Bacterial expression and purification of C1 and C2 domains of protein kinase C isoforms.

Abstract

Protein kinase C (PKC) contains two types of membrane-targeting domains, C1 and C2 domains, in its regulatory region (, , , , ). It has been shown that C1 and C2 domains of conventional PKCs (α, βI, βII, and γ) and C1 domains of novel (δ, ε, θ, and η) play pivotal roles in their subcellular targeting and regulation (, , , , ). C1 domains are small (∼50 amino acids) cysteine-rich structures that contain two structurally important zinc ions (,). In conventional and novel PKCs, C1 domains occur in a tandem repeat (C1A and C1B) and serve as an interaction site for diacylglycerol and its structural analog, phorbol ester (). C1 domains found in atypical PKCs (ζ, and ι/λ), however, do not bind diacylglycerol/phorbol ester because of minor sequence variations and might be involved in protein-protein interactions (). C2 domains (∼130 residues) of PKCs have a common structure in which eight antiparallel β strands are connected by variable loops (,,). For C2 domains of conventional PKCs that bind the membrane in a calcium-dependent manner, three loops located at one side of the domain serve as Ca2+-binding sites. C2 domains of novel PKCs do not bind calcium as a result of the lack of calcium ligands in these loops and might be involved in calcium-independent membrane interactions () and/or protein-protein interactions (,). To elucidate the exact roles of C1 and C2 domains in cellular regulation of PKCs and identify critical residues involved in PKC regulation, it is often necessary to perform in vitro structure-function studies of isolated domains as well as the full-length proteins.