Dataset Information

Identification of PKC-alpha dependent phosphoproteins in mouse retina

ABSTRACT: Adjusting to a wide range of light intensities is an essential feature of retinal rod bipolar cell (RBC) function, and persuasive evidence suggests this modulation involves phosphorylation by protein kinase C-alpha (PKC-alpha). PKC-alpha is a serine/threonine kinase that is strongly expressed in RBCs, but the targets of PKC-alpha phosphorylation in the retina have not been identified. PKC-alpha activity and phosphorylation in RBCs was examined by immunofluorescence confocal microscopy using a conformation-specific PKC-alpha antibody and antibodies to phosphorylated PKC motifs. PKC-alpha activity was dependent on light and expression of TRPM1, and RBC dendrites were the primary sites of light-dependent phosphorylation. PKC-alpha-dependent retinal phosphoproteins were identified using a multiplexed tandem mass tag-based approach to compare total protein and phosphopeptide abundance between phorbol ester-treated wild type and PKC-alpha knockout (PKC-alpha-KO) mouse retinas. Of the 23 proteins showing significant differential abundance between the two groups, most have roles in cytoskeleton/transport, transcriptional regulation, or metabolism/homeostasis. Phosphopeptide mass spectrometry identified over 1100 phosphopeptides in mouse retina, with 12 displaying significantly greater phosphorylation in WT compared to PKC-alpha-KO samples. The differentially phosphorylated proteins fall into the following functional groups: cytoskeleton/transport (4 proteins), ECM/adhesion (2 proteins), signaling (2 proteins), transcriptional regulation (3 proteins), and homeostasis/metabolism (1 protein). Two strongly differentially expressed phosphoproteins, BORG4 and TPBG, were localized to the synaptic layers of the retina, and may play a role in PKC-alpha-dependent modulation of RBC physiology.

INSTRUMENT(S): Orbitrap Fusion ETD

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Retinal Rod Cell, Retina

SUBMITTER: Phillip Wilmarth

LAB HEAD: Dr. Catherine Morgans

PROVIDER: PXD012906 | Pride | 2019-07-15

REPOSITORIES: Pride

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Publications

Identification of PKCα-dependent phosphoproteins in mouse retina.

Wakeham Colin M CM Wilmarth Phillip A PA Cunliffe Jennifer M JM Klimek John E JE Ren Gaoying G David Larry L LL Morgans Catherine W CW

Journal of proteomics 20190628

Adjusting to a wide range of light intensities is an essential feature of retinal rod bipolar cell (RBC) function. While persuasive evidence suggests this modulation involves phosphorylation by protein kinase C-alpha (PKCα), the targets of PKCα phosphorylation in the retina have not been identified. PKCα activity and phosphorylation in RBCs was examined by immunofluorescence confocal microscopy using a conformation-specific PKCα antibody and antibodies to phosphorylated PKC motifs. PKCα activity ...[more]

PMID: 31255707

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Project description:The vertebrate retina uses diverse neuronal cell types arrayed into complex neural circuits to extract, process and relay information from the visual scene to the higher order processing centers of the brain. Amacrine cells, a diverse class of inhibitory interneurons, are thought to mediate the majority of the processing of the visual signal that occurs within the retina. Despite morphological characterization, the number of known molecular markers of amacrine cell types is still much smaller than the 26 morphological types that have been identified. Furthermore, it is not known how this diversity arises during development. Here, we have combined in vivo genetic labeling and single cell genome-wide expression profiling to: 1) Identify specific molecular types of amacrine cells; 2) Demonstrate the molecular diversity of the amacrine cell class. It is difficult to identify new markers of amacrine cells, due to the fact that they only comprise a small percentage of the total cells in the retina. Additionally, given that there are at least 26 distinct types of amacrine cells, population based approaches fail to achieve the precision necessary to discover markers of each type. To facilitate the identification of new markers for different amacrine cell classes and to more fully characterize the molecular signatures of these classes, we isolated single amacrine cells. To accomplish this goal, we introduced genetic reporters (pNdrg4::GFP or pSynapsin::GFP) into the developing retina (P0) by either in vivo or ex vivo electroporation. These reporters were observed to label morphologically distinct sets of amacrine cells at the different timepoints harvested in this study. Electroporated retinas were then dissociated at different time points and single retinal amacrine cells were isolated by virtue of their GFP expression and placed in tubes containing lysis buffer. Then, their mRNAs were reverse transcribed, and the resulting cDNAs were PCR amplified for 35 cycles. Labeled cDNA samples were hybridized to Affymetrix 430 2.0 microarrays and the data was normalized using MAS5.0 software.

Dataset Information

Identification of PKC-alpha dependent phosphoproteins in mouse retina

Publications

Identification of PKCα-dependent phosphoproteins in mouse retina.

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