A Dual Regulatory Role for the Disordered C-Terminus of Protein Kinase Cα

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The protein kinase C (PKC) family of enzymes are involved in numerous regulatory processes in the cell and have been associated with many diseases, including cancer and cardiovascular and neurodegenerative diseases. As such, PKCs are attractive targets for therapeutic intervention (1); however, these efforts have been hindered as detailed structural descriptions of intact PKCs in the inactive state have thus far remained elusive. In their inactive cytosolic state, PKCs adopt an autoinhibited conformation. To phosphorylate their cellular substrates, PKCs must be activated by two second messengers, Ca²⁺ and diacylglycerol (DAG). Activation by Ca²⁺ and DAG triggers a conformational rearrangement that allows PKCs to adopt their membrane-bound, catalytically competent state (2). The exact details regarding the influence of the second messengers on the overall conformational state of PKCs and specific intramolecular interactions between catalytic and regulatory domains of PKCs are not yet known. In this issue of Biophysical Journal, Yang and colleagues present the first direct experimental evidence for intramolecular interactions between the disordered C-terminal V5 domain and the C2 regulatory domain in PKCα (3). Intriguingly, these interactions appear to play an important dual role in maintaining sensitivity to Ca²⁺ fluctuations by simultaneously enhancing Ca²⁺ binding as well as minimizing membrane localization in the absence of activating ligands.

The conventional PKCs (α, βI/II, and γ) feature similar domain organization. The conserved C1A, C1B, and C2 domains are responsible for membrane localization. The C1 domains directly interact with the second messenger DAG, whereas the C2 domain functions primarily as a Ca²⁺ sensor (2). A disordered variable region (V5) at the C-terminus of PKCα features three conserved regions, two of which include phosphorylation sites that are crucial for PKCα activation. Prior studies of PKCβII have suggested regulatory roles for interactions between the conserved motifs of V5 and both the C1B and C2 domains (4, 5). In this study, the authors sought to characterize putative interactions between the hydrophobic motif in the V5 region and the C2 domain of PKCα (3). To do so, they designed a series of peptides containing the hydrophobic motif of V5 and characterized their binding to the C2 domain using NMR spectroscopy. Importantly, a single conserved serine residue in the hydrophobic motif is phosphorylated during PKCα activation, and the authors compared binding of hydrophobic motif peptides in both the phosphorylated and nonphosphorylated states, as well as with a phospho-mimicking Ser to Glu mutation. Although all of the hydrophobic motif peptides bound to C2, the chemical shift changes were largest for binding of the phosphorylated peptide, indicating that phosphorylation of Ser657 in the hydrophobic motif is a driving force behind the interaction between V5 and C2 in PKCα. Mapping the chemical shift changes onto the structure of C2 revealed that the hydrophobic motif of V5 primarily interacts with the calcium- and membrane-binding loops and the lysine-rich cluster. Previous studies by the Igumenova group showed that the lysine-rich cluster was similarly affected by binding of the signaling lipid PtdIns(4,5)P₂ (6), suggesting that the hydrophobic motif may compete with membrane-embedded lipids for a shared binding site.

To further understand the interplay between Ca²⁺ and hydrophobic motif binding, Yang et al. compared chemical shift perturbations for binding of the hydrophobic motif peptide to the apo and Ca²⁺-bound forms of the C2 domain (3). The pattern of chemical shift changes due to hydrophobic motif binding was the same for both forms of the C2 domain, but titration-based affinity measurements indicated that the hydrophobic motif bound more tightly when Ca²⁺ was present. Similarly, the affinity of Ca²⁺ was greater for the hydrophobic-motif-bound C2 than apo C2, suggesting a synergistic effect between hydrophobic motif and Ca²⁺ binding.

The origin of this effect can be explained from the structural model of the hydrophobic motif peptide in complex with C2 (3). The hydrophobic motif peptide binds to C2 in an extended conformation, with salt bridges formed between the phosphoserine and the negatively charged side chains of the hydrophobic motif and the lysine-rich cluster on C2, providing energetic stabilization for peptide binding. By binding to the lysine-rich cluster on C2, the V5 hydrophobic motif peptide changes the surface electrostatic potential of C2, making Ca²⁺ binding more favorable. The stabilizing contributions of charged and aromatic residues at the C2-V5 interface within the context of the full-length PKCα were confirmed using a Förster resonance energy transfer-based assay, further validating the structural model obtained using the isolated domains.

These new structural details regarding interdomain interactions in PKCα certainly enhance our understanding of the conformational arrangement of the enzyme in the inactive and partially activated states. By binding to the lysine-rich cluster on C2, the hydrophobic motif could play an autoinhibitory role. To test this hypothesis, Yang et al. characterized the effect of the hydrophobic motif peptide on the in vitro activity of full-length PKCα (3). If the hydrophobic motif has an inhibitory role, it would be expected to decrease the activity of PKCα under fully activating conditions, as was indeed the case. To determine the validity of the proposed mechanism of synergistic binding between the hydrophobic motif and Ca²⁺, the authors tested the effect of adding the hydrophobic motif peptide to the assay in the absence of Ca²⁺ and phosphatidylserine, both of which function as C2 agonists. In the absence of the C2 agonists, the hydrophobic motif peptide was able to activate PKCα, confirming the synergy between V5 and Ca²⁺ binding interactions even in the intact enzyme. These results confirm the importance of the interactions between the hydrophobic motif of the V5 region and the C2 domain; although the interactions between the isolated domains are weak, it is expected that avidity effects would enhance these interactions in the intact enzyme.

This work highlights the structural consequences of the many necessary events that stimulate activation and membrane localization of PKCs, including phosphorylation of the hydrophobic motif in the V5 region, binding of the hydrophobic motif to the C2 domain, and Ca²⁺ binding to the C2 domain, but the exact ordering of these events in vivo is not yet known. It is intriguing to speculate that perhaps a specific sequence of events is not required because of the intrinsic disorder of the V5 region. Disordered regions often form low-affinity, high-specificity interactions with their binding partners, as is the case here for the hydrophobic motif of V5 binding to C2 (7). The extended conformation of the hydrophobic motif in complex with C2 could facilitate access to modifying enzymes, enabling potential phosphorylation of the conserved serine residue within the hydrophobic motif even while bound to the lysine-rich cluster on C2.

The ability of the hydrophobic motif to mask the lysine-rich cluster and screen interactions between the lysine-rich cluster and negatively charged lipids in the membrane before Ca²⁺ binding is an important new finding that broadens our understanding of PKCα regulation and introduces new strategies for targeting and stabilizing the inactive state of PKCs. The structural and functional details presented by Yang and coworkers (3) provide new insight into the conformation of a complex regulatory enzyme. These details will surely inform future studies of PKCs and may contribute to design of novel therapeutic agents to stabilize the inactive state of PKCα.

Editor: Jeff Peng.