scholarly journals Two PKA RIα holoenzyme states define ATP as an isoform-specific orthosteric inhibitor that competes with the allosteric activator, cAMP

2019 ◽  
Vol 116 (33) ◽  
pp. 16347-16356 ◽  
Author(s):  
Tsan-Wen Lu ◽  
Jian Wu ◽  
Phillip C. Aoto ◽  
Jui-Hung Weng ◽  
Lalima G. Ahuja ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Cross Talk ◽  
Structural Diversity ◽  
Dimer Interface ◽  
Biochemical Studies ◽  
Allosteric Mechanisms ◽  
Allosteric Activator ◽  
Kinase A ◽  
In Cells

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.

scholarly journals Integrin Cross-talk in Endothelial Cells Is Regulated by Protein Kinase A and Protein Phosphatase 1

2008 ◽  
Vol 283 (46) ◽  
pp. 31849-31860 ◽  
Author(s):  
Annette M. Gonzalez ◽  
Jessica Claiborne ◽  
Jonathan C. R. Jones
Keyword(s):  
Endothelial Cells ◽  
Protein Kinase ◽  
Protein Kinase A ◽  
Cross Talk ◽  
Protein Phosphatase ◽  
Protein Phosphatase 1 ◽  
Kinase A

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

2020 ◽  
Vol 477 (17) ◽  
pp. 3453-3469 ◽  
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.

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

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.

Cross-talk between protein kinase A and the MAPK-activated protein kinases RSK1 and MK5

2010 ◽  
Vol 31 (1) ◽  
pp. 1-9 ◽  
Author(s):  
Sergiy Kostenko ◽  
Alexey Shiryaev ◽  
Gianina Dumitriu ◽  
Nancy Gerits ◽  
Ugo Moens
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Protein Kinases ◽  
Cross Talk ◽  
Kinase A

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

2013 ◽  
Vol 288 (20) ◽  
pp. 14158-14169 ◽  
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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