealing an unexpected similarity in how these two essential developmental and cancer pathways signal intracellularly. More broadly, our findings define a new mode of GPCR-PKA communication that may be harnessed by a range of membrane receptors and kinases.

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“…Through its regulation of transcriptional elements, the PKA signaling pathway is involved in liver metabolism (23). The chimeric kinase resulting from the DNAJB1-PRKACA fusion retains the kinase activity of PRKACA (15,24). The DNAJB1-PRKACA fusion protein is expressed at a higher level than wild-type (WT) PRKACA (15,25), and it has been postulated that these higher levels lead to increased cAMPindependent PKA signaling (26).…”

Section: Fibrolamellar Carcinoma (Flc) Represents <1% Of All Liver Cancers In the Unitedmentioning

confidence: 99%

Evaluation of PRKACA as a Therapeutic Target for Fibrolamellar Carcinoma

Schalm

O’Hearn

Wilson

et al. 2022

Preprint

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Background & Aims: Fibrolamellar carcinoma (FLC) is a rare, difficult-to-treat liver cancer primarily affecting pediatric and adolescent patients, and for which precision medicine approaches have historically not been possible. The DNAJB1-PRKACA gene fusion was identified as a driver of FLC pathogenesis. We aimed to assess whether FLC tumors maintain dependency on this gene fusion and determine if PRKACA is a viable therapeutic target. Methods: FLC patient-derived xenograft (PDX) shRNA cell lines were implanted subcutaneously into female NOD-SCID mice and tumors were allowed to develop prior to randomization to doxycycline (to induce knockdown) or control groups. Tumor development was assessed every 2 days. To assess the effect of treatment with novel selective PRKACA small molecule kinase inhibitors, BLU0588 and BLU2864, FLC PDX tumor cells were implanted subcutaneously into NOD-SCID mice and tumors allowed to develop. Mice were randomized to treatment (BLU0588 and BLU2864, orally, once daily) or control groups and tumor size determined as above. Results: Knockdown of DNAJB1-PRKACA reversed a FLC-specific gene signature and reduced PDX tumor growth in mice compared to the control group. Furthermore, FLC PDX tumor growth was significantly reduced with BLU0588 and BLU2864 treatment versus control (P = 0.003 and P = 0.0005, respectively). Conclusions: We demonstrated, using an inducible knockdown and small molecule approaches, that FLC PDX tumors were dependent upon DNAJB1-PRKACA fusion activity. In addition, this study serves as a proof-of-concept that PRKACA is a viable therapeutic target for FLC and warrants further investigation.

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Defective internal allosteric network imparts dysfunctional ATP/substrate-binding cooperativity in oncogenic chimera of protein kinase A

Cited by 26 publications

References 65 publications

Integrated Phosphoproteomics for Identifying Substrates of Human Protein Kinase A (PRKACA) and Its Oncogenic Mutant DNAJB1–PRKACA

Integrated Phosphoproteomics for Identifying Substrates of Human Protein Kinase A (PRKACA) and Its Oncogenic Mutant DNAJB1–PRKACA

A PKA Inhibitor Motif within Smoothened Controls Hedgehog Signal Transduction

Evaluation of PRKACA as a Therapeutic Target for Fibrolamellar Carcinoma

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