TY - JOUR T1 - Identification of PKCα-dependent phosphoproteins in mouse retina JF - bioRxiv DO - 10.1101/589184 SP - 589184 AU - Colin M. Wakeham AU - Phillip A. Wilmarth AU - Jennifer M. Cunliffe AU - John E. Klimek AU - Gaoying Ren AU - Larry L. David AU - Catherine W. Morgans Y1 - 2019/01/01 UR - http://biorxiv.org/content/early/2019/03/27/589184.1.abstract N2 - 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 was dependent on light and expression of TRPM1, and RBC dendrites were the primary sites of light-dependent phosphorylation. PKCα-dependent retinal phosphoproteins were identified using a phosphoproteomics approach to compare total protein and phosphopeptide abundance between phorbol ester-treated wild type and PKCα knockout (PKCα-KO) mouse retinas. Phosphopeptide mass spectrometry identified over 1100 phosphopeptides in mouse retina, with 12 displaying significantly greater phosphorylation in WT compared to PKCα-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 PKCα-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, PKCα, though the specific mechanism by which PKCα modulates RBC physiology is unknown. This study examined PKCα phosophorylation patterns in mouse rod bipolar cells and then used a phosphoproteomics approach to identify PKCα-dependent phosphoproteins in the mouse retina. A small number of retinal proteins showed significant PKCα-dependent phosphorylation, including BORG4 and TPBG, suggesting a potential contribution to PKCα-dependent modulation of RBC physiology.HighlightsPKCα is a major source of phosphorylation in retinal RBC dendrites and its activity in RBCs is light dependent.Proteins showing differential phosphorylation between phorbol ester-treated wild type and PKCα-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).The PKCα-dependent phosphoproteins, BORG4 and TPBG, are present in the synaptic layers of the retina and may be involved in PKCα-dependent modulation of RBC physiology.BORG4Binder of Rho GTPase 4DAGdiacylglycerolERGelectroretinogramDEdifferential expressionFCfold changeFDRfalse discovery rateGCLganglion cell layerINLinner nuclear layerIPLinner plexiform layerKOknockoutLC-MS/MSliquid chromatography tandem mass spectroscopyNHERF1Na+/H+ Exchange Regulatory Factor 1OPLouter plexiform layerPKCα/PrkcaProtein Kinase C-alphaPMAphorbol 12-myristate 13-acetatePSMpeptide spectrum matchRBCrod bipolar cellRPEretinal pigment epitheliumTMTtandem mass tagTPBGTrophoblast GlycoproteinTRPM1Transient Receptor Potential cation channel subfamily M member 1 ER -