scholarly journals Alternative function of a protein kinase homology domain in 2',5'-oligoadenylate dependent RNase L

1999 ◽  
Vol 27 (2) ◽  
pp. 439-445 ◽  
Author(s):  
B. Dong ◽  
R. H. Silverman
Keyword(s):  
Protein Kinase ◽  
Rnase L ◽  
Alternative Function ◽  
Kinase Homology Domain ◽  
Kinase Homology

Conservation of the kinaselike regulatory domain is essential for activation of the natriuretic peptide receptor guanylyl cyclases

1992 ◽  
Vol 12 (6) ◽  
pp. 2581-2590
Author(s):  
K J Koller ◽  
F J de Sauvage ◽  
D G Lowe ◽  
D V Goeddel
Keyword(s):  
Natriuretic Peptide ◽  
Guanylyl Cyclase ◽  
Growth Factor Receptor ◽  
Kinase Domain ◽  
Cyclase Activity ◽  
Guanylyl Cyclases ◽  
Heat Stable ◽  
Guanylyl Cyclase Activity ◽  
Kinase Homology Domain ◽  
Kinase Homology

The natriuretic peptide receptors, NPR-A and NPR-B, are two members of the newly described class of receptor guanylyl cyclases. The kinaselike domain of these proteins is an important regulator of the guanylyl cyclase activity. To begin to understand the molecular nature of this type of regulation, we made complete and partial deletions of the kinase domain in NPR-A and NPR-B. We also made chimeric proteins in which the kinase domains of NPR-A and NPR-B were exchanged or replaced with kinase domains from structurally similar proteins. Complete deletion of the kinase homology domain in NPR-A and NPR-B resulted in constitutive activation of the guanylyl cyclase. Various partial deletions of this region produced proteins that had no ability to activate the enzyme with or without hormone stimulation. The kinase homology domain can be exchanged between the two subtypes with no effect on regulation. However, structurally similar kinaselike domains, such as from the epidermal growth factor receptor or from the heat-stable enterotoxin receptor, another member of the receptor guanylyl cyclase family, were not able to regulate the guanylyl cyclase activity correctly. These findings suggest that the kinaselike domain of NPR-A and NPR-B requires strict sequence conservation to maintain proper regulation of their guanylyl cyclase activity.

scholarly journals A phenolic small molecule inhibitor of RNase L prevents cell death from ADAR1 deficiency

2020 ◽  
Vol 117 (40) ◽  
pp. 24802-24812 ◽  
Author(s):  
Salima Daou ◽  
Manisha Talukdar ◽  
Jinle Tang ◽  
Beihua Dong ◽  
Shuvojit Banerjee ◽  
...  
Keyword(s):  
Cell Death ◽  
Protein Kinase ◽  
Small Molecule ◽  
Small Molecule Inhibitors ◽  
Therapeutic Potential ◽  
Protein Kinase Inhibitors ◽  
Response To Treatment ◽  
Rnase L ◽  
Oligoadenylate Synthetase ◽  
In Cells

The oligoadenylate synthetase (OAS)–RNase L system is an IFN-inducible antiviral pathway activated by viral infection. Viral double-stranded (ds) RNA activates OAS isoforms that synthesize the second messenger 2-5A, which binds and activates the pseudokinase-endoribonuclease RNase L. In cells, OAS activation is tamped down by ADAR1, an adenosine deaminase that destabilizes dsRNA. Mutation of ADAR1 is one cause of Aicardi-Goutières syndrome (AGS), an interferonopathy in children. ADAR1 deficiency in human cells can lead to RNase L activation and subsequent cell death. To evaluate RNase L as a possible therapeutic target for AGS, we sought to identify small-molecule inhibitors of RNase L. A 500-compound library of protein kinase inhibitors was screened for modulators of RNase L activity in vitro. We identified ellagic acid (EA) as a hit with 10-fold higher selectivity against RNase L compared with its nearest paralog, IRE1. SAR analysis identified valoneic acid dilactone (VAL) as a superior inhibitor of RNase L, with 100-fold selectivity over IRE1. Mechanism-of-action analysis indicated that EA and VAL do not bind to the pseudokinase domain of RNase L despite acting as ATP competitive inhibitors of the protein kinase CK2. VAL is nontoxic and functional in cells, although with a 1,000-fold decrease in potency, as measured by RNA cleavage activity in response to treatment with dsRNA activator or by rescue of cell lethality resulting from self dsRNA induced by ADAR1 deficiency. These studies lay the foundation for understanding novel modes of regulating RNase L function using small-molecule inhibitors and avenues of therapeutic potential.

scholarly journals Intact Kinase Homology Domain of Natriuretic Peptide Receptor-B Is Essential for Skeletal Development

2007 ◽  
Vol 92 (10) ◽  
pp. 4009-4014 ◽  
Author(s):  
Rumi Hachiya ◽  
Yuko Ohashi ◽  
Yasutomi Kamei ◽  
Takayoshi Suganami ◽  
Hiroshi Mochizuki ◽  
...  
Keyword(s):  
Short Stature ◽  
Natriuretic Peptide ◽  
Functional Significance ◽  
Skeletal Development ◽  
Natriuretic Peptide Receptor ◽  
Wild Type ◽  
Peptide Receptor ◽  
Cgmp Production ◽  
Kinase Homology Domain ◽  
Kinase Homology

Abstract Context: Natriuretic peptide receptor-B (NPR-B, GC-B in rodents; gene name NPR2) is a guanylyl cyclase-coupled receptor that mediates the effect of C-type natriuretic peptide. Homozygous mutations in human NPR-B cause acromesomelic dysplasia, type Maroteaux (OMIM 602875), an autosomal recessive skeletal dysplasia. NPR-B has an intracellular kinase homology domain (KHD), which has no kinase activity, and its functional significance in vivo is currently unknown. Objective: We examined the functional significance of a novel NPR-B KHD mutation in humans. Patients and Methods: A 28-yr-old Japanese male presented with marked short stature (118.5 cm, −9.3 sd). His limbs showed marked shortening in the middle and distal segments. His parents had relatively short stature with height z-scores of −2.75 and −0.98 (his father and mother, respectively). Direct sequencing of coding region of the NPR2 gene of the family was performed. The mutant receptor activity was investigated by saturation binding assay and cGMP measurement. Additionally, interaction between the mutant and wild type allele was investigated by the titration experiments. Results: We identified a novel missense mutation L658F in KHD of NPR-B in homozygous and heterozygous states in the patient and his parents, respectively. The mutation conferred normal binding affinity for C-type natriuretic peptide but no discernible ligand-induced cGMP production. Furthermore, L658F mutant impaired wild-type NPR-B-mediated cGMP production in a dose-dependent manner, suggesting that short stature found in L658F heterozygote can be caused by its dominant-negative effect. Conclusions: This study provides the first evidence that intact KHD of NPR-B is essential for skeletal development.

scholarly journals RNase L and Double-Stranded RNA-Dependent Protein Kinase Exert Complementary Roles in Islet Cell Defense during Coxsackievirus Infection

2005 ◽  
Vol 174 (3) ◽  
pp. 1171-1177 ◽  
Author(s):  
Malin Flodström-Tullberg ◽  
Monica Hultcrantz ◽  
Alexandr Stotland ◽  
Amy Maday ◽  
Devin Tsai ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Islet Cell ◽  
Rnase L ◽  
Dependent Protein Kinase ◽  
Double Stranded Rna ◽  
Cell Defense ◽  
Dependent Protein

scholarly journals Phosphorylation of the Kinase Homology Domain Is Essential for Activation of the A-Type Natriuretic Peptide Receptor

1998 ◽  
Vol 18 (4) ◽  
pp. 2164-2172 ◽  
Author(s):  
Lincoln R. Potter ◽  
Tony Hunter
Keyword(s):  
Natriuretic Peptide ◽  
Guanylyl Cyclase ◽  
Receptor Activation ◽  
Dominant Negative ◽  
Cyclase Activity ◽  
Atp Binding ◽  
Natriuretic Peptide Receptor ◽  
Peptide Receptor ◽  
Kinase Homology Domain ◽  
Kinase Homology

ABSTRACT Natriuretic peptide receptor A (NPR-A) is the biological receptor for atrial natriuretic peptide (ANP). Activation of the NPR-A guanylyl cyclase requires ANP binding to the extracellular domain and ATP binding to a putative site within its cytoplasmic region. The allosteric interaction of ATP with the intracellular kinase homology domain (KHD) is hypothesized to derepress the carboxyl-terminal guanylyl cyclase catalytic domain, resulting in the synthesis of the second messenger, cyclic GMP. Here, we show that phosphorylation of the KHD is essential for receptor activation. Using a combination of phosphopeptide mapping techniques, we have identified six residues within the ATP-binding domain (S497, T500, S502, S506, S510, and T513) which are phosphorylated when NPR-A is expressed in HEK 293 cells. Mutation of any one of these Ser or Thr residues to Ala caused reductions in the receptor phosphorylation state, the number and pattern of phosphopeptides observed in tryptic maps, and ANP-dependent guanylyl cyclase activity. The reductions were not explained by decreases in NPR-A protein levels, as indicated by immunoblot analysis and determinations of cyclase activity in the presence of detergent. Conversion of Ser-497 to Ala resulted in the most dramatic decrease in cyclase activity (∼20% of wild-type activity), but conversion to an acidic residue (Glu), which mimics the charge of the phosphoserine moiety, had no effect. Simultaneous mutation of five of the phosphorylation sites to Ala resulted in a dephosphorylated receptor which was unresponsive to hormone and had potent dominant negative inhibitory activity. We conclude that phosphorylation of the KHD is absolutely required for hormone-dependent activation of NPR-A.

scholarly journals Activation of p38 Mitogen-Activated Protein Kinase and c-Jun NH2-Terminal Kinase by Double-Stranded RNA and Encephalomyocarditis Virus: Involvement of RNase L, Protein Kinase R, and Alternative Pathways

2000 ◽  
Vol 20 (2) ◽  
pp. 617-627 ◽  
Author(s):  
Mihail S. Iordanov ◽  
Jayashree M. Paranjape ◽  
Aimin Zhou ◽  
John Wong ◽  
Bryan R. G. Williams ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Protein Kinase ◽  
P38 Mapk ◽  
Encephalomyocarditis Virus ◽  
Mitogen Activated Protein Kinase ◽  
Rnase L ◽  
Double Stranded Rna ◽  
Mitogen Activated Protein ◽  
Stimulation Of ◽  
In Cells

ABSTRACT Double-stranded RNA (dsRNA) accumulates in virus-infected mammalian cells and signals the activation of host defense pathways of the interferon system. We describe here a novel form of dsRNA-triggered signaling that leads to the stimulation of the p38 mitogen-activated protein kinase (p38 MAPK) and the c-Jun NH2-terminal kinase (JNK) and of their respective activators MKK3/6 and SEK1/MKK4. The dsRNA-dependent signaling to p38 MAPK was largely intact in cells lacking both RNase L and the dsRNA-activated protein kinase (PKR), i.e., the two best-characterized mediators of dsRNA-triggered antiviral responses. In contrast, activation of both MKK4 and JNK by dsRNA was greatly reduced in cells lacking RNase L (or lacking both RNase L and PKR) but was restored in these cells when introduction of dsRNA was followed by inhibition of ongoing protein synthesis or transcription. These results are consistent with the notion that the role of RNase L and PKR in the activation of MKK4 and JNK is the elimination, via inhibition of protein synthesis, of a labile negative regulator(s) of the signaling to JNK acting upstream of SEK1/MKK4. In the course of these studies, we identified a long-sought site of RNase L-mediated cleavage in the 28S rRNA, which could cause inhibition of translation, thus allowing the activation of JNK by dsRNA. We propose that p38 MAPK is a general participant in dsRNA-triggered cellular responses, whereas the activation of JNK might be restricted to cells with reduced rates of protein synthesis. Our studies demonstrate the existence of alternative (RNase L- and PKR-independent) dsRNA-triggered signaling pathways that lead to the stimulation of stress-activated MAPKs. Activation of p38 MAPK (but not of JNK) was demonstrated in mouse fibroblasts in response to infection with encephalomyocarditis virus (ECMV), a picornavirus that replicates through a dsRNA intermediate. Fibroblasts infected with EMCV (or treated with dsRNA) produced interleukin-6, an inflammatory and pyrogenic cytokine, in a p38 MAPK-dependent fashion. These findings suggest that stress-activated MAPKs participate in mediating inflammatory and febrile responses to viral infections.

Protein Kinase Homology Models: Recent Developments and Results

2011 ◽  
Vol 18 (19) ◽  
pp. 2848-2853 ◽  
Author(s):  
T. Tuccinardi ◽  
Adriano Martinelli
Keyword(s):  
Protein Kinase ◽  
Recent Developments ◽  
Homology Models ◽  
Kinase Homology

scholarly journals A Constitutively “Phosphorylated” Guanylyl Cyclase-linked Atrial Natriuretic Peptide Receptor Mutant Is Resistant to Desensitization

1999 ◽  
Vol 10 (6) ◽  
pp. 1811-1820 ◽  
Author(s):  
Lincoln R. Potter ◽  
Tony Hunter
Keyword(s):  
Atrial Natriuretic Peptide ◽  
Natriuretic Peptide ◽  
Guanylyl Cyclase ◽  
Phosphorylation Sites ◽  
Natriuretic Peptide Receptor ◽  
Wild Type ◽  
Peptide Receptor ◽  
Type Receptor ◽  
Kinase Homology Domain ◽  
Kinase Homology

Dephosphorylation of the natriuretic peptide receptor-A (NPR-A) is hypothesized to mediate its desensitization in response to atrial natriuretic peptide (ANP) binding. Recently, we identified six phosphorylation sites within the kinase homology domain of NPR-A and determined that the conversion of these residues to alanine abolished the ability of the receptor to be phosphorylated or to be activated by ANP and ATP. In an attempt to generate a form of NPR-A that mimics a fully phosphorylated receptor but that is resistant to dephosphorylation, we engineered a receptor variant (NPR-A-6E) containing glutamate substitutions at all six phosphorylation sites. Consistent with the known ability of negatively charged glutamate residues to substitute functionally, in some cases, for phosphorylated residues, we found that NPR-A-6E was activated 10-fold by ANP and ATP. As determined by guanylyl cyclase assays, the hormone-stimulated activity of the wild-type receptor declined over time in membrane preparations in vitro, and this loss was blocked by the serine/threonine protein phosphatase inhibitor microcystin. In contrast, the activity of NPR-A-6E was more linear with time and was unaffected by microcystin. The nonhydrolyzable ATP analogue adenosine 5′-(β,γ-imino)-triphosphate was half as effective as ATP in stimulating the wild-type receptor but was equally as potent in stimulating NPR-A-6E, suggesting that ATP is required to keep the wild-type but not 6E variant phosphorylated. Finally, the desensitization of NPR-A-6E in whole cells was markedly blunted compared with that of the wild-type receptor, consistent with its inability to shed the negative charge from its kinase homology domain via dephosphorylation. These data provide the first direct test of the requirement for dephosphorylation in guanylyl cyclase desensitization and they indicate that it is an essential component of this process.

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