Figure 1.
(A) Schematic representing the organisation of PKCε, highlighting the three serine residues of the V3 domain required for 14-3-3 binding denoted in red alongside the kinases responsible for their phosphorylation. (B) Quantitation of multinucleate cells upon treatment of cells with non-targeting siRNA (control) or PKCε siRNA, with expression of siRNA-resistant mouse GFP-PKCε, GFP-PKCε-D532N, GFP-PKCε-S346A-S368A or GFP-PKCε-S350A. Values plotted are means for n = 3 experiments, with error bars denoting SD. Two-hundred cells were counted per condition per experiment. One-way analysis of variance: **** = P ≤ 0.0001; *** = P ≤ 0.001; ns (not significant) = P > 0.05. (C) Representative immunofluorescence images of DLD1 FRT-TREx cells treated with non-targeting siRNA (control) or PKCε siRNA, with expression of either siRNA-resistant mouse GFP-PKCε, GFP-PKCε-D532N or GFP-PKCε-S346A-S368A induced with doxycycline. Cells stained for DNA (blue), tubulin (red) and Aurora B Ser227 phosphorylation (green), with GFP-PKCε in white if expressed. The Aurora B S227 phosphorylation is highlighted in the lower greyscale panels with the midbody region indicated expanded for each image. Scale bar = 20µm. (D) Quantitation of cells with Aurora B pSer227 signal at the midbody. DLD1 FRT-TREx cells with inducible expression of GFP-PKCε, GFP-PKCε-D532N or GFP-PKCε-S346A-S368A were treated with either non-targeting siRNA (control), PKCε siRNA or PKCε siRNA and doxycycline to induce expression of siRNA-resistant mouse GFP-PKCε constructs, and the Aurora B pSer227 signal at the midbody quantified. Values plotted are means for n = 3 experiments, with error bars denoting SD. Twenty cells with midbodies were counted per condition per experiment. One-way analysis of variance: **** = P ≤ 0.0001; ns (not significant) = P > 0.05. (E) Schematic of incorporation of the diazirine amino acid AbK (red star) into the V3 domain (N371AbK) of PKCε. (F–G) Western blots probed with (F) PKCε antibody and (G) phosphoSer346 antibody, showing cross-linking in HEK293T cells expressing GFP-PKCε-N371AbK, GFP-PKCε-N371AbK-S346A or GFP-PKCε-N371AbK-S350A upon exposure to 365 nm UV for 10 min. The upper and lower arrowheads indicate cross-linked (X-link) PKCε and uncross-linked PKCε, respectively. Western blot images are a representative example of three independent experiments. (H) Table showing the number of PKCε peptide spectrum matches (#PSM) identified in the mass spectrometry analysis in samples containing uncross-linked PKCε (PKCε) vs samples containing PKCε cross-linked to 14-3-3 (PKCε-14-3-3). (I) The percentage of the different types of V3 domain PKCε derived phosphopeptides identified in the uncross-linked and 14-3-3 cross-linked sampled.
A S350 dephosphorylated PKCε-14-3-3 complex is required for Aurora B S227 phosphorylation at the midbody.

(A) Schematic representing the organisation of PKCε, highlighting the three serine residues of the V3 domain required for 14-3-3 binding denoted in red alongside the kinases responsible for their phosphorylation. (B) Quantitation of multinucleate cells upon treatment of cells with non-targeting siRNA (control) or PKCε siRNA, with expression of siRNA-resistant mouse GFP-PKCε, GFP-PKCε-D532N, GFP-PKCε-S346A-S368A or GFP-PKCε-S350A. Values plotted are means for n = 3 experiments, with error bars denoting SD. Two-hundred cells were counted per condition per experiment. One-way analysis of variance: **** = P ≤ 0.0001; *** = P ≤ 0.001; ns (not significant) = P > 0.05. (C) Representative immunofluorescence images of DLD1 FRT-TREx cells treated with non-targeting siRNA (control) or PKCε siRNA, with expression of either siRNA-resistant mouse GFP-PKCε, GFP-PKCε-D532N or GFP-PKCε-S346A-S368A induced with doxycycline. Cells stained for DNA (blue), tubulin (red) and Aurora B Ser227 phosphorylation (green), with GFP-PKCε in white if expressed. The Aurora B S227 phosphorylation is highlighted in the lower greyscale panels with the midbody region indicated expanded for each image. Scale bar = 20µm. (D) Quantitation of cells with Aurora B pSer227 signal at the midbody. DLD1 FRT-TREx cells with inducible expression of GFP-PKCε, GFP-PKCε-D532N or GFP-PKCε-S346A-S368A were treated with either non-targeting siRNA (control), PKCε siRNA or PKCε siRNA and doxycycline to induce expression of siRNA-resistant mouse GFP-PKCε constructs, and the Aurora B pSer227 signal at the midbody quantified. Values plotted are means for n = 3 experiments, with error bars denoting SD. Twenty cells with midbodies were counted per condition per experiment. One-way analysis of variance: **** = P ≤ 0.0001; ns (not significant) = P > 0.05. (E) Schematic of incorporation of the diazirine amino acid AbK (red star) into the V3 domain (N371AbK) of PKCε. (F–G) Western blots probed with (F) PKCε antibody and (G) phosphoSer346 antibody, showing cross-linking in HEK293T cells expressing GFP-PKCε-N371AbK, GFP-PKCε-N371AbK-S346A or GFP-PKCε-N371AbK-S350A upon exposure to 365 nm UV for 10 min. The upper and lower arrowheads indicate cross-linked (X-link) PKCε and uncross-linked PKCε, respectively. Western blot images are a representative example of three independent experiments. (H) Table showing the number of PKCε peptide spectrum matches (#PSM) identified in the mass spectrometry analysis in samples containing uncross-linked PKCε (PKCε) vs samples containing PKCε cross-linked to 14-3-3 (PKCε-14-3-3). (I) The percentage of the different types of V3 domain PKCε derived phosphopeptides identified in the uncross-linked and 14-3-3 cross-linked sampled.

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