Identification of PKCα-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. While persuasive evidence suggests this modulation involves phosphorylation by protein kinase C-alpha (PKC), the targets of PKC phosphorylation in the retina have not been identified. PKC activity and phosphorylation in RBCs was examined by immunofluorescence confocal microscopy using a conformation-specific PKC antibody and antibodies to phosphorylated PKC motifs. PKC activity was dependent on light and expression of TRPM1, and RBC dendrites were the primary sites of light-dependent phosphorylation. PKC-dependent retinal phosphoproteins were identified using a phosphoproteomics approach to compare total protein and phosphopeptide abundance between phorbol ester-treated wild type and PKC knockout (PKC-KO) mouse retinas. Phosphopeptide mass spectrometry identified over 1100 phosphopeptides in mouse retina, with 12 displaying significantly greater phosphorylation in WT compared to PKC-KO samples. The differentially phosphorylated proteins fall into the following functional groups: cytoskeleton/trafficking (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-dependent modulation of RBC physiology. Data are available via ProteomeXchange with identifier PXD012906.\n\nSignificanceRetinal rod bipolar cells (RBCs), the second-order neurons of the mammalian rod visual pathway, are able to modulate their sensitivity to remain functional across a wide range of light intensities, from starlight to daylight. Evidence suggests that this modulation requires the serine/threonine kinase, PKC, though the specific mechanism by which PKC modulates RBC physiology is unknown. This study examined PKC phosophorylation patterns in mouse rod bipolar cells and then used a phosphoproteomics approach to identify PKC-dependent phosphoproteins in the mouse retina. A small number of retinal proteins showed significant PKC-dependent phosphorylation, including BORG4 and TPBG, suggesting a potential contribution to PKC-dependent modulation of RBC physiology.\n\nHighlightsO_LIPKC is a major source of phosphorylation in retinal RBC dendrites and its activity in RBCs is light dependent.\nC_LIO_LIProteins showing differential phosphorylation between phorbol ester-treated wild type and PKC-KO retinas belong to the following major functional groups: cytoskeleton/trafficking (4 proteins), ECM/adhesion (2 proteins), signaling (2 proteins), transcriptional regulation (3 proteins), and homeostasis/metabolism (1 protein).\nC_LIO_LIThe PKC-dependent phosphoproteins, BORG4 and TPBG, are present in the synaptic layers of the retina and may be involved in PKC-dependent modulation of RBC physiology.\nC_LI