Latent agonist activity of the steroid antagonist, RU486, is unmasked in cells treated with activators of protein kinase A

Protein kinase A (PKA) holoenzyme, comprised of a cAMP-binding regulatory (R)-subunit dimer and 2 catalytic (C)-subunits, is the master switch for cAMP-mediated signaling. Of the 4 R-subunits (RIα, RIβ, RIIα, RIIβ), RIα is most essential for regulating PKA activity in cells. Our 2 RIα2C2 holoenzyme states, which show different conformations with and without ATP, reveal how ATP/Mg2+ functions as a negative orthosteric modulator. Biochemical studies demonstrate how the removal of ATP primes the holoenzyme for cAMP-mediated activation. The opposing competition between ATP/cAMP is unique to RIα. In RIIβ, ATP serves as a substrate and facilitates cAMP-activation. The isoform-specific RI-holoenzyme dimer interface mediated by N3A–N3A′ motifs defines multidomain cross-talk and an allosteric network that creates competing roles for ATP and cAMP. Comparisons to the RIIβ holoenzyme demonstrate isoform-specific holoenzyme interfaces and highlights distinct allosteric mechanisms for activation in addition to the structural diversity of the isoforms.

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Protein kinase A negatively regulates VEGF-induced AMPK activation by phosphorylating CaMKK2 at serine 495

Biochemical Journal ◽

10.1042/bcj20200555 ◽

2020 ◽

Vol 477 (17) ◽

pp. 3453-3469 ◽

Cited By ~ 2

Author(s):

Katrin Spengler ◽

Darya Zibrova ◽

Angela Woods ◽

Christopher G. Langendorf ◽

John W. Scott ◽

...

Keyword(s):

Endothelial Cells ◽

Protein Kinase ◽

Protein Kinase A ◽

Ampk Activation ◽

Dependent Protein ◽

And Function ◽

Amp Activated Protein Kinase ◽

Camp Elevation ◽

Kinase A ◽

In Cells

Activation of AMP-activated protein kinase (AMPK) in endothelial cells by vascular endothelial growth factor (VEGF) via the Ca2+/calmodulin-dependent protein kinase kinase 2 (CaMKK2) represents a pro-angiogenic pathway, whose regulation and function is incompletely understood. This study investigates whether the VEGF/AMPK pathway is regulated by cAMP-mediated signalling. We show that cAMP elevation in endothelial cells by forskolin, an activator of the adenylate cyclase, and/or 3-isobutyl-1-methylxanthine (IBMX), an inhibitor of phosphodiesterases, triggers protein kinase A (PKA)-mediated phosphorylation of CaMKK2 (serine residues S495, S511) and AMPK (S487). Phosphorylation of CaMKK2 by PKA led to an inhibition of its activity as measured in CaMKK2 immunoprecipitates of forskolin/IBMX-treated cells. This inhibition was linked to phosphorylation of S495, since it was not seen in cells expressing a non-phosphorylatable CaMKK2 S495C mutant. Phosphorylation of S511 alone in these cells was not able to inhibit CaMKK2 activity. Moreover, phosphorylation of AMPK at S487 was not sufficient to inhibit VEGF-induced AMPK activation in cells, in which PKA-mediated CaMKK2 inhibition was prevented by expression of the CaMKK2 S495C mutant. cAMP elevation in endothelial cells reduced basal and VEGF-induced acetyl-CoA carboxylase (ACC) phosphorylation at S79 even if AMPK was not inhibited. Together, this study reveals a novel regulatory mechanism of VEGF-induced AMPK activation by cAMP/PKA, which may explain, in part, inhibitory effects of PKA on angiogenic sprouting and play a role in balancing pro- and anti-angiogenic mechanisms in order to ensure functional angiogenesis.

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A tripartite cooperative mechanism confers resistance of the protein kinase A catalytic subunit to dephosphorylation

Journal of Biological Chemistry ◽

10.1074/jbc.ra119.010004 ◽

2020 ◽

Vol 295 (10) ◽

pp. 3316-3329

Author(s):

Tung O. Chan ◽

Roger S. Armen ◽

Santosh Yadav ◽

Sushrut Shah ◽

Jin Zhang ◽

...

Keyword(s):

Protein Kinase ◽

Protein Kinase A ◽

Catalytic Subunit ◽

Site Directed Mutagenesis ◽

Airway Smooth Muscle Cells ◽

Activation Loop ◽

Allosteric Sites ◽

And Migration ◽

Kinase A ◽

In Cells

Phosphorylation of specific residues in the activation loops of AGC kinase group (protein kinase A, G, and C families) is required for activity of most of these kinases, including the catalytic subunit of PKA (PKAc). Although many phosphorylated AGC kinases are sensitive to phosphatase-mediated dephosphorylation, the PKAc activation loop uniquely resists dephosphorylation, rendering it “constitutively” phosphorylated in cells. Previous biophysical experiments and structural modeling have suggested that the N-terminal myristoylation signal and the C-terminal FXXF motif in PKAc regulate its thermal stability and catalysis. Here, using site-directed mutagenesis, molecular modeling, and in cell-free and cell-based systems, we demonstrate that substitutions of either the PKAc myristoylation signal or the FXXF motif only modestly reduce phosphorylation and fail to affect PKAc function in cells. However, we observed that these two sites cooperate with an N-terminal FXXW motif to cooperatively establish phosphatase resistance of PKAc while not affecting kinase-dependent phosphorylation of the activation loop. We noted that this tripartite cooperative mechanism of phosphatase resistance is functionally relevant, as demonstrated by changes in morphology, adhesion, and migration of human airway smooth muscle cells transfected with PKAc variants containing amino acid substitutions in these three sites. These findings establish that three allosteric sites located at the PKAc N and C termini coordinately regulate the phosphatase sensitivity of this enzyme. This cooperative mechanism of phosphatase resistance of AGC kinase opens new perspectives toward therapeutic manipulation of kinase signaling in disease.

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Protein Kinase A Regulates MYC Protein through Transcriptional and Post-translational Mechanisms in a Catalytic Subunit Isoform-specific Manner

Journal of Biological Chemistry ◽

10.1074/jbc.m112.432377 ◽

2013 ◽

Vol 288 (20) ◽

pp. 14158-14169 ◽

Cited By ~ 24

Author(s):

Achuth Padmanabhan ◽

Xiang Li ◽

Charles J. Bieberich

Keyword(s):

Protein Kinase ◽

Protein Kinase A ◽

Term Effect ◽

Catalytic Subunit ◽

Short Term ◽

Catalytic Subunits ◽

Multiple Substrates ◽

Pka Catalytic Subunit ◽

Kinase A ◽

In Cells

MYC levels are tightly regulated in cells, and deregulation is associated with many cancers. In this report, we describe the existence of a MYC-protein kinase A (PKA)-polo-like kinase 1 (PLK1) signaling loop in cells. We report that sequential MYC phosphorylation by PKA and PLK1 protects MYC from proteasome-mediated degradation. Interestingly, short term pan-PKA inhibition diminishes MYC level, whereas prolonged PKA catalytic subunit α (PKACα) knockdown, but not PKA catalytic subunit β (PKACβ) knockdown, increases MYC. We show that the short term effect of pan-PKA inhibition on MYC is post-translational and the PKACα-specific long term effect on MYC is transcriptional. These data also reveal distinct functional roles among PKA catalytic isoforms in MYC regulation. We attribute this effect to differential phosphorylation selectivity among PKA catalytic subunits, which we demonstrate for multiple substrates. Further, we also show that MYC up-regulates PKACβ, transcriptionally forming a proximate positive feedback loop. These results establish PKA as a regulator of MYC and highlight the distinct biological roles of the different PKA catalytic subunits.

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Cyclic AMP-Independent Regulation of Protein Kinase A Substrate Phosphorylation by Kelch Repeat Proteins

Eukaryotic Cell ◽

10.1128/ec.4.11.1794-1800.2005 ◽

2005 ◽

Vol 4 (11) ◽

pp. 1794-1800 ◽

Cited By ~ 32

Author(s):

Ailan Lu ◽

Jeanne P. Hirsch

Keyword(s):

Protein Kinase ◽

Cyclic Amp ◽

Protein Kinase A ◽

Intracellular Camp ◽

Invasive Growth ◽

Camp Phosphodiesterase ◽

Α Subunit ◽

Substrate Phosphorylation ◽

Kinase A ◽

In Cells

ABSTRACT Pseudohyphal and invasive growth in the yeast Saccharomyces cerevisiae is regulated by the kelch repeat-containing proteins Gpb1p and Gpb2p, which act downstream of the G protein α-subunit Gpa2p. Here we show that deletion of GPB1 and GPB2 causes increased haploid invasive growth in cells containing any one of the three protein kinase A (PKA) catalytic subunits, suggesting that Gpb1p and Gpb2p are able to inhibit each of these kinases. Cells containing gpb1Δ gpb2Δ mutations also display increased phosphorylation of the PKA substrates Sfl1p and Msn2p, indicating that Gpb1p and Gpb2p are negative regulators of PKA substrate phosphorylation. Stimulation of PKA-dependent signaling by gpb1Δ gpb2Δ mutations occurs in cells that lack both adenylyl cyclase and the high-affinity cyclic AMP (cAMP) phosphodiesterase. This effect is also seen in cells that lack the low-affinity cAMP phosphodiesterase. Given that these three enzymes control the synthesis and degradation of cAMP, these results indicate that the effect of Gpb1p and Gpb2p on PKA substrate phosphorylation does not occur by regulating the intracellular cAMP concentration. These findings suggest that Gpb1p and Gpb2p mediate their effects on the cAMP/PKA signaling pathway either by inhibiting the activity of PKA in a cAMP-independent manner or by activating phosphatases that act on PKA substrates.

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Phosphorylation of protein kinase A (PKA) regulatory subunit RIα by protein kinase G (PKG) primes PKA for catalytic activity in cells

Journal of Biological Chemistry ◽

10.1074/jbc.m117.809988 ◽

2018 ◽

Vol 293 (12) ◽

pp. 4411-4421 ◽

Cited By ~ 9

Author(s):

Kristofer J. Haushalter ◽

Darren E. Casteel ◽

Andrea Raffeiner ◽

Eduard Stefan ◽

Hemal H. Patel ◽

...

Keyword(s):

Catalytic Activity ◽

Protein Kinase ◽

Protein Kinase A ◽

Protein Kinase G ◽

Regulatory Subunit ◽

Pka Regulatory Subunit ◽

Kinase A ◽

In Cells

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Synthetic design of farnesyl-electrostatic peptides for development of a protein kinase A membrane translocation switch

Scientific Reports ◽

10.1038/s41598-021-95840-8 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Allen K. Kim ◽

Helen D. Wu ◽

Takanari Inoue

Keyword(s):

Protein Kinase ◽

Protein Kinase A ◽

Single Molecule ◽

Plasma Membranes ◽

Modular Design ◽

Molecular Switch ◽

Dependent Manner ◽

Sensors And Actuators ◽

Kinase A ◽

In Cells

AbstractMolecular switches that respond to a biochemical stimulus in cells have proven utility as a foundation for developing molecular sensors and actuators that could be used to address important biological questions. Developing a molecular switch unfortunately remains difficult as it requires elaborate coordination of sensing and actuation mechanisms built into a single molecule. Here, we rationally designed a molecular switch that changes its subcellular localization in response to an intended stimulus such as an activator of protein kinase A (PKA). By arranging the sequence for Kemptide in tandem, we designed a farnesylated peptide whose localization can dramatically change upon phosphorylation by PKA. After testing a different valence number of Kemptide as well as modulating the linker sequence connecting them, we identified an efficient peptide switch that exhibited dynamic translocation between plasma membranes and internal endomembranes in a PKA activity dependent manner. Due to the modular design and small size, our PKA switch can have versatile utility in future studies as a platform for visualizing and perturbing signal transduction pathways, as well as for performing synthetic operations in cells.

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