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Identification of Novel Substrates for cGMP Dependent Protein Kinase (PKG) through Kinase Activity Profiling to Understand Its Putative Role in Inherited Retinal Degeneration

Inherited retinal degenerative diseases (IRDs), which ultimately lead to photoreceptor cell death, are characterized by high genetic heterogeneity. Many IRD-associated genetic defects affect 3′,5′-cyclic guanosine monophosphate (cGMP) levels. cGMP-dependent protein kinases (PKGI and PKGII) have emer...

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Autores principales: Roy, Akanksha, Groten, John, Marigo, Valeria, Tomar, Tushar, Hilhorst, Riet
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7865299/
https://www.ncbi.nlm.nih.gov/pubmed/33503999
http://dx.doi.org/10.3390/ijms22031180
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author Roy, Akanksha
Groten, John
Marigo, Valeria
Tomar, Tushar
Hilhorst, Riet
author_facet Roy, Akanksha
Groten, John
Marigo, Valeria
Tomar, Tushar
Hilhorst, Riet
author_sort Roy, Akanksha
collection PubMed
description Inherited retinal degenerative diseases (IRDs), which ultimately lead to photoreceptor cell death, are characterized by high genetic heterogeneity. Many IRD-associated genetic defects affect 3′,5′-cyclic guanosine monophosphate (cGMP) levels. cGMP-dependent protein kinases (PKGI and PKGII) have emerged as novel targets, and their inhibition has shown functional protection in IRDs. The development of such novel neuroprotective compounds warrants a better understanding of the pathways downstream of PKGs that lead to photoreceptor degeneration. Here, we used human recombinant PKGs in combination with PKG activity modulators (cGMP, 3′,5′-cyclic adenosine monophosphate (cAMP), PKG activator, and PKG inhibitors) on a multiplex peptide microarray to identify substrates for PKGI and PKGII. In addition, we applied this technology in combination with PKG modulators to monitor kinase activity in a complex cell system, i.e. the retinal cell line 661W, which is used as a model system for IRDs. The high-throughput method allowed quick identification of bona fide substrates for PKGI and PKGII. The response to PKG modulators helped us to identify, in addition to ten known substrates, about 50 novel substrates for PKGI and/or PKGII which are either specific for one enzyme or common to both. Interestingly, both PKGs are able to phosphorylate the regulatory subunit of PKA, whereas only PKGII can phosphorylate the catalytic subunit of PKA. In 661W cells, the results suggest that PKG activators cause minor activation of PKG, but a prominent increase in the activity of cAMP-dependent protein kinase (PKA). However, the literature suggests an important role for PKG in IRDs. This conflicting information could be reconciled by cross-talk between PKG and PKA in the retinal cells. This must be explored further to elucidate the role of PKGs in IRDs.
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spelling pubmed-78652992021-02-07 Identification of Novel Substrates for cGMP Dependent Protein Kinase (PKG) through Kinase Activity Profiling to Understand Its Putative Role in Inherited Retinal Degeneration Roy, Akanksha Groten, John Marigo, Valeria Tomar, Tushar Hilhorst, Riet Int J Mol Sci Article Inherited retinal degenerative diseases (IRDs), which ultimately lead to photoreceptor cell death, are characterized by high genetic heterogeneity. Many IRD-associated genetic defects affect 3′,5′-cyclic guanosine monophosphate (cGMP) levels. cGMP-dependent protein kinases (PKGI and PKGII) have emerged as novel targets, and their inhibition has shown functional protection in IRDs. The development of such novel neuroprotective compounds warrants a better understanding of the pathways downstream of PKGs that lead to photoreceptor degeneration. Here, we used human recombinant PKGs in combination with PKG activity modulators (cGMP, 3′,5′-cyclic adenosine monophosphate (cAMP), PKG activator, and PKG inhibitors) on a multiplex peptide microarray to identify substrates for PKGI and PKGII. In addition, we applied this technology in combination with PKG modulators to monitor kinase activity in a complex cell system, i.e. the retinal cell line 661W, which is used as a model system for IRDs. The high-throughput method allowed quick identification of bona fide substrates for PKGI and PKGII. The response to PKG modulators helped us to identify, in addition to ten known substrates, about 50 novel substrates for PKGI and/or PKGII which are either specific for one enzyme or common to both. Interestingly, both PKGs are able to phosphorylate the regulatory subunit of PKA, whereas only PKGII can phosphorylate the catalytic subunit of PKA. In 661W cells, the results suggest that PKG activators cause minor activation of PKG, but a prominent increase in the activity of cAMP-dependent protein kinase (PKA). However, the literature suggests an important role for PKG in IRDs. This conflicting information could be reconciled by cross-talk between PKG and PKA in the retinal cells. This must be explored further to elucidate the role of PKGs in IRDs. MDPI 2021-01-25 /pmc/articles/PMC7865299/ /pubmed/33503999 http://dx.doi.org/10.3390/ijms22031180 Text en © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Roy, Akanksha
Groten, John
Marigo, Valeria
Tomar, Tushar
Hilhorst, Riet
Identification of Novel Substrates for cGMP Dependent Protein Kinase (PKG) through Kinase Activity Profiling to Understand Its Putative Role in Inherited Retinal Degeneration
title Identification of Novel Substrates for cGMP Dependent Protein Kinase (PKG) through Kinase Activity Profiling to Understand Its Putative Role in Inherited Retinal Degeneration
title_full Identification of Novel Substrates for cGMP Dependent Protein Kinase (PKG) through Kinase Activity Profiling to Understand Its Putative Role in Inherited Retinal Degeneration
title_fullStr Identification of Novel Substrates for cGMP Dependent Protein Kinase (PKG) through Kinase Activity Profiling to Understand Its Putative Role in Inherited Retinal Degeneration
title_full_unstemmed Identification of Novel Substrates for cGMP Dependent Protein Kinase (PKG) through Kinase Activity Profiling to Understand Its Putative Role in Inherited Retinal Degeneration
title_short Identification of Novel Substrates for cGMP Dependent Protein Kinase (PKG) through Kinase Activity Profiling to Understand Its Putative Role in Inherited Retinal Degeneration
title_sort identification of novel substrates for cgmp dependent protein kinase (pkg) through kinase activity profiling to understand its putative role in inherited retinal degeneration
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7865299/
https://www.ncbi.nlm.nih.gov/pubmed/33503999
http://dx.doi.org/10.3390/ijms22031180
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