Research ArticleBiochemistry

Activation of atypical protein kinase C by sphingosine 1-phosphate revealed by an aPKC-specific activity reporter

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Science Signaling  01 Jan 2019:
Vol. 12, Issue 562, eaat6662
DOI: 10.1126/scisignal.aat6662
  • Fig. 1 Development of an aPKC-selective activity reporter.

    (A) The architecture of aCKAR is based on that of the pan-PKC reporter CKAR and consists of monomeric CFP (cyan), the FHA2 domain of Rad53p (blue), an aPKC-selective substrate peptide (red), and monomeric YFP (yellow). In the unphosphorylated state, monomeric CFP and monomeric YFP are in proximity and in an orientation resulting in FRET. Once phosphorylated by aPKC at the Thr within the substrate sequence (highlighted in yellow), the FHA2 domain binds the phosphorylated sequence, resulting in a conformational change that alters the FRET ratio. Ile at the P+3 position (highlighted in green) is critical for the binding of phospho-Thr to the FHA2 domain. The substrate peptide of CKAR was replaced with an aPKC-selective substrate peptide with a sequence corresponding to residues 74 to 87 of IRAP [rat IRAP (U76997)], except Ser80 at the phosphoacceptor site was replaced with Thr (yellow) and the Asn at the P+3 position was replaced with Ile (green). (B) COS7 cells were cotransfected with aCKAR (left) or CKAR (right) and mCherry (Vector) or mCherry-PKMζ. The CFP/YFP FRET (C/Y) emission ratio was quantified as a function of time after the addition of PZ09 (5 μM). The drop in FRET ratio upon addition of inhibitor represents the basal (unstimulated) activity of endogenous aPKC (blue); the additional drop in cells overexpressing PKMζ (red) reflects the basal activity of PKMζ. Data are mean (± SE) C/Y emission ratios normalized to the starting point (1.0) from n ≥ 25 cells. (C) As in (B), except that cells were cotransfected with the indicated reporters and either red fluorescent protein (RFP) or RFP tagged to a construct of the catalytic domain of PKCλ [PKCλ(Cat)]. Data are means ± SE from n ≥ 21 cells. (D) COS7 cells were cotransfected with aCKAR (left) or CKAR (right) and mCherry, mCherry-PKCα, mCherry-PKCβII, mCherry-PKCδ, or mCherry-PKCε. The normalized C/Y emission ratio was quantified as a function of time after the addition of PDBu (200 nM). The increase in FRET ratio represents the agonist-induced activity of these PKC isozymes. Data are means ± SE from n ≥ 16 cells (left) or ≥ 21 cells (right). (E) Knockdown of endogenous expression of PKCζ and PKCι in HeLa cells using siRNAs for human PKCζ and PKCι. HeLa cells were transfected for 48 hours with nontargeting control siRNA, human PKCζ siRNA, or human PKCι siRNA individually. Equal aliquots of lysate were blotted and probed with antibodies against PKCζ, PKCι, and actin (protein loading control). (F) HeLa cells were cotransfected with aCKAR and control siRNA or both PKCζ siRNA and PKCι siRNA. The normalized C/Y emission ratio was quantified as a function of time after the addition of PZ09 (5 μM). Data represent the means ± SE from n ≥ 21 cells. (G) COS7 cells were cotransfected with aCKAR or a construct in which the phosphoacceptor site is mutated to Ala [aCKAR(T/A)] and mCherry or mCherry-PKMζ. The normalized C/Y emission ratio was quantified as a function of time after the addition of PZ09 (5 μM). Data are means ± SE from n ≥ 28 cells. (H) COS7 cells were transfected with aCKAR or the PKA reporter AKAR. The normalized C/Y emission ratio was quantified as a function of time after the addition of forskolin (10 μM) to increase the production of cAMP. Data are means ± SE from n ≥ 28 cells. (I) COS7 cells were cotransfected with aCKAR or the AKT/PKB reporter BKAR and mCherry-Akt1. The normalized C/Y emission ratio was quantified as a function of time after the addition of EGF (50 ng/ml) to activate AKT/PKB. Data are means ± SE from n ≥ 28 cells. (J) COS7 cells were cotransfected with aCKAR or the PKD reporter DKAR and mCherry-PKD1. The normalized C/Y emission ratio was quantified as a function of time after the addition of PDBu (200 nM) to activate PKD (and PKC, which does not phosphorylate this reporter). Data are means ± SE from n ≥ 25 cells.

  • Fig. 2 Basal activity of aPKC is regulated by S1P signaling.

    (A) COS7, HEK293, HeLa, HepG2, MCF7, MDA-MB-231, or SH-SY5Y cells were transfected with aCKAR. The basal activity of endogenous aPKC was measured after the addition of PZ09 (5 μM); dimethyl sulfoxide (DMSO) vehicle was added as a control. The normalized C/Y emission ratio was quantified as a function of time after the addition of PZ09. Data are means ± SE from n ≥ 20 cells. The arrow indicates the point of DMSO vehicle or PZ09 addition. (B) The relative basal activity of endogenous aPKC was quantified from the data in (A) and represents the difference between the C/Y emission ratios summed between 10 and 12 min for the vehicle versus PZ09 treatments. (C) Western blot of lysates from 2.0 × 105 cells of the indicated cell lines probed with antibodies to PKCζ or PKCι. The endogenous abundance of β-actin was also detected using an anti–β-actin antibody. (D) Normalized abundance of PKCζ (left) or PKCι (right) was quantified from the result of (C) and represents the intensity of PKC divided by the intensity of β-actin for each cell type. (E) HeLa cells were transfected with aCKAR and pretreated for 16 hours with DMSO vehicle, LY294002 (20 μM), SKI-II (5 μM), or LY294002 (20 μM) + SKI-II (5 μM). These cells were subsequently (“→” in graph legend) stimulated with DMSO vehicle or 5 μM PZ09 (addition indicated by vertical arrow) to assess the effect of these pretreatments on basal aPKC activity. The normalized C/Y emission ratio was quantified as a function of time. Data are means ± SE from n ≥ 22 cells. (F) As in (E), except experiments were conducted in serum-free media. Data are means ± SE from n ≥ 22 cells. (G) HeLa cells were cotransfected with CKAR fused to the PB1 domain of Par6 (CKAR-PB1Par6; left) or CKAR (right) and mCherry (Vector) or mCherry-PKCζ (PKCζ). Cells were pretreated with DMSO vehicle or SKI-II (5 μM) for 16 hours and then treated with PZ09 (5 μM). The normalized C/Y emission ratio was quantified as a function of time. Data are means ± SE from n ≥ 22 cells. (H) As in (G), except cells were transfected with mCherry-PKCζ (PB1 deletion mutant) [PKCζ(ΔPB1)] where indicated. Data are means ± SE from n ≥ 17 cells.

  • Fig. 3 Basal activity of aPKC is regulated by intracellular S1P.

    (A) HeLa cells were transfected with aCKAR and then pretreated with DMSO vehicle or 5 μM SKI-II for 16 hours; these cells were then loaded with 1 μM caged S1P (C-S1P) for 30 min, washed of extracellular caged S1P, exposed to UV light as described in Materials and Methods, and incubated for another 5 min after photolysis (+hν). Cells were subsequently treated with DMSO vehicle or 5 μM PZ09 to measure the basal activity of endogenous aPKC. The normalized C/Y emission ratio was quantified as a function of time after DMSO vehicle or PZ09 treatment. Data are means ± SE from n ≥ 27 cells. The arrow indicates the point of DMSO vehicle or PZ09 addition. (B) Knockdown of endogenous expression of SphK1 and SphK2 in HeLa cells using siRNAs for human SphK1 and SphK2. HeLa cells were transfected for 48 hours with nontargeting control siRNA, human SphK1 siRNA, or human SphK2 siRNA individually. Relative mRNA expression of SPHK1 and SPHK2 in HeLa cells were analyzed by real-time quantitative polymerase chain reaction (RT-qPCR). Data are means ± SE from at least three independent experiments. (C) HeLa cells were cotransfected with aCKAR and control siRNA, SphK1 siRNA, SphK2 siRNA, or both SphK1 siRNA and SphK2 siRNA. The normalized C/Y emission ratio was quantified as a function of time after the addition of PZ09 (5 μM). Data are means ± SE from n ≥ 20 cells. (D) HeLa cells were first cotransfected with aCKAR and control siRNA (Control) or both SphK1 siRNA and SphK2 siRNA (SphK1/2). Where indicated in the legend, cells were then loaded with 1 μM caged S1P for 30 min, washed of extracellular caged S1P, exposed to UV light, incubated for another 5 min after photolysis (+hν), and, lastly, treated with DMSO vehicle or 5 μM PZ09 to measure basal activity. The normalized C/Y emission ratio was quantified as a function of time after DMSO or PZ09 treatment. Data are means ± SE from n ≥ 57 cells. The arrow indicates the point of DMSO (Vehicle) or PZ09 addition. (E) HeLa cells were transfected with aCKAR, then exposed to vehicle or loaded with 1 μM caged S1P for 30 min, washed of extracellular caged S1P, and pretreated with 10 μM VPC23019 for 5 min before live-cell imaging. Cells were then photolyzed to detect intracellular S1P-induced activation of endogenous aPKC and, lastly, treated with 5 μM PZ09. The normalized C/Y emission ratio was quantified as a function of time after photolysis. Data are means ± SE from n ≥ 37 cells. (F) mRNA expression of S1PR1, S1PR2, S1PR3, S1PR4, and S1PR5 in HeLa cells were analyzed by RT-qPCR and normalized to that of glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Data are means ± SE from at least three independent experiments. (G) HeLa cells were transfected with aCKAR and then pretreated with or without 10 μM VPC23019 (VPC) for 5 min before live-cell imaging. Cells were then stimulated with DMSO vehicle or S1P (100 nM or 10 μM, as indicated) during live-cell imaging and, lastly, treated with 5 μM PZ09. The normalized C/Y emission ratio was quantified as a function of time after photolysis. Data are means ± SE from n ≥ 16 cells.

  • Fig. 4 Direct activation of aPKC by S1P.

    (A) Effect of S1P on activation of PKCζ assessed by an in vitro kinase assay. Kinase activity of purified GST (glutathione S-transferase)–PKCζ was measured in the absence (Vehicle) or presence of 30 μM S1P at the indicated time points. Data are means ± SE from at least three independent experiments. *P < 0.05 and **P < 0.01 versus vehicle at the same time point by Student’s t test. (B) Dose-dependent effects of S1P on activation of PKCζ assessed by an in vitro kinase assay. Kinase activity of purified GST-PKCζ was measured for 60 min in the absence or presence of the indicated concentrations of S1P. Data are means ± SE from at least three independent experiments. *P < 0.05 and **P < 0.01 versus vehicle. (C) Kinase activity of purified GST-PKCζ was measured in the absence or presence of 30 μM S1P or 30 μM S1P + 10 μM PZ09. Data are means ± SE from at least three independent experiments. **P < 0.01. (D) Kinase activity of purified GST-PKCζ was measured in the absence or presence of 30 μM DH-S1P or 30 μM DH-S1P + 10 μM PZ09. Data are means ± SE from at least three independent experiments. *P < 0.05 and **P < 0.01. (E) Kinase activity of purified GST-PKCζ was measured in the absence or presence of 30 μM S1P or 30 μM Sph. Data are means ± SE from at least three independent experiments. **P < 0.01. (F) Kinase activity of purified GST-PKCζ was measured in the presence of Triton X-100 mixed micelles containing 0 to 15 mol % PS and 0 or 5 mol % S1P. Data are means ± SE from three independent experiments. *P < 0.05 and **P < 0.01. (G) Kinase activity of purified GST-PKCζ was measured in the absence or presence of PS (140 μM) with various concentrations of S1P or 10 μM PZ09. Data are means ± SE from at least three independent experiments. **P < 0.01. (H) HeLa cells were cotransfected with CKAR and mCherry-PKCζ or mCherry-PKMζ. Cells were pretreated with DMSO vehicle or 5 μM SKI-II for 16 hours and then treated with 5 μM PZ09. The normalized C/Y emission ratio was quantified as a function of time after PZ09 treatment. Data are means ± SE from n ≥ 25 cells.

  • Fig. 5 Direct binding of S1P to aPKC.

    (A) COS7 cells were transfected with green-fluorescent protein (GFP), GFP-PKCα, GFP-PKCγ, GFP-PKCδ, or GFP-PKCζ. Left: S1P-PKC binding ability was measured by a protein-lipid binding assay with NBD-labeled S1P. Data are means ± SE from at least three independent experiments (*P < 0.05 versus GFP vector control). Right: Immunoprecipitated GFP or GFP-PKC was run on SDS–polyacrylamide gel electrophoresis (SDS-PAGE), and the amount of exogenously expressed GFP or GFP-PKC was detected via Western blot analysis using an anti-GFP antibody. (B) The binding affinity of PKCζ for S1P was assessed using the PLO assay. The interactions between purified GST-PKCζ and various concentrations of S1P were detected using an anti-GST antibody. Data are means ± SE from at least three independent experiments. *P < 0.05 and **P < 0.01 versus 0.03 pmol of S1P by Student’s t test. (C) The binding affinity of PKCζ or the RING domain of TRAF2 for S1P was assessed by a PLO assay. The interaction between GFP, GFP-PKCζ, or GFP-tagged RING domain of TRAF2 (GFP-RING) and 30 pmol of S1P was detected using an anti-GST antibody. Data are means ± SE from at least three independent experiments. **P < 0.05 versus GFP vector control. (D) The binding affinity of PKCζ for related lipids was assessed using the PLO assay. The interactions between purified GST-PKCζ and 30 pmol of S1P, Sph, C16-ceramide (Ceramide), PS, or LPA were detected using an anti-GST antibody. Data are means ± SE from at least three independent experiments. *P < 0.05. (E) Domain schematic of deletion mutants of PKCζ. Structures of full-length PKCζ and six deletion mutants are shown. PB1, PB1 domain; Pseudo, pseudosubstrate segment; C1, C1 domain; PDZ, PDZ domain; Reg, regulatory domain; Cat, catalytic domain; N, N terminus; C, C terminus. (F) The binding affinity of domain deletion mutants of PKCζ for S1P was assessed using the PLO assay. The interactions between RFP- or mCherry-fused full-length PKCζ [RFP-PKCζ (wild-type) or mCherry-PKCζ (wild-type)], PB1 deletion mutant (ΔPB1), pseudosubstrate deletion mutant (ΔPseudo), C1 deletion mutant (ΔC1), regulatory domain deletion mutant (ΔReg) (PKMζ), catalytic domain deletion mutant (ΔCat), or PDZ deletion mutant (ΔPDZ) and 30 pmol of S1P were detected using an anti-GST antibody. The S1P-PKCζ binding affinity was compared to the RFP or mCherry vector control. Data are means ± SE from at least three independent experiments. *P < 0.05 and **P < 0.01 versus vector control.

  • Fig. 6 Identification of critical sites and amino acids for binding of aPKC to S1P.

    (A) Flowchart of the strategy for identifying the critical sites and amino acids of PKCζ for S1P binding and S1P-induced activation is shown. The blue boxes indicate in silico assays, and the red boxes indicate cellular assays. (B) Potential ligand-binding pockets on the surface of the homology model of the catalytic domain of PKCζ predicted using the Schrödinger’s SiteMap algorithm. (C) S1P or Sph was docked to the center of mass position for pocket 1, pocket 2, or pocket 3 on the catalytic domain of PKCζ using the induced fit docking protocol in the Schrödinger package. The five lowest docking scores from the induced fit docking for each pocket are shown in the bar graph (kcal/mol). IFD, induced fit docking. (D) The induced fit docking poses of S1P (carbon atoms in pink) to pocket 1, pocket 2, and pocket 3 of the catalytic domain of human PKCζ are shown with corresponding docking scores. These docking poses have the lowest docking score (kcal/mol) in each pocket. (E) Two-dimensional interaction diagram of the S1P-PKCζ binding as in (D). Negatively charged, positively charged, polar, hydrophobic, and glycine residues at the active site are represented by red, purple, cyan, green, and white spheres, respectively. Hydrogen bonds between the S1P and backbone or side chains are shown in solid pink arrows or dashed pink arrows, respectively. Salt bridges are shown in red-blue lines. Lys82, Lys101, and Arg142 in pocket 1 correspond to Lys265, Lys284, and Arg325 of full-length PKCζ. Arg192 and Lys216 in pocket 2 correspond to Arg375 and Lys399 of full-length PKCζ. Lys330 in pocket 3 corresponds to Lys513 of full-length PKCζ. (F) HeLa cells were cotransfected with aCKAR and mCherry (vector), mCherry-PKCζ (wild-type), mCherry-PKCζ(K265Q/K284Q/R325Q) (mutant for pocket 1; Pocket1mt), mCherry-PKCζ(R375Q/K399Q) (mutant for pocket 2; Pocket2mt), or mCherry-PKCζ(K513Q) (mutant for pocket 3; Pocket3mt). Cells were then loaded with 1 μM caged S1P for 30 min, washed, and pretreated with 10 μM VPC23019 for 5 min before live-cell imaging. Cells were photolyzed to detect intracellular S1P-induced activation of exogenous mCherry-PKCζ. The normalized C/Y emission ratio was quantified as a function of time after photolysis. Data are means ± SE from n ≥ 27 cells. (G) As in (F), except HeLa cells were cotransfected with aCKAR and mCherry (Vector), mCherry-PKCζ (Wild-type), mCherry-PKCζ(R375Q/K399Q) (Pocket2mt), mCherry-PKCζ(R375Q), or mCherry-PKCζ(K399Q) mutant. Data are means ± SE from n ≥ 17 cells. (H) Kinase activity of purified GST-PKCζ (Wild-type) or GST-PKCζ(R375Q/K399Q) (Pocket2mt) was measured in the absence or presence of 30 μM S1P or 140 μM PS. Data are means ± SE from at least three independent experiments. *P < 0.05 by Student’s t test. (I) HeLa cells were cotransfected with aCKAR and mCherry-PKCζ (Wild-type; left) or mCherry-PKCζ(R375Q/K399Q) (Pocket2mt; right). Cells were then pretreated with DMSO vehicle, 20 μM LY294002, or 5 μM SKI-II for 16 hours and then treated with 5 μM PZ09 during live-cell imaging to detect basal activity of exogenous mCherry-PKCζ. The normalized C/Y emission ratio was quantified as a function of time after PZ09 treatment. Data are means ± SE from n ≥ 25 cells. (J) HeLa cells were cotransfected with CKAR-PB1Par6 and mCherry (Vector), mCherry-PKCζ (Wild-type), or mCherry-PKCζ(R375Q/K399Q) (Pocket2mt). Cells were then treated with 5 μM PZ09 during live-cell imaging to detect basal activity of exogenous mCherry-PKCζ. The normalized C/Y emission ratio was quantified as a function of time after PZ09 treatment. Data are means ± SE from n ≥ 16 cells.

  • Fig. 7 S1P-induced basal activity of aPKC is involved in apoptosis resistance.

    (A) HeLa cells were treated with DMSO vehicle, 5 μM SKI-II, 20 μM LY294002 (singly or combined), or 5 μM PZ09 for 12 hours. Cells were then stained with Hoechst 33342, Apopxin Green (Apoptosis), and 7-AAD (Necrosis) and observed by fluorescence microscopy. Scale bar, 20 μm. (B) Percentage of the Apopxin Green+ or 7-AAD+ cells was quantified respectively from the results represented in (A). Data are means ± SE from at least three independent experiments. **P < 0.01 versus vehicle by Student’s t test. (C) HeLa cells were treated with the agents described in (A) for 24 hours. DNA condensation was then observed using Hoechst 33342 staining under fluorescence microscopy, and the percentage of condensed cells was quantified. Data are means ± SE from at least three independent experiments. **P < 0.01 versus vehicle. (D) HeLa cells were treated with DMSO vehicle or 5 μM SKI-II in the presence or absence of serum for 12 hours. Cells were then stained with Hoechst 33342, Apopxin Green, and 7-AAD and then observed by fluorescence microscopy. Scale bar, 20 μm. (E) Percentage of the Apopxin Green+ or 7-AAD+ cells was quantified respectively from the results in (D). Data are means ± SE from at least three independent experiments. *P < 0.05 and **P < 0.01. (F) HeLa cells were treated with DMSO vehicle or 5 μM SKI-II in the presence or absence of serum for 24 hours. DNA condensation was observed using Hoechst 33342 staining under fluorescence microscopy, and the percentage of condensed cells was quantified. Data are means ± SE from at least three independent experiments. *P < 0.05 and **P < 0.01. (G) HeLa cells were transfected with mCherry vector, mCherry-PKCζ, or mCherry-PKMζ. Cells were treated with DMSO vehicle, 5 μM SKI-II, or 5 μM SKI-II + 5 μM PZ09 in serum-free conditions for 12 hours, then stained with Apopxin Green, and observed by fluorescence microscopy. Arrows indicate costained cells. Scale bar, 20 μm. (H) Percentage of the Apopxin Green+ cells within the population of mCherry+ cells was quantified from the results in (G). Data are means ± SE from at least three independent experiments. *P < 0.05 and **P < 0.01. (I) HeLa cells were transfected with mCherry vector, mCherry-PKCζ, or mCherry-PKMζ. Cells were treated as described in (G) for 24 hours. DNA condensation was observed using Hoechst 33342 staining under fluorescence microscopy, and the percentage of condensed cells was quantified. Data are means ± SE from at least three independent experiments. *P < 0.05 and **P < 0.01. (J) HeLa cells were cotransfected with control siRNA or both PKCζ siRNA and PKCι siRNA and mCherry (vector), mCherry-PKCζ, or mCherry-PKCζ(R375Q/K399Q). Cells were then serum-starved with serum-free medium for 12 hours. Cells were stained with Apopxin Green and then observed using fluorescence microscopy. Arrows indicate costained cells. Scale bar, 20 μm. (K) Percentage of the Apopxin Green+ cells within the population of mCherry+ cells was quantified from the results in (J). Data are means ± SE from at least three independent experiments. **P < 0.01. (L) Model depicting the proposed mechanism of S1P-mediated activation of aPKC that promotes a basal signaling output that suppresses apoptosis. aPKC is autoinhibited by interaction of the pseudosubstrate (Pseudo) with the substrate-binding cavity of the kinase domain (blue circle, top left). S1P, constitutively produced from Sph by SphK, binds to a pocket with basic amino acids (++), Arg375 and Lys399, close to the substrate-binding site in the kinase domain; this interaction displaces the pseudosubstrate to allow substrate binding and downstream signaling. This S1P-induced basal activation of aPKC promotes resistance to apoptosis. P, phosphate; ADP, adenosine 5′-diphosphate.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/12/562/eaat6662/DC1

    Fig. S1. Phosphatase sensitivity of aCKAR is greater than that of CKAR.

    Fig. S2. S1P signaling does not affect the Par6-regulated basal activity of PKCζ in breast cancer cells.

    Fig. S3. Identification of critical sites and amino acids for PKCι-S1P binding in silico.

    Fig. S4. Apoptotic nuclear morphology images.

  • This PDF file includes:

    • Fig. S1. Phosphatase sensitivity of aCKAR is greater than that of CKAR.
    • Fig. S2. S1P signaling does not affect the Par6-regulated basal activity of PKCζ in breast cancer cells.
    • Fig. S3. Identification of critical sites and amino acids for PKCι-S1P binding in silico.
    • Fig. S4. Apoptotic nuclear morphology images.

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