Allosteric pluripotency as revealed by protein kinase A

The functional response of a signaling system to an allosteric stimulus often depends on subcellular conditions, a phenomenon known as pluripotent allostery. For example, a single allosteric modulator, Rp-cAMPS, of the prototypical protein kinase A (PKA) switches from antagonist to agonist depending on MgATP levels. However, the mechanism underlying such pluripotent allostery has remained elusive for decades. Using nuclear magnetic resonance spectroscopy, ensemble models, kinase assays, and molecular dynamics simulations, we show that allosteric pluripotency arises from surprisingly divergent responses of highly homologous tandem domains. The differential responses perturb domain-domain interactions and remodel the free-energy landscape of inhibitory excited states sampled by the regulatory subunit of PKA. The resulting activation threshold values are comparable to the effective free energy of regulatory and catalytic subunit binding, which depends on metabolites, substrates, and mutations, explaining pluripotent allostery and warranting a general redefinition of allosteric targets to include specific subcellular environments.

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Unidirectional allostery in the regulatory subunit RIα facilitates efficient deactivation of protein kinase A

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1610142113 ◽

2016 ◽

Vol 113 (44) ◽

pp. E6776-E6785 ◽

Cited By ~ 10

Author(s):

Cong Guo ◽

Huan-Xiang Zhou

Keyword(s):

Protein Kinase ◽

Protein Kinase A ◽

Structural Similarity ◽

Community Analysis ◽

Catalytic Subunit ◽

Regulatory Subunit ◽

Allosteric Communication ◽

Dynamics Simulations ◽

A Site ◽

Kinase A

The holoenzyme complex of protein kinase A is in an inactive state; activation involves ordered cAMP binding to two tandem domains of the regulatory subunit and release of the catalytic subunit. Deactivation has been less studied, during which the two cAMPs unbind from the regulatory subunit to allow association of the catalytic subunit to reform the holoenzyme complex. Unbinding of the cAMPs appears ordered as indicated by a large difference in unbinding rates from the two sites, but the cause has remained elusive given the structural similarity of the two tandem domains. Even more intriguingly, NMR data show that allosteric communication between the two domains is unidirectional. Here, we present a mechanism for the unidirectionality, developed from extensive molecular dynamics simulations of the tandem domains in different cAMP-bound forms. Disparate responses to cAMP releases from the two sites (A and B) in conformational flexibility and chemical shift perturbation confirmed unidirectional allosteric communication. Community analysis revealed that the A-site cAMP, by forming across-domain interactions, bridges an essential pathway for interdomain communication. The pathway is impaired when this cAMP is removed but remains intact when only the B-site cAMP is removed. Specifically, removal of the A-site cAMP leads to the separation of the two domains, creating room for binding the catalytic subunit. Moreover, the A-site cAMP, by maintaining interdomain coupling, retards the unbinding of the B-site cAMP and stalls an unproductive pathway of cAMP release. Our work expands the perspective on allostery and implicates functional importance for the directionality of allostery.

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Expression and mutation analysis of regulatory subunit Iα of protein kinase A in undifferentiated and anaplastic thyroid carcinoma

Experimental and Clinical Endocrinology & Diabetes ◽

10.1055/s-2004-819071 ◽

2004 ◽

Vol 112 (S 1) ◽

Author(s):

M Lippert ◽

SY Sheu ◽

K Worm ◽

M Wichert ◽

KW Schmid ◽

...

Keyword(s):

Protein Kinase ◽

Thyroid Carcinoma ◽

Protein Kinase A ◽

Mutation Analysis ◽

Anaplastic Thyroid Carcinoma ◽

Regulatory Subunit ◽

Kinase A

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Targeting the Regulatory Subunit R2Alpha of Protein Kinase A in Human Glioblastoma through shRNA-Expressing Lentiviral Vectors

Viruses ◽

10.3390/v13071361 ◽

2021 ◽

Vol 13 (7) ◽

pp. 1361

Author(s):

Maira Zorzan ◽

Claudia Del Vecchio ◽

Stefania Vogiatzis ◽

Elisa Saccon ◽

Cristina Parolin ◽

...

Keyword(s):

Protein Kinase ◽

Protein Kinase A ◽

Lentiviral Vectors ◽

Brain Parenchyma ◽

Regulatory Subunit ◽

Glioblastoma Cells ◽

Cellular Models ◽

Therapeutic Setting ◽

Promising Target ◽

Kinase A

Glioblastoma is the most malignant and most common form of brain tumor, still today associated with a poor 14-months median survival from diagnosis. Protein kinase A, particularly its regulatory subunit R2Alpha, presents a typical intracellular distribution in glioblastoma cells compared to the healthy brain parenchyma and this peculiarity might be exploited in a therapeutic setting. In the present study, a third-generation lentiviral system for delivery of shRNA targeting the regulatory subunit R2Alpha of protein kinase A was developed. Generated lentiviral vectors are able to induce an efficient and stable downregulation of R2Alpha in different cellular models, including non-stem and stem-like glioblastoma cells. In addition, our data suggest a potential correlation between silencing of the regulatory subunit of protein kinase A and reduced viability of tumor cells, apparently due to a reduction in replication rate. Thus, our findings support the role of protein kinase A as a promising target for novel anti-glioma therapies.

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The crystal structure of yeast regulatory subunit reveals key evolutionary insights into Protein Kinase A oligomerization

Journal of Structural Biology ◽

10.1016/j.jsb.2021.107732 ◽

2021 ◽

pp. 107732

Author(s):

Nicolás González Bardeci ◽

Enzo Tofolón ◽

Felipe Trajtenberg ◽

Julio Caramelo ◽

Nicole Larrieux ◽

...

Keyword(s):

Crystal Structure ◽

Protein Kinase ◽

Protein Kinase A ◽

Regulatory Subunit ◽

Kinase A

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Overexpression of the regulatory subunit of protein kinase A increases heterologous protein expression in Pichia pastoris

Biotechnology Letters ◽

10.1007/s10529-020-02977-z ◽

2020 ◽

Vol 42 (12) ◽

pp. 2685-2692 ◽

Cited By ~ 1

Author(s):

Xihao Liao ◽

Wenyang Lin ◽

Nanzhu Chen ◽

Lu Li ◽

Dafu Huang ◽

...

Keyword(s):

Protein Kinase ◽

Pichia Pastoris ◽

Protein Expression ◽

Protein Kinase A ◽

Heterologous Protein ◽

Regulatory Subunit ◽

Heterologous Protein Expression ◽

Kinase A

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The Role of Uncoupling Protein 1 in the Metabolism and Adiposity of RIIβ-Protein Kinase A-Deficient Mice

Molecular Endocrinology ◽

10.1210/me.2004-0194 ◽

2004 ◽

Vol 18 (9) ◽

pp. 2302-2311 ◽

Cited By ~ 21

Author(s):

Michael A. Nolan ◽

Maria A. Sikorski ◽

G. Stanley McKnight

Keyword(s):

Adipose Tissue ◽

Oxygen Consumption ◽

Protein Kinase ◽

Protein Kinase A ◽

Uncoupling Protein ◽

Mrna Level ◽

Regulatory Subunit ◽

Uncoupling Protein 1 ◽

Brown Adipose ◽

Kinase A

Abstract Mice lacking the RIIβ regulatory subunit of protein kinase A exhibit a 50% reduction in white adipose tissue stores compared with wild-type littermates and are resistant to diet-induced obesity. RIIβ−/− mice also have an increase in resting oxygen consumption along with a 4-fold increase in the brown adipose-specific mitochondrial uncoupling protein 1 (UCP1). In this study, we examined the basis for UCP1 induction and tested the hypothesis that the induced levels of UCP1 in RIIβ null mice are essential for the lean phenotype. The induction of UCP1 occurred at the protein but not the mRNA level and correlated with an increase in mitochondria in brown adipose tissue. Mice lacking both RIIβ and UCP1 (RIIβ−/−/Ucp1−/−) were created, and the key parameters of metabolism and body composition were studied. We discovered that RIIβ−/− mice exhibit nocturnal hyperactivity in addition to the increased oxygen consumption at rest. Disruption of UCP1 in RIIβ−/− mice reduced basal oxygen consumption but did not prevent the nocturnal hyperactivity. The double knockout animals also retained the lean phenotype of the RIIβ null mice, demonstrating that induction of UCP1 and increased resting oxygen consumption is not the cause of leanness in the RIIβ mutant mice.

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Complex effects of kinase localization revealed by compartment-specific regulation of protein kinase A activity

10.1101/2021.04.08.439038 ◽

2021 ◽

Author(s):

Rebecca LaCroix ◽

Benjamin Lin ◽

Andre Levchenko

Keyword(s):

Cell Migration ◽

Protein Kinase ◽

Protein Kinase A ◽

Kinase Activity ◽

Single Cells ◽

Regulatory Subunit ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

Specific Regulation ◽

Kinase A

SummaryKinase activity in signaling networks frequently depends on regulatory subunits that can both inhibit activity by interacting with the catalytic subunits and target the kinase to distinct molecular partners and subcellular compartments. Here, using a new synthetic molecular interaction system, we show that translocation of a regulatory subunit of the protein kinase A (PKA-R) to the plasma membrane has a paradoxical effect on the membrane kinase activity. It can both enhance it at lower translocation levels, even in the absence of signaling inputs, and inhibit it at higher translocation levels, suggesting its role as a linker that can both couple and decouple signaling processes in a concentration-dependent manner. We further demonstrate that superposition of gradients of PKA-R abundance across single cells can control the directionality of cell migration, reversing it at high enough input levels. Thus complex in vivo patterns of PKA-R localization can drive complex phenotypes, including cell migration.

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