Abstract
Conventional and novel isoenzymes of PKC are activated by the membrane-embedded second messenger diacylglycerol (DAG) through its interactions with the C1 regulatory domain. The affinity of C1 domains to DAG varies considerably among PKCs. To gain insight into the origin of differential DAG affinities, we conducted high-resolution NMR studies of C1B domain from PKCδ (C1Bδ) and its W252Y variant. The W252Y mutation was previously shown to render C1Bδ less responsive to DAG (Dries, D. R., Gallegos, L. L., and Newton, A. C. (2007) A single residue in the C1 domain sensitizes novel protein kinase C isoforms to cellular diacylglycerol production. J. Biol. Chem. 282, 826-830) and thereby emulate the behavior of C1B domains from conventional PKCs that have a conserved Tyr at the equivalent position. Our data revealed that W252Y mutation did not perturb the conformation of C1Bδ in solution but significantly reduced its propensity to partition into a membrane-mimicking environment in the absence of DAG. Using detergent micelles doped with a paramagnetic lipid, we determined that both the residue identity at position 252 and complexation with diacylglycerol influence the geometry of C1Bδ-micelle interactions. In addition, we identified the C-terminal helix α1 of C1Bδ as an interaction site with the head groups of phosphatidylserine, a known activator of PKCδ. Taken together, our studies (i) reveal the identities of C1Bδ residues involved in interactions with membrane-mimicking environment, DAG, and phosphatidylserine, as well as the affinities associated with each event and (ii) suggest that the initial ligand-independent membrane recruitment of C1B domains, which is greatly facilitated by the interfacial partitioning of Trp-252, is responsible, at least in part, for the differential DAG affinities.
Highlights
The activity of protein kinase C isoenzymes (PKCs) is regulated by diacylglycerol (DAG)
W252Y Mutation Decreases the Affinity of C1B␦ to DOG without Significant Perturbations of the Structure—It has been demonstrated that the identity of residue at position 22 has a profound influence on the affinity of conserved region 1 (C1) domains to DAG [35]
One distinct feature of the C1B␦ 15N-1H HSQC spectrum is that the cross-peaks for some residues of the loops 12 and 34 are missing, among them the backbone 1HN-15N resonance of the mutation site Trp-252
Summary
The activity of protein kinase C isoenzymes (PKCs) is regulated by diacylglycerol (DAG). Results: Aromatic residue that tunes DAG affinity influences the recruitment of the conserved region 1 (C1) regulatory domain to a membrane mimic. Significance: This work offers insight into the origin of differential DAG affinities in PKCs. Conventional and novel isoenzymes of PKC are activated by the membrane-embedded second messenger diacylglycerol (DAG) through its interactions with the C1 regulatory domain. Our studies (i) reveal the identities of C1B␦ residues involved in interactions with membrane-mimicking environment, DAG, and phosphatidylserine, as well as the affinities associated with each event and (ii) suggest that the initial ligand-independent membrane recruitment of C1B domains, which is greatly facilitated by the interfacial partitioning of Trp-252, is responsible, at least in part, for the differential DAG affinities
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