Integrative Kinase Activity Profiling and Phosphoproteomics of rd10 Mouse Retina during cGMP-Dependent Retinal Degeneration
(2024) In International Journal of Molecular Sciences 25(6).- Abstract
Inherited retinal degenerative diseases (IRDs) are a group of rare diseases that lead to a progressive loss of photoreceptor cells and, ultimately, blindness. The overactivation of cGMP-dependent protein kinase G (PKG), one of the key effectors of cGMP-signaling, was previously found to be involved in photoreceptor cell death and was studied in murine IRD models to elucidate the pathophysiology of retinal degeneration. However, PKG is a serine/threonine kinase (STK) with several hundred potential phosphorylation targets and, so far, little is known about the specificity of the target interaction and downstream effects of PKG activation. Here, we carried out both the kinome activity and phosphoproteomic profiling of organotypic retinal... (More)
Inherited retinal degenerative diseases (IRDs) are a group of rare diseases that lead to a progressive loss of photoreceptor cells and, ultimately, blindness. The overactivation of cGMP-dependent protein kinase G (PKG), one of the key effectors of cGMP-signaling, was previously found to be involved in photoreceptor cell death and was studied in murine IRD models to elucidate the pathophysiology of retinal degeneration. However, PKG is a serine/threonine kinase (STK) with several hundred potential phosphorylation targets and, so far, little is known about the specificity of the target interaction and downstream effects of PKG activation. Here, we carried out both the kinome activity and phosphoproteomic profiling of organotypic retinal explant cultures derived from the rd10 mouse model for IRD. After treating the explants with the PKG inhibitor CN03, an overall decrease in peptide phosphorylation was observed, with the most significant decrease occurring in seven peptides, including those from the known PKG substrate cyclic-AMP-response-element-binding CREB, but also Ca2+/calmodulin-dependent kinase (CaMK) peptides and TOP2A. The phosphoproteomic data, in turn, revealed proteins with decreased phosphorylation, as well as proteins with increased phosphorylation. The integration of both datasets identified common biological networks altered by PKG inhibition, which included kinases predominantly from the so-called AGC and CaMK families of kinases (e.g., PKG1, PKG2, PKA, CaMKs, RSKs, and AKTs). A pathway analysis confirmed the role of CREB, Calmodulin, mitogen-activated protein kinase (MAPK) and CREB modulation. Among the peptides and pathways that showed reduced phosphorylation activity, the substrates CREB, CaMK2, and CaMK4 were validated for their retinal localization and activity, using immunostaining and immunoblotting in the rd10 retina. In summary, the integrative analysis of the kinome activity and phosphoproteomic data revealed both known and novel PKG substrates in a murine IRD model. This data establishes a basis for an improved understanding of the biological pathways involved in cGMP-mediated photoreceptor degeneration. Moreover, validated PKG targets like CREB and CaMKs merit exploration as novel (surrogate) biomarkers to determine the effects of a clinical PKG-targeted treatment for IRDs.
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- author
- Roy, Akanksha ; Zhou, Jiaming LU ; Nolet, Merijn ; Welinder, Charlotte LU ; Zhu, Yu ; Paquet-Durand, François ; Groten, John ; Tomar, Tushar and Ekström, Per LU
- organization
- publishing date
- 2024-03
- type
- Contribution to journal
- publication status
- published
- subject
- keywords
- CaMK, CREB, kinome activity profiling, phosphoproteomics, PKG, rd10
- in
- International Journal of Molecular Sciences
- volume
- 25
- issue
- 6
- article number
- 3446
- publisher
- MDPI AG
- external identifiers
-
- pmid:38542418
- scopus:85189084340
- ISSN
- 1661-6596
- DOI
- 10.3390/ijms25063446
- language
- English
- LU publication?
- yes
- id
- 4ac39fdb-e716-4a00-a6b9-fc5564c0ba6c
- date added to LUP
- 2024-04-17 15:04:50
- date last changed
- 2024-04-18 03:00:03
@article{4ac39fdb-e716-4a00-a6b9-fc5564c0ba6c,
abstract = {{<p>Inherited retinal degenerative diseases (IRDs) are a group of rare diseases that lead to a progressive loss of photoreceptor cells and, ultimately, blindness. The overactivation of cGMP-dependent protein kinase G (PKG), one of the key effectors of cGMP-signaling, was previously found to be involved in photoreceptor cell death and was studied in murine IRD models to elucidate the pathophysiology of retinal degeneration. However, PKG is a serine/threonine kinase (STK) with several hundred potential phosphorylation targets and, so far, little is known about the specificity of the target interaction and downstream effects of PKG activation. Here, we carried out both the kinome activity and phosphoproteomic profiling of organotypic retinal explant cultures derived from the rd10 mouse model for IRD. After treating the explants with the PKG inhibitor CN03, an overall decrease in peptide phosphorylation was observed, with the most significant decrease occurring in seven peptides, including those from the known PKG substrate cyclic-AMP-response-element-binding CREB, but also Ca<sup>2+</sup>/calmodulin-dependent kinase (CaMK) peptides and TOP2A. The phosphoproteomic data, in turn, revealed proteins with decreased phosphorylation, as well as proteins with increased phosphorylation. The integration of both datasets identified common biological networks altered by PKG inhibition, which included kinases predominantly from the so-called AGC and CaMK families of kinases (e.g., PKG1, PKG2, PKA, CaMKs, RSKs, and AKTs). A pathway analysis confirmed the role of CREB, Calmodulin, mitogen-activated protein kinase (MAPK) and CREB modulation. Among the peptides and pathways that showed reduced phosphorylation activity, the substrates CREB, CaMK2, and CaMK4 were validated for their retinal localization and activity, using immunostaining and immunoblotting in the rd10 retina. In summary, the integrative analysis of the kinome activity and phosphoproteomic data revealed both known and novel PKG substrates in a murine IRD model. This data establishes a basis for an improved understanding of the biological pathways involved in cGMP-mediated photoreceptor degeneration. Moreover, validated PKG targets like CREB and CaMKs merit exploration as novel (surrogate) biomarkers to determine the effects of a clinical PKG-targeted treatment for IRDs.</p>}},
author = {{Roy, Akanksha and Zhou, Jiaming and Nolet, Merijn and Welinder, Charlotte and Zhu, Yu and Paquet-Durand, François and Groten, John and Tomar, Tushar and Ekström, Per}},
issn = {{1661-6596}},
keywords = {{CaMK; CREB; kinome activity profiling; phosphoproteomics; PKG; rd10}},
language = {{eng}},
number = {{6}},
publisher = {{MDPI AG}},
series = {{International Journal of Molecular Sciences}},
title = {{Integrative Kinase Activity Profiling and Phosphoproteomics of rd10 Mouse Retina during cGMP-Dependent Retinal Degeneration}},
url = {{http://dx.doi.org/10.3390/ijms25063446}},
doi = {{10.3390/ijms25063446}},
volume = {{25}},
year = {{2024}},
}