Design and development of Opto-PKCɛ

(a) Schematic of the ideal properties of an optically-controlled PKCɛ. Small molecule activators possess off-target effects, without spatiotemporal specificity. Direct optogenetic control of PKCɛ could allow dissection of the pathway if the challenges listed could be overcome.

(b) Schematic of the regulatory and catalytic domains of PKCɛ. The regulatory domain of PKCɛ was truncated and replaced by the blue light sensitive cryptochrome domains tagged with mCherry fluorescent protein.

(c) Representative Western blots of phosphorylation levels of PKCɛ substrates, PKCɛ and GAPDH for HEK293T cells transfected with constructs listed. These cells were either subjected to 5 minutes of dark condition or blue light.

(d) Quantification (n=3) of normalized phosphorylation levels of PKCɛ substrates relative to GAPDH. The data is presented as mean +/− SD.

Computational simulations to aid in rationalization of Opto-PKCɛ dark-light activity.

(a) Superposition of the final trajectory structures from representative aMD simulations of phosphorylated (cyan) and T566A mutant (yellow) PKCε. The activation loops of phosphorylated and T566A mutant PKCε were shown in purple and orange cartoon, respectively.

(b) AlphaFold3 models of the monomeric (left) and tetrameric (right) Opto-PKCɛ reveal the Glu293-Arg858 interaction as a potential gatekeeper for dark-light activity. The PKCɛ domain is shown in white while the rest of the protein is shown in green. Other monomers in the tetramer are shown in yellow and cyan. Glu293, Arg858 and ATP are shown in sticks.

(c) Representative Western blots of phosphorylation levels of PKCɛ substrates, PKCɛ and GAPDH for HEK293T cells transfected with constructs listed. These cells were either subjected to 5 minutes of dark condition or blue light.

(d) Quantification (n=3) of normalized phosphorylation levels of PKCɛ substrates relative to GAPDH. The data is presented as mean +/− SD.

Opto-PKCɛ maintains largely similar interactome as that of PKCɛ.

(a) Schematic of the workflow comparing interactome of Flag-Opto-PKCε and Flag-PKCε. Untransfected HEK293T cells was used as a negative control, with proteins found at least 10 times more in abundance considered for analysis.

(b) Mass spectroscopy of proteins associated with Flag-Opto-PKCε or Flag-PKCε demonstrates that Flag-Opto-PKCε has a largely similar interactome as Flag-PKCε. Key proteins belonging to exclusively each subset or overlapping set is detailed.

Opto-PKCɛ enables dissection of PKCɛ signaling.

(a) Schematic of the experiments comparing signaling dynamics arising from 15 minutes of Opto-PKCɛ activation and PMA induction respectively.

(b) Representative Western blots of phosphorylation levels of PKC and PKA substrates, PKCɛ and GAPDH for HEK293T cells subjected to both conditions. Quantification (n=3) of normalized phosphorylation levels of PKC and PKA substrates relative to GAPDH.

(c) Venn Diagram of phosphopeptides quantifying the number of phosphopeptides appearing in both subsets or in either subset. Some of the main phosphopeptides known to be activated by PMA or classical PKCɛ phosphosites are highlighted.

(d) KSEA revealed that CSNK and GSK3 substrates were enriched in opto-PKCε activation group while PKA pathways and other PKC pathways were enriched under PMA treatment.

(e) KEA validated the increased phosphorylation of CSNK and GSK3 substrates under opto-PKCε activation (in purple) and those of PKA substrates (in red) under PMA treatment. Common substrates are highlighted in blue.

The data in (b) are presented as mean +/− SD.

Opto-PKCɛ enables subcellular dissection of PKCɛ signaling.

(a) Representative images of HEK293T cells transfected with Opto-PKCɛ and CIBN-GFP-CAAX from initial 5 min blue light to 15 minutes dark and then back to blue light exposure. CIBN-GFP-CAAX could be found to colocalize with Opto-PKCɛ after 30 seconds of blue light exposure. Scale bar = 10 µm

(b) Representative images of HEK293T cells transfected with Opto-PKCɛ and CIBN-GFP-miro1 from initial 5 min blue light to 15 minutes dark and then back to blue light exposure. CIBN-GFP-miro1 could be found to colocalize with Opto-PKCɛ after 30 seconds of blue light exposure. Scale bar = 10 µm

(c) Western blot analysis of subcellular fractionation experiment depicting phosphorylation of PKC substrates, E-cadherin, Cox7a and GAPDH of cells containing Opto-PKCɛ with CIBN-GFP-miro1 or CIBN-GFP-CAAX and subjected to 15 minutes of dark or blue light.

(d) Western blot analysis of subcellular fractionation experiment depicting phosphorylation of PKC substrates, ZO-1 and GAPDH of cells containing myr-Opto-PKCɛ and subjected to 15 minutes of dark or blue light.

Sustained PKCɛ activation at the plasma membrane of hepatocytes results in phosphorylation of Insulin Receptor at Thr-1160.

(a) Western blot analysis of subcellular fractionation experiment depicting phosphorylation of PKC substrates, ZO-1 and GAPDH of primary hepatocytes containing transfected plasmids as listed and subjected to 15 minutes of dark or blue light.

(b) Representative Western blots of phosphorylation levels of phosphor-IRK (T1160/T1161), phosphor-IRK (T1158/T1162/T1163), Insulin Receptor, PKCɛ and GAPDH for primary hepatocytes subjected to stated conditions. Quantification (n=3) of normalized phosphorylation levels relative to insulin receptor levels.

(c) Representative Western blots and phosphorylation levels of phosphor-IRK (T1160/T1161), Insulin Receptor, PKCɛ and GAPDH for primary hepatocytes subjected to stated conditions. Quantification (n=3) of phosphor-IRK (T1160/T1161) relative to Insulin Receptor levels.

The data in (b) and (c) are presented as mean +/− SD.

PKCɛ activation at the mitochondria results in reduced oxygen consumption rate.

(a) HEK293T cells transfected with Opto-PKCɛ and CIBN-GFP-miro were subjected to 15 minutes of either dark or blue light. Oxygen Consumption Rate (OCR) measurements were then obtained with an extracellular flux analyser (Seahorse Bioscience). 4um of Oligomycin, 0.5um of FCCP, 1um of Antimycin A and Rotenone were injected into the cells at different time points preset on the machine and OCR response was recorded. 5 sets of experiments are represented.

(b) Quantification (n=5) of the OCR of HEK293T cells transfected with Opto-PKCɛ and CIBN-GFP-miro under different conditions.

(c) HEK293T cells transfected with CIBN-GFP-miro were subjected to either dark or 15 minutes of blue light. Oxygen Consumption Rate (OCR) measurements were then obtained with an extracellular flux analyser (Seahorse Bioscience). Similar inhibitor concentrations were used as in (a).

(d) List of top phosphorylated substrates by PKCɛ that are known to be localized at mitochondria.

(e) Western blots of co-immunoprecipitation experiments of NDUFS4 looking at phosphor-Ser and NDUFS4 levels subjected to stated conditions. Forskolin was added as a positive control.

The data in (a) and (b) are presented as mean +/− SD.