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.

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.

scholarly journals Activity, expression and function of a second Drosophila protein kinase A catalytic subunit gene.

Genetics ◽  
1995 ◽  
Vol 141 (4) ◽  
pp. 1507-1520 ◽  
Author(s):  
A Meléndez ◽  
W Li ◽  
D Kalderon
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Essential Gene ◽  
Sequence Similarity ◽  
Catalytic Subunit ◽  
Subunit Gene ◽  
Drosophila Protein ◽  
Pka Catalytic Subunit ◽  
Kinase A

Abstract The DC2 gene was isolated previously on the basis of sequence similarity to DC0, the major Drosophila protein kinase A (PKA) catalytic subunit gene. We show here that the 67-kD Drosophila DC2 protein behaves as a PKA catalytic subunit in vitro. DC2 is transcribed in mesodermal anlagen of early embryos. This expression depends on dorsal but on neither twist nor snail activity. DC2 transcriptional fusions mimic this embryonic expression and are also expressed in subsets of cells in the optic lamina, wing disc and leg discs of third instar larvae. A saturation screen of a small deficiency interval containing DC2 for recessive lethal mutations yielded no DC2 alleles. We therefore isolated new deficiencies to generate deficiency trans-heterozygotes that lacked DC2 activity. These animals were viable and fertile. The absence of DC2 did not affect the viability or phenotype of imaginal disc cells lacking DC0 activity or embryonic hatching of animals with reduced DC0 activity. Furthermore, transgenes expressing DC2 from a DC0 promoter did not efficiently rescue a variety of DC0 mutant phenotypes. These observations indicate that DC2 is not an essential gene and is unlikely to be functionally redundant with DC0, which has multiple unique functions during development.

scholarly journals Protein Kinase A-Mediated Septin7 Phosphorylation Disrupts Septin Filaments and Ciliogenesis

Cells ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 361
Author(s):  
Han-Yu Wang ◽  
Chun-Hsiang Lin ◽  
Yi-Ru Shen ◽  
Ting-Yu Chen ◽  
Chia-Yih Wang ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Primary Cilia ◽  
Cell Growth And Migration ◽  
Filament Dynamics ◽  
Gtp Binding ◽  
Cilia Formation ◽  
And Migration ◽  
Septin Filament ◽  
Kinase A

Septins are GTP-binding proteins that form heteromeric filaments for proper cell growth and migration. Among the septins, septin7 (SEPT7) is an important component of all septin filaments. Here we show that protein kinase A (PKA) phosphorylates SEPT7 at Thr197, thus disrupting septin filament dynamics and ciliogenesis. The Thr197 residue of SEPT7, a PKA phosphorylating site, was conserved among different species. Treatment with cAMP or overexpression of PKA catalytic subunit (PKACA2) induced SEPT7 phosphorylation, followed by disruption of septin filament formation. Constitutive phosphorylation of SEPT7 at Thr197 reduced SEPT7‒SEPT7 interaction, but did not affect SEPT7‒SEPT6‒SEPT2 or SEPT4 interaction. Moreover, we noted that SEPT7 interacted with PKACA2 via its GTP-binding domain. Furthermore, PKA-mediated SEPT7 phosphorylation disrupted primary cilia formation. Thus, our data uncover the novel biological function of SEPT7 phosphorylation in septin filament polymerization and primary cilia formation.

scholarly journals Protein Kinase A Catalytic Subunit Primed for Action: Time-Lapse Crystallography of Michaelis Complex Formation

Structure ◽  
2015 ◽  
Vol 23 (12) ◽  
pp. 2331-2340 ◽  
Author(s):  
Amit Das ◽  
Oksana Gerlits ◽  
Jerry M. Parks ◽  
Paul Langan ◽  
Andrey Kovalevsky ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Complex Formation ◽  
Protein Kinase A ◽  
Time Lapse ◽  
Catalytic Subunit ◽  
Action Time ◽  
Kinase A

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 Meprin-β activity modulates the β-catalytic subunit of protein kinase A in ischemia-reperfusion-induced acute kidney injury

2020 ◽  
Vol 318 (5) ◽  
pp. F1147-F1159
Author(s):  
Faihaa Ahmed ◽  
Jean-Marie Mwiza ◽  
Mizpha Fernander ◽  
Ismaila Yahaya ◽  
Shaymaa Abousaad ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Signaling Pathway ◽  
Kinase Activity ◽  
Ischemia Reperfusion ◽  
Fluorescent Microscopy ◽  
Kidney Tissue ◽  
Kidney Injury ◽  
Catalytic Subunit ◽  
Kinase A

Meprin metalloproteases have been implicated in the progression of kidney injury. Previous work from our group has shown that meprins proteolytically process the catalytic subunit of protein kinase A (PKA-C), resulting in decreased PKA-C kinase activity. The goal of the present study was to determine the PKA-C isoforms impacted by meprin-β and whether meprin-β expression affects downstream mediators of the PKA signaling pathway in ischemia-reperfusion (IR)-induced kidney injury. IR was induced in 12-wk-old male wild-type (WT) and meprin-β knockout (βKO) mice. Madin-Darby canine kidney cells transfected with meprin-β cDNA were also subjected to 2 h of hypoxia. Western blot analysis was used to evaluate levels of total PKA-C, PKA-Cα, PKA-Cβ, phosphorylated (p-)PKA-C, and p-ERK1/2. Meprin-β expression enhanced kidney injury as indicated by levels of neutrophil gelatinase-associated lipocalin and cystatin C. IR-associated decreases were observed in levels of p-PKA-C in kidney tissue from WT mice but not βKO mice, suggesting that meprin-β expression/activity is responsible for the in vivo reduction in kinase activity. Significant increases in levels of PKA-Cβ were observed in kidney lysates for WT mice but not βKO mice at 6 h post-IR. Proximal tubule PKA-Cβ increases in WT but not βKO kidneys were demonstrated by fluorescent microscopy. Furthermore, IR-induced injury was associated with significant increases in p-ERK levels for both genotypes. The present data demonstrate that meprin-β enhances IR-induced kidney injury in part by modulating mediators of the PKA-Cβ signaling pathway.

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