Abstract
The C-terminal V5 domain is one of the most variable domains in Protein Kinase C isoforms (PKCs). V5 confers isoform specificity on its parent enzyme through interactions with isoform-specific adaptor proteins and possibly through specific intra-molecular interactions with other PKC domains. The structural information about V5 domains in solution is sparse. The objective of this work was to determine the conformational preferences of the V5 domain from the α isoform of PKC (V5α) and evaluate its ability to associate with membrane mimetics. We show that V5α and its phosphorylation-mimicking variant, dmV5α, are intrinsically disordered protein domains. Phosphorylation-mimicking mutations do not alter the overall conformation of the polypeptide backbone, as evidenced by the local nature of chemical shift perturbations and the secondary structure propensity scores. However, the population of the “cis-trans” conformer of the Thr638-Pro639-Pro640 turn motif, which has been implicated in the down-regulation of PKCα via peptidyl-prolyl isomerase Pin1, increases in dmV5α, along with the conformational flexibility of the region between the turn and hydrophobic motifs. Both wild type and dmV5α associate with micelles made of a zwitterionic detergent, n-dodecylphosphocholine. Upon micelle binding, V5α acquires a higher propensity to form helical structures at the conserved “NFD” motif and the entire C-terminal third of the domain. The ability of V5α to partition into the hydrophobic micellar environment suggests that it may serve as a membrane anchor during the PKC maturation process.
Highlights
Protein Kinase C isoenzymes (PKCs) are serine/threonine kinases that play key roles in a multitude of signal transduction pathways [1,2]
Ten mammalian PKCs are divided into three classes: conventional (a, bI, bII, and c, Figure 1(A)), which are activated by diacylglycerol and Ca2+; novel (d, e, g, and h), which are activated by diacylglycerol only; and atypical (i/l and f), which are activated by neither cofactor
Analysis of the existing literature suggests that V5 plays at least three roles within its parent protein: (i) it stabilizes the kinase sub-domain through direct interactions with its N-lobe [13,14,15]; (ii) it participates in the auto-inhibitory interactions with the components of the N-terminal regulatory domain [7,13,16,17,18]; and (iii) it mediates sub-cellular localization of PKC isoforms by interacting with the isoform-specific adaptor proteins, receptors for activated C kinases [19]
Summary
Protein Kinase C isoenzymes (PKCs) are serine/threonine kinases that play key roles in a multitude of signal transduction pathways [1,2]. The catalytic domains of PKCs are highly conserved, with the exception of the most C-terminal region comprising 60–70 amino acids. This region is referred to as the ‘‘Variable 5’’, or V5, domain. Analysis of the existing literature suggests that V5 plays at least three roles within its parent protein: (i) it stabilizes the kinase sub-domain through direct interactions with its N-lobe [13,14,15]; (ii) it participates in the auto-inhibitory interactions with the components of the N-terminal regulatory domain [7,13,16,17,18]; and (iii) it mediates sub-cellular localization of PKC isoforms by interacting with the isoform-specific adaptor proteins, receptors for activated C kinases [19]
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