Integrative Kinase Activity Profiling and Phosphoproteomics of rd10 Mouse Retina during cGMP-Dependent Retinal Degeneration

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 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.

Keywords: CREB; CaMK; PKG; kinome activity profiling; phosphoproteomics; rd10.

Figure 1

CN03-mediated photoreceptor protection in rd10 retinal explant cultures. Retinal cross-sections of wild-type (WT) and rd10 explant cultures. Retinas were explanted at post-natal day (P) 8 and cultivated with/without 50 µM of CN03 from P10 to P18. (a) The TUNEL assay (red) highlighted dying cells, especially in the outer nuclear layer (ONL). DAPI (grey) was used as a nuclear counterstain. INL = inner nuclear layer; GCL = ganglion cell layer. (b) Bar graph showing percentages of TUNEL-positive cells for WT, rd10-untreated, and CN03-treated rd10 retinal explants. n = three different retinal explants per genotype/condition; error bars indicate standard deviation. Significance levels were determined via one-way ANOVA with Dunnett multiple comparisons; significance levels: * (p ≤ 0.05), ** (p ≤ 0.01).

Figure 2

Serine/threonine kinase (STK) activity in rd10 retinal explants after PKG inhibition. Retinal explant cultures were either non-treated (NT) or treated with 50 µM of CN03 (rd10 NT, n  =  10; rd10 CN03, n  =  10). The kinase activity of retinal lysates was measured on PamChip^® Serine/Threonine Kinase (STK) arrays. (a) Violin plot representing the phosphorylation signal intensity of all the peptides on PamChip^® STK arrays as a Log2 signal intensity and their intensity value distribution when comparing rd10 to rd10 CN03-treated retinal explants. (b) Volcano plot showing the Log Fold Change (LFC) and −Log₁₀ p value of the phosphorylated peptides. Red dots represent peptides with significant differences in phosphorylation (p < 0.05) as assessed by a paired t-test. Black dots represent peptides with no significant phosphorylation change. (c) Peptides displaying significant changes in phosphorylation are shown as a bar plot with their respective LFC. (d) The predicted kinases whose activities were most likely to be altered by the CN03 treatment (see Table 1) are visualized as a kinome phylogenetic tree where the branch and node color are encoded by the kinase statistic, with values < 0 (in blue) representing decreased kinase activity in rd10 CN03-treated retinal explants. The node size is indicated by the kinase score, which is based on the specificity and selectivity of that kinase for a particular peptide.

Figure 3

Phosphorylated sites in rd10 retinal explants in response to PKG inhibition. Retinal explant cultures were treated or not with 50 µM of CN03, followed by phosphopeptide enrichment and mass spectrometry analysis. (a) Heatmap representing proteins with significantly different phosphorylations as their Log₂ signal intensity. A red color indicates the relatively lower Log₂ signal intensity of the protein, while a green color indicates one that is relatively higher. (b) Volcano plot showing the Log Fold Change (LFC) and −Log₁₀ p-value of the phosphorylated sites. Red dots represent peptides/sites with significant changes in phosphorylation (p < 0.05), black dots indicate peptides with no significant phosphorylation change. (c) Kinases predicted to have significantly altered activity are visualized as a kinome phylogenetic tree, with their branch and node color encoded by the kinase statistic and values < 0 (in blue) and >0 (in red) representing the kinase activity as decreased or increased, respectively, in rd10 CN03 retinal explants. The node size is indicated by the kinase score which is based on the specificity and selectivity of that kinase for a particular peptide.

Figure 4

Pathway and network analysis for integrated kinome activity and phosphoproteomic data of CN03-treated rd10 retinal explants. (a) Network of kinases with potentially altered activity in CN03-treated rd10 retina. (b) Key biological pathways with potentially altered activity in the CN03-treated rd10 situation. The false discovery rate (FDR) is the p-value adjusted for multiple tests using the Benjamini–Hochberg procedure, measuring the significance of enrichment. Pathways are ranked according to their FDR values and colored by their strength. Strength describes how large the enrichment effect is and is calculated as Log₁₀ (no. observed proteins/no. of expected proteins). The node size indicates the gene ratio, i.e., the percentage of total genes or proteins in the given Reactome pathways (only input genes or proteins with at least one Reactome pathway were included in the calculation).

Figure 5

Presence and localization of PKG target proteins in the WT and rd10 retinas. Immunostaining for Ca²⁺/calmodulin-dependent kinases (CaMK) and phosphorylated cyclic-AMP-response-element-binding (pCREB). The micrograph panels show retinal cross-sections derived from wild-type (WT; top row) and rd10 (bottom row) P18 mice and stained with antibodies for CAMK2, pCAMK2, CAMK4, pCaMK4, or pCREB. Bar diagrams under the micrographs indicate the fluorescence intensities of the respective antibody staining, which were calculated as arbitrary fluorescence units/µm² in the indicated area of interest (ONL). The fluorescence was adjusted with overexposure in the images for presentation purposes. OPL = outer plexiform layer, INL = inner nuclear layer, IPL = inner plexiform layer, GCL = ganglion cell layer. Significance levels: * (p ≤ 0.05), ** (p ≤ 0.01), ns = not significant.

Figure 6

Confirmation of PKG target proteins in retina by Western blot. (a) Representative Western blots showing protein levels for the PKG targets pCREB (43 kD), pCaMK2 (54 kD), and pCaMK4 (51 kD) in the retina. First row of each blot is a marker lane. (b) Quantitative analysis with n ≥ 3 retinal tissues for each group. (c) Protein levels for PKG targets pCREB and pCaMK4 in retinal explants treated or not with CN03 (50 µM). First row of each blot is a marker lane. (d) Quantitative analysis of ≥ 5 retinal explants for each group. The significant differences between the groups were determined via unpaired Student’s t-test with significance indicated as ** (p ≤ 0.01) and **** (p ≤ 0.0001).