Identification of PKCα-dependent phosphoproteins in mouse retina.

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 the targets of phosphorylation in the retina have not been identified. duanyu1531α activity and phosphorylation in RBCs was examined by immunofluorescence confocal microscopy using a conformation-specific duanyu1531α antibody and antibodies to phosphorylated motifs. duanyu1531α activity was dependent on light and expression of TRPM1, and RBC dendrites were the primary sites of light-dependent phosphorylation. retinal phosphoproteins were identified using a phosphoproteomics approach to compare total protein and phosphopeptide abundance between phorbol ester-treated wild type and duanyu1531α knockout mouse retinas. Phosphopeptide mass spectrometry identified over 1100 phosphopeptides in mouse retina, with 12 displaying significantly greater phosphorylation in WT compared to 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 duanyu1531α-dependent modulation of RBC physiology. Data are available via ProteomeXchange with identifier PXD012906. SIGNIFICANCE: Retinal 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, though the specific mechanism by which duanyu1531α modulates RBC physiology is unknown. This study examined duanyu1531α phosophorylation patterns in mouse rod bipolar cells and then used a phosphoproteomics approach to identify duanyu1531α-dependent phosphoproteins in the mouse retina. A small number of retinal proteins showed significant duanyu1531α-dependent phosphorylation, including BORG4 and TPBG, suggesting a potential contribution to duanyu1531α-dependent modulation of RBC physiology.

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