Phase Separation of a PKA Regulatory Subunit Controls cAMP Compartmentation and Oncogenic Signaling

Zhang, Jason Z.; Lu, Tsan Wen; Stolerman, Lucas M.; Tenner, Brian; Yang, Jessica R.; Zhang, Jin Fan; Falcke, Martin; Rangamani, Padmini; Taylor, Susan S.; Mehta, Sohum

doi:10.1016/j.cell.2020.07.043

Cited by 194 publications

(200 citation statements)

References 89 publications

Supporting

Mentioning

187

Contrasting

Unclassified

Order By: Relevance

“…The RIIβ holoenzyme formed with the fusion protein is easier to activate with cAMP than WT holoenzyme. This is in contrast to RIα where the PKAc and DnaJB1-P-KAc RIα holoenzymes show similar cAMP activation [11]; however, DnaJB1-PKAc can disrupt RIα localization and cAMP signaling compartmentation [54]. Each of these PKA signaling networks is highly regulated, and perturbation of that fine tuning can have profound consequences.…”

Section: Dnajb1-pkac Disrupts Pka Function In An Isoform-specific Mannermentioning

confidence: 94%

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

Aoto

Weng

et al. 2020

PLoS Biol

Self Cite

View full text Add to dashboard Cite

When the J-domain of the heat shock protein DnaJB1 is fused to the catalytic (C) subunit of cAMP-dependent protein kinase (PKA), replacing exon 1, this fusion protein, J-C subunit (J-C), becomes the driver of fibrolamellar hepatocellular carcinoma (FL-HCC). Here, we use cryo-electron microscopy (cryo-EM) to characterize J-C bound to RIIβ, the major PKA regulatory (R) subunit in liver, thus reporting the first cryo-EM structure of any PKA holoenzyme. We report several differences in both structure and dynamics that could not be captured by the conventional crystallography approaches used to obtain prior structures. Most striking is the asymmetry caused by the absence of the second cyclic nucleotide binding (CNB) domain and the J-domain in one of the RIIβ:J-C protomers. Using molecular dynamics (MD) simulations, we discovered that this asymmetry is already present in the wild-type (WT) RIIβ2C2 but had been masked in the previous crystal structure. This asymmetry may link to the intrinsic allosteric regulation of all PKA holoenzymes and could also explain why most disease mutations in PKA regulatory subunits are dominant negative. The cryo-EM structure, combined with small-angle X-ray scattering (SAXS), also allowed us to predict the general position of the Dimerization/Docking (D/D) domain, which is essential for localization and interacting with membrane-anchored A-Kinase-Anchoring Proteins (AKAPs). This position provides a multivalent mechanism for interaction of the RIIβ holoenzyme with membranes and would be perturbed in the oncogenic fusion protein. The J-domain also alters several biochemical properties of the RIIβ holoenzyme: It is easier to activate with cAMP, and the cooperativity is reduced. These results provide new insights into how the finely tuned allosteric PKA signaling network is disrupted by the oncogenic J-C subunit, ultimately leading to the development of FL-HCC.

show abstract

Section: Dnajb1-pkac Disrupts Pka Function In An Isoform-specific Mannermentioning

confidence: 94%

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

Aoto

Weng

et al. 2020

PLoS Biol

Self Cite

View full text Add to dashboard Cite

show abstract

“…However, only a limited number of cAMP-binding proteins have been identified and they are not expected to substantially buffer cAMP, at least globally within the cell. A recent study from the Zhang’s laboratory 8 provides an exciting solution to this long-standing problem by unveiling a novel mechanism through which cAMP can be compartmentalized within the cell.…”

mentioning

confidence: 99%

“…The RIα condensates contain a concentration of cAMP that is significantly higher than in the surrounding cytosol, thereby acting as a local buffer for the second messenger. 8 …”

mentioning

confidence: 99%

“…With these, they demonstrated that cAMP itself promotes LLPS of RIα, suggesting that the liquid condensates can form in response to cAMP-generating stimuli and provide a dynamic buffer for cAMP. 8 Using a cAMP reporter directly fused to the catalytic portion of PDE4D2, 10 they showed that in the presence of RIα condensates, effective degradation of stimulated cAMP takes place in the space immediately surrounding the hydrolytic moiety of PDE4D2, thus providing signal compartmentalization. In contrast, when cells were treated with 1,6-exanediol, an alcohol that disrupts liquid–liquid assemblies, or when RIα was knocked out, PDE4D2 was no longer able to cope with the increasing levels of cAMP and compartmentalization was lost.…”

mentioning

confidence: 99%

“…In contrast, when cells were treated with 1,6-exanediol, an alcohol that disrupts liquid–liquid assemblies, or when RIα was knocked out, PDE4D2 was no longer able to cope with the increasing levels of cAMP and compartmentalization was lost. 8 …”

mentioning

confidence: 99%

See 2 more Smart Citations

cAMP Buffering via Liquid–Liquid Phase Separation

Zaccolo

2020

Function

View full text Add to dashboard Cite

Emerging Implications of Phase Separation in Cancer

et al. 2022

View full text Add to dashboard Cite

In eukaryotic cells, biological activities are executed in distinct cellular compartments or organelles. Canonical organelles with membrane‐bound structures are well understood. Cells also inherently contain versatile membrane‐less organelles (MLOs) that feature liquid or gel‐like bodies. A biophysical process termed liquid–liquid phase separation (LLPS) elucidates how MLOs form through dynamic biomolecule assembly. LLPS‐related molecules often have multivalency, which is essential for low‐affinity inter‐ or intra‐molecule interactions to trigger phase separation. Accumulating evidence shows that LLPS concentrates and organizes desired molecules or segregates unneeded molecules in cells. Thus, MLOs have tunable functional specificity in response to environmental stimuli and metabolic processes. Aberrant LLPS is widely associated with several hallmarks of cancer, including sustained proliferative signaling, growth suppressor evasion, cell death resistance, telomere maintenance, DNA damage repair, etc. Insights into the molecular mechanisms of LLPS provide new insights into cancer therapeutics. Here, the current understanding of the emerging concepts of LLPS and its involvement in cancer are comprehensively reviewed.

show abstract

Phase Separation of a PKA Regulatory Subunit Controls cAMP Compartmentation and Oncogenic Signaling

Cited by 194 publications

References 89 publications

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

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

cAMP Buffering via Liquid–Liquid Phase Separation

Emerging Implications of Phase Separation in Cancer

Contact Info

Product

Resources

About