Intramolecular conformational changes optimize protein kinase C signaling

Abstract

Optimal tuning of enzyme signaling is critical for cellular homeostasis. We use fluorescence resonance energy transfer reporters in live cells to follow conformational transitions that tune the affinity of a multidomain signal transducer, protein kinase C (PKC), for optimal response to second messengers. This enzyme comprises two diacylglycerol sensors, the C1A and C1B domains, that have a sufficiently high intrinsic affinity for ligand so that the enzyme would be in a ligand-engaged, active state if not for mechanisms that mask its domains. We show that both diacylglycerol sensors are exposed in newly synthesized PKC and that conformational transitions following priming phosphorylations mask the domains so that the lower affinity sensor, the C1B domain, is the primary diacylglycerol binder. The conformational rearrangements of PKC serve as a paradigm for how multimodule transducers optimize their dynamic range of signaling.

Figure 1

Maturation of PKC retards agonist-dependent membrane translocation kinetics. (a) Schematic of cPKCs and nPKCs showing domain composition with the C1A and C1B domains (orange) and the C2 domain (yellow). The W and Y are the Trp and Tyr residues at position 22 within the C1A (W58 in PKCβII) or C1B (Y123 in PKCβII) domains of cPKCs that dictate membrane affinity. nPKCs contain Trp at both of these sites. The kinase domain (cyan) and the three priming phosphorylations are shown: the activation loop, turn motif, and hydrophobic motif. (B) Ribbon structure of the kinase domain of PKCβII (PDB ID 2I0E) showing the three priming phosphorylations in stick form (Thr500, activation loop; Thr641, turn motif; Ser600, hydrophobic motif) and the kinase-inactivating mutations (Lys371 and AspD466 in the active site and Pro616 and Pro619 in the PXXP motif) in ball form. (C) Representative images displaying localization of the indicated YFP- and RFP-tagged PKCs, co-transfected into COS7 cells, before (top), after 2.5 min (middle), or after 12.5 min (bottom) of PDBu treatment are shown. (D) Western blot displaying whole-cell lysates of COS7 cells transfected with the indicated PKCβII constructs. The asterisk denotes the position of mature, fully phosphorylated PKCβII, and the dash denotes the position of unphosphorylated PKCβII.

Figure 2

An intramolecular FRET reporter reads conformational transitions of PKC in live cells. (A) Diagram of Kinameleon showing CFP on N-terminus and YFP on C-terminus of PKC; schematic of how very low FRET reflects an unprimed conformation (upper panel), intermediate FRET reflects a mature (phosphorylated at the activation loop in pink, turn motif in orange, and hydrophobic motif in green) but inactive conformation (middle panel), and high FRET reflects an active conformation (lower panel). (B) Pseudocolor FRET ratio images (left) and localization (right) of MDCK cells transiently expressing Kinameleon-K371R (representing unprocessed PKC), Kinameleon-WT (representing mature, phosphorylated, but inactive PKC), Kinameleon-WT after 15 min of PDBu treatment (representing mature, active PKC), and Kinameleon-WT after 12 hrs of PDBu treatment (representing dephosphorylated PKC), report different PKC conformations. (C) Quantitation of the FRET ratios ± SEM of Kinameleon-WT post PDBu treatment of cells and of Kinameleon-K371R in the absence of PDBu treatment. (D) Kinameleon expressed in MDCK cells and stimulated with 200 nM PDBu results in increased FRET, shown as a change in the FRET ratio (upper panel). The increasing FRET change with higher expression levels (linear regression in red, with 95% confidence bands in green) indicates an intermolecular (concentration dependent) interaction. The non-zero y-intercept indicates an intramolecular (concentration-independent) interaction, consistent with a conformational change upon translocation. In contrast, co-expression of CFP-PKCβII-CFP and YFP-PKCβII-YFP show only an intermolecular interaction after 200 nM PDBu (lower panel).

Figure 3

Translocation kinetics of the isolated C1A-C1B domain of PKCβII can be tuned by a single residue. (A) Ribbon structure of the C1B domain of PKCα (PDB ID 2ELI) showing the DAG affinity toggle, Tyr at position 22 in the domain (Tyr123 in PKCα and PKCβ). This residue is present as Trp (Trp58) in the C1A domain of PKCα and PKCβ. (B) COS7 cells transfected with the indicated C1A-C1B constructs flanked by CFP and YFP were monitored for their intermolecular FRET ratio ± SEM upon PDBu stimulation. (C) Representative YFP images of the basal localization of wild-type or mutant C1A-C1B domains. (D) Trace showing translocation kinetics of the C1A-C1B domain ± SEM with sub-saturating levels of phorbol esters (50 nM PDBu), followed by saturating amounts of PDBu to yield a final concentration of 200 nM.

Figure 4

Unprimed cPKCs have an exposed C1A-C1B tandem module that is masked upon maturation. (A) The FRET ratios of COS7 cells co-transfected with membrane-targeted CFP and the indicated full-length, YFP-tagged PKCβII constructs were monitored upon PDBu treatment. Plots show data normalized to 100% for the maximal FRET response ± SEM. (B) Western blot of whole-cell lysates of COS7 cells transfected with the indicated HA-PKCα constructs. The asterisk denotes the position of mature, fully phosphorylated PKCα, and the dash denotes the position of unphosphorylated PKCα. (C) FRET ratios ± SEM of COS7 cells co-transfected with membrane-targeted CFP and the indicated full-length, YFP-tagged PKCα constructs were monitored upon PDBu stimulation. (D) FRET ratios ± SEM of COS7 cells co-transfected with membrane-targeted CFP and the indicated full-length, YFP-tagged PKCβII constructs were monitored upon stimulation with a sub-saturating PDBu concentration for wild-type PKCβII (50 nM), followed by treatment with another 150 nM PDBu to evoke a maximal response.

Figure 5

Both the C1A and C1B domains of unphosphorylated PKCs are exposed and become masked upon priming of PKC. (A-D) FRET ratios ± SEM of COS7 cells co-transfected with membrane-targeted CFP and the indicated full-length, YFP-tagged PKCβII constructs were monitored upon PDBu treatment. (E) Representative YFP images of localization of the indicated PKCβII or PKCδ mutants before (top) or after (bottom) PDBu treatment.

Figure 6

Both the C1A and C1B domains are involved in membrane binding, but the C1B domain dominates. The FRET ratios ± SEM of COS7 cells co-transfected with membrane-targeted CFP and the indicated full-length, YFP-tagged PKCβII constructs were monitored upon stimulation with the PKC agonists DiC8 and PDBu.

Figure 7

Model showing how maturation of cPKC masks C1 domains to increase the dynamic range of DAG sensing and thus PKC output. (A) Unprimed PKC is in an open conformation that associates with membranes via weak interactions from the C2 domain, both C1A and C1B domains, the exposed pseudosubstrate, and the C-terminal tail. In this conformation, both C1A and C1B domains are fully exposed. (B) Upon ordered phosphorylation of PKC at its activation loop (pink), turn motif (orange), and hydrophobic motif (green) sites, PKC matures into its closed conformation, in which both the C1A and C1B domains become masked, the pseudosubstrate binds the substrate binding site, and the enzyme localizes to the cytosol. This masking of the C1 domains prevents pretargeting of PKC to membranes in the absence of agonist-evoked increases in DAG, thus decreasing basal signaling. (C) In response to agonists that promote PIP₂ hydrolysis, Ca²⁺-dependent binding of the C2 domain of cPKCs to the plasma membrane allows the low-affinity DAG sensor to find its membrane-embedded ligand, DAG. (D) Binding of DAG, predominantly to the C1B domain of PKCβII, expels the pseudosubstrate from the substrate-binding cavity and activates PKC. Use primarily of the lower-affinity C1B domain increases the dynamic range of PKC output as the signal does not saturate as readily using the lower affinity module and allows cPKCs to signal at the plasma membrane as opposed to the Golgi. (E) Dephosphorylation of activated PKC allows it to regain the exposed (open) conformation of unprimed PKC.