Identification of PKC-alpha dependent phosphoproteins in mouse retina
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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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