Structural basis of protein kinase C isoform function

Abstract

Protein kinase C (PKC) isoforms comprise a family of lipid-activated enzymes that have been implicated in a wide range of cellular functions. PKCs are modular enzymes comprised of a regulatory domain (that contains the membrane-targeting motifs that respond to lipid cofactors, and in the case of some PKCs calcium) and a relatively conserved catalytic domain that binds ATP and substrates. These enzymes are coexpressed and respond to similar stimulatory agonists in many cell types. However, there is growing evidence that individual PKC isoforms subserve unique (and in some cases opposing) functions in cells, at least in part as a result of isoform-specific subcellular compartmentalization patterns, protein-protein interactions, and posttranslational modifications that influence catalytic function. This review focuses on the structural basis for differences in lipid cofactor responsiveness for individual PKC isoforms, the regulatory phosphorylations that control the normal maturation, activation, signaling function, and downregulation of these enzymes, and the intra-/intermolecular interactions that control PKC isoform activation and subcellular targeting in cells. A detailed understanding of the unique molecular features that underlie isoform-specific posttranslational modification patterns, protein-protein interactions, and subcellular targeting (i.e., that impart functional specificity) should provide the basis for the design of novel PKC isoform-specific activator or inhibitor compounds that can achieve therapeutically useful changes in PKC signaling in cells.

FIG. 1

Domain structure of protein kinase C (PKC) isoforms. Top: PKCs have a conserved kinase domain (depicted in teal) and more variable regulatory domains. All PKC regulatory domains have a pseudosubstrate motif (shown in green) NH₂ terminal to the C1 domain (shown in pink). Tandem C1 domains are the molecular sensors of phorbol 12-myristate 13-acetate (PMA)/diacylglycerol (DAG) in cPKC and nPKC isoforms, whereas the single aPKC C1 domain does not bind DAG/PMA. The C2 domains (in yellow) function as calcium-dependent phospholipid binding modules in cPKCs. nPKC C2 domains do not bind calcium; the PKCδ-C2-like domain is a phosphotyrosine interaction module. PKC isoform variable regions are shown in gray. Bottom: ribbon diagrams of PKC C1B domain, C2 domain, and kinase domain structures. Kinase domain phosphorylation sites are discussed in the text. (Figure courtesy of Dr. Alexandra Newton.)

FIG. 2

Alignment of PKC C1 domains. Numbering is based on PKCα. Conserved cysteine and histidine residues are in blue. Other structural determinants of C1 domain function are as indicated on the figure.

FIG. 3

Alignment of PKC C2 domains.

FIG. 4

Alignment of the ATP binding site, invariant lysine, and gatekeeper residues in PKC kinase domains. NES sequences NH₂ terminal to aPKC ATP binding sites are depicted.

FIG. 5

Alignment of kinase domain activation loops. A: Mg positioning loop (in green), activation loop phosphorylation site, invariant tyrosines that are phosphorylated in certain PKCs, and the unique structural features of PKCδ that recently have been considered a mechanism to render this isoform catalytically competent without activation loop phosphorylation are depicted. B: sequence alignment for the A-helix in PKCδ and protein kinase A (PKA) (see text). C: activation loop phosphorylation mechanisms and functions.

FIG. 6

Alignment of kinase domain V5 domain.

FIG. 7

PKCε overexpression leads to PKCδ-Tyr³¹¹/Tyr³³² phosphorylation and increased PKCδ-Thr⁵⁰⁵ autophosphorylation; the mechanisms that control activation loop phosphorylation on native PKCδ do not influence the heterologously overexpressed enzyme. A: schematic of PKCε-PKCδ cross-talk in cells (see text). B: green fluorescent protein (GFP)-tagged PKCδ was heterologously overexpressed in COS7 cells (lanes 2–4). PKCδ overexpression was without (lane 2) or with PKCε overexpression for 24 h (lane 3) or 48 h (lane 4). Samples were subjected to SDS-PAGE and immunblotting for PKCδ protein and PKCδ-Thr⁵⁰⁵ phosphorylation. The heterologously overexpressed protein (labeled in blue) is constitutively Thr⁵⁰⁵-phosphorylated; GFP-PKCδ-Thr⁵⁰⁵ phosphorylation is not increased by PKCε overexpression. Note, while this experiment shows a minor decrease in PKCδ protein and Thr⁵⁰⁵ phosphorylation in the context of PKCε overexpression for 24 h, this was not a consistent finding in replicates of this experiment. In contrast, native PKCδ (labeled in red, which migrates more rapidly than the GFP-tagged PKCδ transgene) is not phosphorylated at Thr⁵⁰⁵ in resting cultures that do not overexpress PKCε; PKCε overexpression induces Thr⁵⁰⁵ (and Tyr³¹¹) phosphorylation on native PKCδ.

FIG. 8

Src-dependent PKCδ tyrosine phosphorylation leads to changes in PKCδ phosphorylation of cardiac troponin I; PKCδ-Tyr³¹¹ and Thr⁵⁰⁵ critically regulate PKCδ substrate specificity.

FIG. 9

Evolving concepts of signaling pathway activation. A: the original concepts of linear signal transduction pathways held that stimuli evoke responses via the actions of transducers (such as G proteins or PKC isoforms). B: models of signal transduction were first revised to allow for the presence of multiple stimuli that can converge on a single signaling pathway and alter the amplitude of a signaling response. This could reflect the activation of different pools of a single enzyme. Alternatively, amplitude control might be due to the differential stimulatory properties of full or partial agonists. C: more current models of signaling transduction allow for a high level of signal integration by proteins (such as PKCδ) that are regulated through conformational changes, translocation events, and various posttranslational modifications (phosphorylation, oxidation, tyrosine nitration, etc.) that can underlie stimulus-specific signaling responses.