Abstract
Purpose :
Protein kinase C alpha (PKCα) is abundant in rod bipolar cells (RBCs), yet its physiological role in RBCs is not well understood. To gain insight into the biochemical mechanisms by which PKCα influences RBC physiology, we sought to identify RBC proteins that undergo PKCα-dependent phosphorylation.
Methods :
An antibody mix that binds phosphorylated PKC substrate motifs (anti-pSer) was used for immunoblotting and immunofluorescence microscopy of mouse retinas. To identify phosphopeptides, proteins were extracted from WT and PKCα-KO retinas, digested with trypsin, and the phosphopeptides enriched on TiO2 beads. Total peptides and phosphopeptides were analyzed by TMT multiplex labeling and Orbitrap Fusion mass spectrometry.
Results :
Comparison of anti-pSer immunofluorescence between WT and PKCα-KO retina sections indicates that RBC dendrites are the main sites of PKCα phosphorylation in the mouse retina. Anti-pSer immunofluorescence was reduced in TRPM1-KO retina, and was absent in dark-adapted WT retina. Comparison of protein abundance by mass spectrometry between WT and PKCα-KO retinas indicated that 95% of proteins differed by less than 1.25-fold. Of the 23 proteins that showed significant differences in abundance, most have roles in cytoskeleton/transport, transcriptional regulation, or metabolism. Phosphopeptide mass spectrometry identified over 1000 phosphoproteins, with 12 displaying significantly greater phosphorylation in WT compared to PKCα-KO retinas. Of these 12 phosphoproteins, only one differed significantly in total protein abundance between WT and PKCα-KO samples. The differentially phosphorylated proteins fall into the following functional groups: cytoskeleton/transport (4), ECM/adhesion (2), signaling (2), transcriptional regulation (3), and metabolism (1). Five phosphopeptides belonging to three proteins showed the largest and most significantly increased phosphorylation in WT over PKCα-KO retinas. Of the three proteins, TPBG, a leucine-rich repeat glycoprotein, was shown to be highly expressed in RBCs.
Conclusions :
Taken together, anti-pSer labeling in WT and PKCα-KO mouse retina support a model in which PKCα is activated in RBC dendrites in the light, possibly by calcium ions entering through TRPM1 channels. Phosphoproteomics data indicate that TPBG is a major target of PKCα-dependent phosphorylation in RBCs; therefore, it may mediate some of the effects of PKCα on RBC physiology.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.