scholarly journals Structural insights into inhibitor regulation of the DNA repair protein DNA-PKcs

Nature ◽  
2022 ◽  
Author(s):  
Shikang Liang ◽  
Sherine E. Thomas ◽  
Amanda K. Chaplin ◽  
Steven W. Hardwick ◽  
Dimitri Y. Chirgadze ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Inhibitory Effect ◽  
Rational Drug Design ◽  
Dna Double Strand Breaks ◽  
Mobility Shift ◽  
Dependent Protein Kinase ◽  
Drug Candidates ◽  
Nuclear Extracts ◽  
Dependent Protein ◽  
Mobility Shift Assay

AbstractThe DNA-dependent protein kinase catalytic subunit (DNA-PKcs) has a central role in non-homologous end joining, one of the two main pathways that detect and repair DNA double-strand breaks (DSBs) in humans1,2. DNA-PKcs is of great importance in repairing pathological DSBs, making DNA-PKcs inhibitors attractive therapeutic agents for cancer in combination with DSB-inducing radiotherapy and chemotherapy3. Many of the selective inhibitors of DNA-PKcs that have been developed exhibit potential as treatment for various cancers4. Here we report cryo-electron microscopy (cryo-EM) structures of human DNA-PKcs natively purified from HeLa cell nuclear extracts, in complex with adenosine-5′-(γ-thio)-triphosphate (ATPγS) and four inhibitors (wortmannin, NU7441, AZD7648 and M3814), including drug candidates undergoing clinical trials. The structures reveal molecular details of ATP binding at the active site before catalysis and provide insights into the modes of action and specificities of the competitive inhibitors. Of note, binding of the ligands causes movement of the PIKK regulatory domain (PRD), revealing a connection between the p-loop and PRD conformations. Electrophoretic mobility shift assay and cryo-EM studies on the DNA-dependent protein kinase holoenzyme further show that ligand binding does not have a negative allosteric or inhibitory effect on assembly of the holoenzyme complex and that inhibitors function through direct competition with ATP. Overall, the structures described in this study should greatly assist future efforts in rational drug design targeting DNA-PKcs, demonstrating the potential of cryo-EM in structure-guided drug development for large and challenging targets.

Protein kinase A acts at multiple points to inhibit Xenopus oocyte maturation

1994 ◽  
Vol 14 (7) ◽  
pp. 4419-4426
Author(s):  
W Matten ◽  
I Daar ◽  
G F Vande Woude
Keyword(s):  
Protein Kinase ◽  
Xenopus Oocytes ◽  
Negative Regulator ◽  
Mitogen Activated Protein Kinase ◽  
Mobility Shift ◽  
Dependent Protein Kinase ◽  
Multiple Points ◽  
Mitogen Activated Protein ◽  
Dependent Protein ◽  
Phosphatase Activation

In Xenopus oocytes, initiation of maturation is dependent on reduction of cyclic AMP-dependent protein kinase (PKA) activity and the synthesis of the mos proto-oncogene product. Mos is required during meiosis I for the activation of both maturation-promoting factor (MPF) and mitogen-activated protein kinase (MAPK). Here we show that injection of the catalytic subunit of PKA (PKAc) prevented progesterone-induced synthesis of endogenous Mos as well as downstream MPF and MAPK activation. However, PKAc did not prevent injected soluble Mos product from activating MAPK. While MAPK is activated during Mos-PKAc coinjection, attendant MPF activation is blocked. Additionally, PKAc caused a potent block in the electrophoretic mobility shift of cdc25 that is associated with phosphatase activation. This inhibition of cdc25 activity was not reversed by progesterone, Mos, or MPF. We conclude that PKAc acts as a negative regulator at several points in meiotic maturation by preventing both Mos translation and MPF activation.

Characterization of the proteins of the intestinal Na(+)-K(+)-2Cl- cotransporter

1994 ◽  
Vol 267 (2) ◽  
pp. C375-C384 ◽  
Author(s):  
W. Suvitayavat ◽  
P. B. Dunham ◽  
M. Haas ◽  
M. C. Rao
Keyword(s):  
Protein Kinase ◽  
Subcellular Fractionation ◽  
Inhibitory Effect ◽  
Polyclonal Antiserum ◽  
Calyculin A ◽  
Dependent Protein Kinase ◽  
Cyclic Monophosphate ◽  
Microsomal Membranes ◽  
Dependent Protein

Absorptive intestinal epithelia, such as that of the winter flounder, absorb salt via a bumetanide-sensitive Na(+)-K(+)-2Cl- cotransport mechanism on the brush-border membrane (BBM). The present study demonstrates the first molecular characterization of the intestinal Na(+)-K(+)-2Cl- cotransporter and its unique regulation. The photoaffinity bumetanide analogue, 4-[3H]benzoyl-5-sulfamoyl-3- (3-thenyloxy)benzoic acid, specifically labeled three groups of proteins in flounder intestinal microsomal membranes (MM): a approximately 180-kDa peptide, prominently labeled, and diffuse bands at approximately 110-70 and 50 kDa, less intensely labeled. Subcellular fractionation revealed a single prominently labeled protein of approximately 170 kDa in BBM but not in basolateral membranes (BLM) and little or no labeling of proteins of approximately 110-70 or 50 kDa. Polyclonal antiserum raised against the Ehrlich ascites cell cotransporter identified a 180-kDa peptide in MM and a 175-kDa peptide (pI approximately 5.4) in BBM but none in BLM or in the cytosol of flounder intestine. As predicted from the regulation of cotransport in this tissue, phosphorylation of this protein is increased by guanosine 3',5'-cyclic monophosphate (cGMP)-dependent but not by adenosine 3',5'-cyclic monophosphate-dependent protein kinase. In addition, phosphorylation of the protein is not increased by protein kinase C or Ca2+/calmodulin-dependent protein kinase but is increased by the phosphatase inhibitor calyculin A. Finally, calyculin A preserves the inhibitory effect of cGMP on ion transport, even in the absence of the nucleotide, suggesting that phosphorylation-dephosphorylation mechanisms are crucial in cotransporter regulation. Thus the flounder intestinal cotransporter is a approximately 175-kDa BBM protein that can be regulated by phosphorylation.

Ca2+ and lipolysis in adipocytes from exercise-trained rats

1994 ◽  
Vol 77 (6) ◽  
pp. 2618-2624 ◽  
Author(s):  
T. Izawa ◽  
T. Komabayashi
Keyword(s):  
Protein Kinase ◽  
Adrenocorticotropic Hormone ◽  
Kinase Activity ◽  
Inhibitory Effect ◽  
Protein Kinase Activity ◽  
Dibutyryl Camp ◽  
Inhibitory Effects ◽  
Dependent Protein Kinase ◽  
Camp Dependent Protein Kinase ◽  
Dependent Protein

The effects of Ca2+ on lipolysis and protein kinase activity in adipocytes from exercise-trained rats were investigated. Chronic exercise significantly increased lipolytic responses to norepinephrine and dibutyryl adenosine 3′,5′-cyclic monophosphate (cAMP). The inhibitory effects of N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide (W-7), a calumodulin inhibitor, on norepinephrine- and dibutyryl cAMP-stimulated lipolysis were significantly greater in trained than in sedentary rats. Training did not alter cAMP-dependent protein kinase activity. However, the inhibitory effect of W-7 on cAMP-dependent protein kinase activity was much greater in trained than in sedentary rats. The basal intracellular free Ca2+ concentration ([Ca2+]i) was significantly higher in trained than in sedentary rats. The rapid and transient increases in [Ca2+]i due to adrenocorticotropic hormone and phenylephrine from basal levels were significantly lower in trained than in sedentary rats. However, the higher basal [Ca2+]i level in trained rats led to increases in sustained [Ca2+]i levels after stimulation. We concluded that in trained rats the regulation of protein kinase activity by cAMP depends to a greater degree on Ca(2+)-calmodulin complex than it does in sedentary rats and that training alters adipocyte intracellular Ca2+ homeostasis, including [Ca2+]i responsiveness to hormones.

scholarly journals Involvement of calcium-sensitive phospholipid-dependent protein kinase in control of acid secretion by isolated rat parietal cells

1985 ◽  
Vol 232 (2) ◽  
pp. 609-611 ◽  
Author(s):  
N G Anderson ◽  
P J Hanson
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Acid Secretion ◽  
Phorbol Esters ◽  
Inhibitory Effect ◽  
Dependent Protein Kinase ◽  
Kinase C ◽  
Protein Kinase C Activity ◽  
Dependent Protein ◽  
Isolated Rat

The relative potency with which phorbol esters inhibited histamine-stimulated aminopyrine accumulation (an index of acid secretion) paralleled that which has been established for the activation of purified protein kinase C. The inhibitory effect of 1-oleoyl-2-acetylglycerol on aminopyrine accumulation stimulated by various secretagogues was similar to that of 12-O-tetradecanoylphorbol 13-acetate. Protein kinase C activity was present in a parietal-cell-enriched fraction. In conclusion, protein kinase C could be involved in mechanisms regulating gastric acid secretion.

scholarly journals Cell Cycle Dependence of DNA-dependent Protein Kinase Phosphorylation in Response to DNA Double Strand Breaks

2005 ◽  
Vol 280 (15) ◽  
pp. 14709-14715 ◽  
Author(s):  
Benjamin P. C. Chen ◽  
Doug W. Chan ◽  
Junya Kobayashi ◽  
Sandeep Burma ◽  
Aroumougame Asaithamby ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Protein Kinase ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Dependent Protein Kinase ◽  
Strand Breaks ◽  
Dependent Protein ◽  
Cell Cycle Dependence ◽  
Kinase Phosphorylation ◽  
Cycle Dependence

Involvement of cAMP-Dependent Protein Kinase in μ-Opioid Modulation of NMDA-Mediated Synaptic Currents

1997 ◽  
Vol 78 (2) ◽  
pp. 759-766 ◽  
Author(s):  
Cui-Wei Xie ◽  
Darrell V. Lewis
Keyword(s):  
Nmda Receptor ◽  
Protein Kinase ◽  
G Proteins ◽  
Inhibitory Action ◽  
Granule Cells ◽  
Inhibitory Effect ◽  
Dependent Protein Kinase ◽  
Camp Dependent Protein Kinase ◽  
Dependent Protein ◽  
Camp Cascade

Xie, Cui-Wei and Darrell V. Lewis. Involvement of cAMP-dependent protein kinase in μ-opioid modulation of NMDA-mediated synaptic currents. J. Neurophysiol. 78: 759–766, 1997. We have previously reported dual effects of μ-opioids on N-methyl-d-aspartate (NMDA)-receptor-mediated synaptic events in the hippocampal dentate gyrus: an indirect facilitating effect via suppression of GABAergic interneurons (disinhibition) and a direct inhibitory effect in the presence of γ-aminobutyric acid-A (GABAA) antagonists. The cellular mechanism underlying the inhibitory effect of μ-opioids remains to be determined. In the present study we examine the role of adenosine 3′,5′-cyclic monophosphate (cAMP)-dependent protein kinase (PKA) in μ-opioid-induced inhibition of NMDA currents in rat hippocampal slices. NMDA-receptor-mediated excitatory postsynaptic currents (NMDA EPSCs) were evoked by stimulating the lateral perforant path and were recorded from dentate granule cells with the use of whole cell voltage-clamp techniques in the presence of the GABAA antagonist and a non-NMDA type of glutamate receptor antagonist. Two selective μ-agonists, [N-MePhe3, D-Pro4]-morphiceptin and [D-Ala2, N-MePhe4, Gly-ol5]-enkephalin, induced dose-dependent inhibition of NMDA EPSCs in a concentration range of 0.3–10 μM. This inhibitory effect could be completely reversed by the opioid antagonists naloxone or prevented by a selective μ-antagonist cyprodime, but was not affected by removal of Mg2+ from the external perfusion medium. Intracellular application of pertussis toxin (PTX) into the granule cell via whole cell recording pipettes completely prevented μ-opioid-induced reduction in NMDA currents, suggesting that a postsynaptic mechanism involving PTX-sensitive G proteins might be responsible for the inhibitory action of μ-opioids. Further studies were conducted to identify the intracellular messengers that coupled with G proteins and transduced the effect of μ-opioids in granule cells. The adenylate cyclase activator forskolin was found to enhance NMDA-receptor-mediated synaptic responses and to reverse the inhibitory effect of μ-opioids. Sp-cAMPS, a specific PKA activator, also enhanced NMDA EPSCs, whereas the PKA inhibitor Rp-cAMPS reduced NMDA EPSCs and occluded further inhibition of the current by μ-opioids. These findings strongly suggest that NMDA receptor function is subject to the modulation by PKA, and that μ-opioids can inhibit NMDA currents through suppression of the cAMP cascade in the postsynaptic neuron. Combined with our previous findings, the present results also indicate that μ-opioids can modulate NMDA-receptor-mediated synaptic activity in a complex manner. The net effect of μ-opioids in the dentate gyrus may depend on the interplay between its disinhibitory action, which facilitates NMDA-receptor-mediated responses, and its inhibitory action on the cAMP cascade.

Protein kinase inhibitors reduce SR Ca transport in permeabilized cardiac myocytes

1994 ◽  
Vol 267 (2) ◽  
pp. H812-H820 ◽  
Author(s):  
A. Mattiazzi ◽  
L. Hove-Madsen ◽  
D. M. Bers
Keyword(s):  
Protein Kinase ◽  
Kinase Inhibitors ◽  
Inhibitory Effect ◽  
Ruthenium Red ◽  
Protein Kinase Inhibitors ◽  
Inhibitory Peptide ◽  
Dependent Protein Kinase ◽  
Ca Transport ◽  
Ca Uptake ◽  
Dependent Protein

Phosphorylation of the sarcoplasmic reticulum (SR) protein phospholamban by adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase (PKA) and Ca-calmodulin-dependent protein kinase (CaM-KII) stimulates Ca-adenosinetriphosphatase (ATPase) activity and SR Ca transport, but the role of CaM-KII-dependent phosphorylation is not well defined. We studied the PKA- and CaM-KII-dependent regulation of SR Ca transport in digitonin-permeabilized rabbit ventricular myocytes. SR Ca uptake and free Ca concentration were measured on line with indo 1 and Ca electrodes in the presence of 20 microM ruthenium red and 10 mM oxalate. neither N5,2'-w-dibutyryl-cAMP (up to 500 microM) nor the nonhydrolyzable cAMP agonist adenosine 3'5'-cyclic monophosphorothioate sodium salt (Sp-cAMP[S]; up to 275 microM) affected the maximum uptake rate (Vmax) or the dissociation constant (Kd) for Ca uptake. However, the PKA inhibitor H-89 significantly increased Kd (e.g., from 307 +/- 67 to 826 +/- 62 nM Ca at 40-65 microM H-89) without significantly affecting Vmax. Both CaM-KII inhibitors, KN-62 (60 microM) and a CaM-KII inhibitory peptide (10 microM), significantly decreased Vmax from 11.95 +/- 0.5 to 9.48 +/- 0.6 nmol.mg-1.min-1 and from 10.95 +/- 1.72 to 7.37 +/- 0.94 nmol.mg-1.min-1, respectively, without consistently changing Kd. The effects of H-89 on Kd and of KN-62 on Vmax were prevented by a monoclonal antibody to phospholamban 2D12 (consistent with the antibody removing the inhibitory effect of phospholamban on the SR Ca-ATPase).(ABSTRACT TRUNCATED AT 250 WORDS)

Interaction of acetylcholine and epinephrine on heart cyclic AMP-dependent protein kinase

1978 ◽  
Vol 234 (4) ◽  
pp. H432-H438
Author(s):  
S. L. Keely ◽  
T. M. Lincoln ◽  
J. D. Corbin
Keyword(s):  
Protein Kinase ◽  
Kinase Activity ◽  
Inhibitory Effect ◽  
Contractile Force ◽  
Protein Kinase Activity ◽  
Dependent Protein Kinase ◽  
Kinase Activation ◽  
Camp Dependent Protein Kinase ◽  
Cholinergic Agent ◽  
Dependent Protein

In the isolated perfused rat heart, epinephrine produced a rapid, concentration-dependent increase in cyclic adenosine 3',5'-monophosphate (cAMP), activation of cAMP-dependent protein kinase, activation of phosphorylase, and increase in contractile force. At epinephrine concentrations of 1 micron or less, acetylcholine antagonized all these beta-adrenergic effects and also increased cyclic guanosine 3',5'-monophosphate (cGMP) levels. When used alone, acetylcholine produced a rapid elevation of cGMP and markedly diminished contractile force but did not significantly lower basal cAMP levels or cAMP-dependent protein kinase activity. The data suggest that changes in cAMP-dependent protein kinase activity can explain the antagonism of epinephrine-induced activation of phosphorylase by acetylcholine, but cannot completely account for the inhibitory effect of the cholinergic agent on contractile force.

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